ALBERT

Beschreibung: Publication year: 2012 Source: FEBS Open Bio Shoji Ohuchi, Yusuke Mori, Yoshikazu Nakamura Aptamers are promising gene components that can be used for the construction of synthetic gene circuits. In this study, we isolated an RNA aptamer that specifically inhibits transcription of T7 RNA polymerase (RNAP). The 38-nucleotide aptamer, which was a shortened variant of an initial SELEX isolate, showed moderate inhibitory activity. By stepwise doped-SELEX, we isolated evolved variants with strong inhibitory activity. A 29-nucleotide variant of a doped-SELEX isolate showed 50% inhibitory concentration at 11 nM under typical in vitro transcription conditions. Pull-down experiments revealed that the aptamer inhibited the association of T7 RNAP with T7 promoter DNA. Highlights ▸ An inhibitory RNA aptamer against T7 RNA polymerase was isolated. ▸ Evolved aptamers with strong inhibitory activity were isolated by doped-SELEX. ▸ Aptamer sequence/structure requirements were elucidated based on phylogeny. ▸ A 29-nucleotide variant of an evolved aptamer has an IC 50 of 11 nM. ▸ The aptamer inhibits the association of T7 RNA polymerase with T7 promoter DNA.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio Zhanxiang Wang, Debbie C. Thurmond Human type 2 diabetes is associated with β-cell apoptosis, and human islets from diabetic donors are ∼80% deficient in PAK1 protein. Toward addressing linkage of PAK1 to β-cell survival, PAK1-siRNA targeted MIN6 β-cells were found to exhibit increased caspase-3 cleavage, cytosolic cytochrome-C and the pro-apoptotic protein Bad. PAK1 +/− heterozygous mouse islets recapitulated the upregulation of Bad protein expression, as did hyperglycemic treatment of human or mouse islets; Bad levels were exacerbated most in PAK1+/- islets subjected to hyperglycemic stress. These data implicate PAK1 in β-cell survival via quenching of Bad protein expression, and suggest PAK1 as potential molecular target to preserve β-cell mass. Highlights ▸ Bad expression in islets was increased by chronic exposure to hyperglycemic conditions. ▸ Depletion of PAK1 in the absence of glucose induced Bad expression. ▸ Bad expression increased most in PAK1 +/− heterozygous islets subjected to hyperglycemia. ▸ PAK1 is required to protect against β-cell apoptosis under hyperglycemic conditions.

Beschreibung: Background: Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed. Results: Screening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100. Conclusions: The present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens.

Beschreibung: Background: Sialic acid (N-acetylneuraminic acid; NeuNAc) is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung. Results: In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc) were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells. Conclusions: The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media.

Beschreibung: Background: Y. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic. Variation and the population structure among the clinical Y. enterocolitica BT 1A isolates have been poorly characterized. We used multi-locus sequence typing (MLST), 16S rRNA gene sequencing, PCR for ystA and ystB, lipopolysaccharide analysis, phage typing, human serum complement killing assay and analysis of the symptoms of the patients to characterize 298 clinical Y. enterocolitica BT 1A isolates in order to evaluate their relatedness and pathogenic potential. Results: A subset of 71 BT 1A strains, selected based on their varying LPS patterns, were subjected to detailed genetic analyses. The MLST on seven house-keeping genes (adk, argA, aroA, glnA, gyrB, thrA, trpE) conducted on 43 of the strains discriminated them into 39 MLST-types. By Bayesian analysis of the population structure (BAPS) the strains clustered conclusively into two distinct lineages, i.e. Genetic groups 1 and 2. The strains of Genetic group 1 were more closely related (97% similarity) to the pathogenic bio/serotype 4/O:3 strains than Genetic group 2 strains (95% similarity). Further comparison of the 16S rRNA genes of the BT 1A strains indicated that altogether 17 of the 71 strains belong to Genetic group 2. On the 16S rRNA analysis, these 17 strains were only 98% similar to the previously identified subspecies of Y. enterocolitica. The strains of Genetic group 2 were uniform in their pathogenecity-related properties: they lacked the ystB gene, belonged to the same LPS subtype or were of rough type, were all resistant to the five tested yersiniophages, were largely resistant to serum complement and did not ferment fucose. The 54 strains in Genetic group 1 showed much more variation in these properties. The most commonly detected LPS types were similar to the LPS types of reference strains with serotypes O:6,30 and O:6,31 (37%), O:7,8 (19%) and O:5 (15%). Conclusions: The results of the present study strengthen the assertion that strains classified as Y. enterocolitica BT 1A represent more than one subspecies. Especially the BT 1A strains in our Genetic group 2 commonly showed resistance to human serum complement killing, which may indicate pathogenic potential for these strains. However, their virulence mechanisms remain unknown.

Beschreibung: Background: Adaptive responses in fungi result from the interaction of membrane receptors and extracellular ligands. Many different classes of receptors have been described in eukaryotic cells. Recently a new family of receptors classified as belonging to the progesterone-adiponectin receptor (PAQR) family has been identified. These receptors have the seven transmembrane domains characteristic of G-protein coupled receptors, but their activity has not been associated directly to G proteins. They share sequence similarity to the eubacterial hemolysin III proteins. Results: A new receptor, SsPAQR1 (Sporothrix schenckii progesterone-adiponectinQ receptor1), was identified as interacting with Sporothrix schenckii G protein alpha subunit SSG-2 in a yeast two-hybrid assay. The receptor was identified as a member of the PAQR family. The cDNA sequence revealed a predicted ORF of 1542 bp encoding a 514 amino acids protein with a calculated molecular weight of 57.8 kDa. Protein domain analysis of SsPAQR1 showed the 7 transmembrane domains (TM) characteristic of G protein coupled receptors and the presence of the distinctive motifs that characterize PAQRs. A yeast-based assay specific for PAQRs identified progesterone as the agonist. S. schenckii yeast cells exposed to progesterone (0.50 mM) showed an increase in intracellular levels of 3[PRIME], 5[PRIME] cyclic adenosine monophosphate (cAMP) within the first min of incubation with the hormone. Different progesterone concentrations were tested for their effect on the growth of the fungus. Cultures incubated at 35[DEGREE SIGN]C did not grow at concentrations of progesterone of 0.05 mM or higher. Cultures incubated at 25[DEGREE SIGN]C grew at all concentrations tested (0.01 mM-0.50 mM) with growth decreasing gradually with the increase in progesterone concentration. Conclusion: This work describes a receptor associated with a G protein alpha subunit in S. schenckii belonging to the PAQR family. Progesterone was identified as the ligand. Exposure to progesterone increased the levels of cAMP in fungal yeast cells within the first min of incubation suggesting the connection of this receptor to the cAMP signalling pathway. Progesterone inhibited the growth of both the yeast and mycelium forms of the fungus, with the yeast form being the most affected by the hormone.

Beschreibung: Background: Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. Results: Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 epsilon-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum DeltacrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum DeltacrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 +/- 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum DeltacrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 +/- 0.3 mg/g CDW were obtained. Conclusion: C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.

Beschreibung: Background: Pockmarks (depressions in the seabed) have been discovered throughout the world's oceans and are often related to hydrocarbon seepage. Although high concentrations of pockmarks are present in the seabed overlaying the Troll oil and gas reservoir in the northern North Sea, geological surveys have not detected hydrocarbon seepage in this area at the present time. In this study we have used metagenomics to characterize the prokaryotic communities inhabiting the surface sediments in the Troll area in relation to geochemical parameters, particularly related to hydrocarbon presence. We also investigated the possibility of increased potential for methane oxidation related to the pockmarks. Five metagenomes from pockmarks and plain seabed sediments were sequenced by pyrosequencing (Roche/454) technology. In addition, two metagenomes from seabed sediments geologically unlikely to be influenced by hydrocarbon seepage (the Oslofjord) were included. The taxonomic distribution and metabolic potential of the metagenomes were analyzed by multivariate analysis and statistical comparisons to reveal variation within and between the two sampling areas. Results: The main difference identified between the two sampling areas was an overabundance of predominantly autotrophic nitrifiers, especially Nitrosopumilus, and oligotrophic marine Gammaproteobacteria in the Troll metagenomes compared to the Oslofjord. Increased potential for degradation of hydrocarbons, especially aromatic hydrocarbons, was detected in two of the Troll samples: one pockmark sample and one from the plain seabed. Although presence of methanotrophic organisms was indicated in all samples, no overabundance in pockmark samples compared to the Oslofjord samples supports no, or only low level, methane seepage in the Troll pockmarks at the present time. Conclusions: Given the relatively low content of total organic carbon and great depths of hydrocarbon containing sediments in the Troll area, it is possible that at least part of the carbon source available for the predominantly autotrophic nitrifiers thriving in this area originates from sequential prokaryotic degradation and oxidation of hydrocarbons to CO2. By turning CO2 back into organic carbon this subcommunity could play an important environmental role in these dark oligotrophic sediments. The oxidation of ammonia to nitrite and nitrate in this process could further increase the supply of terminal electron acceptors for hydrocarbon degradation.

Beschreibung: Background: Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between the use of peptides by lactic acid bacteria and biogenic amine synthesis. Results: Here we demonstrate for the first time that a Lactobacillus plantarum strain isolated from a red wine can produce the biogenic amine tyramine from peptides containing tyrosine. In our conditions, most of the tyramine was produced during the late exponential growth phase, coinciding with the expression of the tyrDC and tyrP genes. The DNA sequences of tyrDC and tyrP in this strain share 98% identity with those in Lactobacillus brevis consistent with horizontal gene transfer from L. brevis to L. plantarum. Conclusion: Peptides amino acids are precursors of biogenic amines for Lactobacillus plantarum strain IR BL0076.

Beschreibung: Background: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. Results: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). Conclusions: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.

Beschreibung: Background: Interstitial Cystitis (IC) is a chronic inflammatory condition of the bladder with unknown etiology. The aim of this study was to characterize the microbial community present in the urine from IC female patients by 454 high throughput sequencing of the 16S variable regions V1V2 and V6. The taxonomical composition, richness and diversity of the IC microbiota were determined and compared to the microbial profile of asymptomatic healthy female (HF) urine. Results: The composition and distribution of bacterial sequences differed between the urine microbiota of IC patients and HFs. Reduced sequence richness and diversity were found in IC patient urine, and a significant difference in the community structure of IC urine in relation to HF urine was observed. More than 90% of the IC sequence reads were identified as belonging to the bacterial genus Lactobacillus, a marked increase compared to 60% in HF urine. Conclusion: The 16S rDNA sequence data demonstrates a shift in the composition of the bacterial community in IC urine. The reduced microbial diversity and richness is accompanied by a higher abundance of the bacterial genus Lactobacillus, compared to HF urine. This study demonstrates that high throughput sequencing analysis of urine microbiota in IC patients is a powerful tool towards a better understanding of this enigmatic disease.

Beschreibung: Background: Biofilm production is an important mechanism for bacterial survival and its association with antimicrobial resistance represents a challenge for the patient treatment. In this study we evaluated the in vitro action of macrolides in combination with anti-pseudomonal agents on biofilm-grown Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patient. Results: A total of 64 isolates were analysed. The (BIC) results were consistently higher than those minimal inhibitory concentration (MIC) for most anti-pseudomonal agents tested (ceftazidime: P = 0.001, ciprofloxacin: P = 0.234, tobramycin: P = 0.001, imipenem: P 〈 0.001, meropenem: P = 0.005). When macrolides were associated with the anti-pseudomonal agents, the BIC values were reduced significantly for ceftazidime (P 〈 0.001) and tobramycin (P 〈 0.001), regardless the concentration of macrolides. Strong (IQ) was observed when azithromycin at 8 mg/L was associated with all anti-pseudomonal agents tested in biofilm conditions. Conclusions: P. aeruginosa from CF patients within biofilms are highly resistant to antibiotics but macrolides proved to augment the in vitro activity of anti-pseudomonal agents.

Beschreibung: Background: Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a beta-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. Results: A potential pelgipeptin synthetase gene cluster (plp) was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPS), with one, seven, and one module(s), respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1) provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. Conclusions: In this study, a gene cluster (plp) responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.

Beschreibung: Background: Bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics. Results: A small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118). Conclusions: Small RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.

Beschreibung: Background: Small size eukaryotes play a fundamental role in the functioning of coastal ecosystems, however, the way in which these micro-organisms respond to combined effects of water temperature, UVB radiations (UVBR) and nutrient availability is still poorly investigated. Results: We coupled molecular tools (18S rRNA gene sequencing and fingerprinting) with microscope-based identification and counting to experimentally investigate the short-term responses of small eukaryotes (

Beschreibung: Background: In the past decade, researchers have proposed that the pldA gene for outer membrane phospholipase A (OMPLA) is important for bacterial colonization of the human gastric ventricle. Several conserved Helicobacter pylori genes have distinct genotypes in different parts of the world, biogeographic patterns that can be analyzed through phylogenetic trees. The current study will shed light on the importance of the pldA gene in H. pylori. In silico sequence analysis will be used to investigate whether the bacteria are in the process of preserving, optimizing, or rejecting the pldA gene. The pldA gene will be phylogenetically compared to other housekeeping (HK) gene, and a possible origin via horizontal gene transfer (HGT) will be evaluated through both at intra- and inter-species evolutionary analyses. Results: In this study, pldA gene sequences were phylogenetically analyzed and compared with a large reference set of concatenated HK gene sequences. A total of 246 pldA nucleotide sequences were used; 207 were from Norwegian isolates, 20 were from Korean isolates, and 19 were from the NCBI database. Best-fit evolutionary models were determined with MEGA5 ModelTest for the pldA (K80 + I + G) and HK (GTR + I + G) sequences, and maximum likelihood trees were constructed. Both HK and pldA genes showed biogeographic clustering. Horizontal gene transfer was inferred based on significantly different GC contents, the codon adaptation index, and a phylogenetic conflict between a tree of OMPLA protein sequences representing 171 species and a tree of the AtpA HK protein for 169 species. Although a vast majority of the residues in OMPLA were predicted to be under purifying selection, sites undergoing positive selection were also found. Conclusions: Our findings indicate that the pldA gene could have been more recently acquired than seven of the HK genes found in H. pylori. However, the common biogeographic patterns of both the HK and pldA sequences indicated that the transfer occurred long ago. Our results indicate that the bacterium is preserving the function of OMPLA, although some sites are still being evolutionarily optimized.

Beschreibung: Background: The compatible solute trehalose is involved in the osmostress response of Rhizobium etli, the microsymbiont of Phaseolus vulgaris. In this work, we reconstructed trehalose metabolism in R. etli, and investigated its role in cellular adaptation and survival to heat and desiccation stress under free living conditions. Results: Besides trehalose as major compatible solute, R. etli CE3 also accumulated glutamate and, if present in the medium, mannitol. Putative genes for trehalose synthesis (otsAB/treS/treZY), uptake (aglEFGK/thuEFGK) and degradation (thuAB/treC) were scattered among the chromosome and plasmids p42a, p42c, p42e, and p42f, and in some instances found redundant. Two copies of the otsA gene, encoding trehalose-6-P-synthase, were located in the chromosome (otsAch) and plasmid p42a (otsAa), and the latter seemed to be acquired by horizontal transfer. High temperature alone did not influence growth of R. etli, but a combination of high temperature and osmotic stress was more deleterious for growth than osmotic stress alone. Although high temperature induced some trehalose synthesis by R. etli, trehalose biosynthesis was mainly triggered by osmotic stress. However, an otsAch mutant, unable to synthesize trehalose in minimal medium, showed impaired growth at high temperature, suggesting that trehalose plays a role in thermoprotection of R. etli. Desiccation tolerance by R. etli wild type cells was dependent of high trehalose production by osmotic pre-conditioned cells. Cells of the mutant strain otsAch showed ca. 3-fold lower survival levels than the wild type strain after drying, and a null viability after 4 days storage. Conclusions: Our findings suggest a beneficial effect of osmotic stress in R. etli tolerance to desiccation, and an important role of trehalose on the response of R. etli to high temperature and desiccation stress.Mercedes Reina-Bueno and Montserrat Argandona contributed equally to this work

Beschreibung: Background: The cell envelope of a bacterial pathogen can be damaged by harsh conditions in theenvironment outside a host and by immune factors during infection. Cell envelope stressresponses preserve the integrity of this essential compartment and are often required forvirulence. Bordetella species are important respiratory pathogens that possess a large number of putative transcription factors. However, no cell envelope stress responses have beendescribed in these species. Among the putative Bordetella transcription factors are a numberof genes belonging to the extracytoplasmic function (ECF) group of alternative sigma factors,some of which are known to mediate cell envelope stress responses in other bacteria. Here weinvestigate the role of one such gene, sigE, in stress survival and pathogenesis of Bordetellabronchiseptica. Results: We demonstrate that sigE encodes a functional sigma factor that mediates a cell envelopestress response. Mutants of B. bronchiseptica strain RB50 lacking sigE are more sensitive tohigh temperature, ethanol, and perturbation of the envelope by SDS-EDTA and certain beta-lactam antibiotics. Using a series of immunocompromised mice deficient in differentcomponents of the innate and adaptive immune responses, we show that SigE plays animportant role in evading the innate immune response during lethal infections of mice lackingB cells and T cells. SigE is not required, however, for colonization of the respiratory tract ofimmunocompetent mice. The sigE mutant is more efficiently phagocytosed and killed byperipheral blood polymorphonuclear leukocytes (PMNs) than RB50, and exhibits decreasedcytotoxicity toward macrophages. These altered interactions with phagocytes couldcontribute to the defects observed during lethal infection. Conclusions: Much of the work on transcriptional regulation during infection in B. bronchiseptica hasfocused on the BvgAS two-component system. This study reveals that the SigE regulon alsomediates a discrete subset of functions associated with virulence. SigE is the first cellenvelope stress-sensing system to be described in the bordetellae. In addition to its roleduring lethal infection of mice deficient in adaptive immunity, our results indicate that SigEis likely to be important for survival in the face of stresses encountered in the environmentbetween hosts.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio Johann M. Gostner, Sebastian Schröcksnadel, Kathrin Becker, Marcel Jenny, Harald Schennach, Florian Überall, Dietmar Fuchs Antimalarial chloroquine is also used for the treatment of immune-mediated diseases. The interference of chloroquine with interferon-γ-induced tryptophan breakdown and neopterin production has been investigated in human peripheral blood mononuclear cells (PBMC) in vitro . Micromolar concentrations (2–50 μM) of chloroquine dose-dependently suppressed mitogen-induced tryptophan breakdown in PBMC but not in the myelomonocytic THP-1 -Blue cell line, after 48 h of treatment. In stimulated PBMC, neopterin production was super-induced by 10 μM chloroquine, while it was significantly suppressed at a concentration of 50 μM. These anti-inflammatory effects may relate to the therapeutic benefit of chloroquine in inflammatory conditions and may widen the spectrum of its clinical applications. Highlights ▸ 2–50 μM chloroquine suppresses mitogen-induced tryptophan breakdown in human PBMCs. ▸ The same effect was not seen in the myelomonocytic THP-1 Blue cell line. ▸ The anti-inflammatory property of chloroquine targets T cells more than monocytes. ▸ This anti-inflammatory effect may explain the drug's therapeutic benefit for malaria. ▸ Chloroquine treatment could be of benefit for other chronic inflammatory conditions.

Beschreibung: Background: The accumulation of thick stagnant mucus provides a suitable environment for the growth of Pseudomonas aeruginosa and Staphylococcus aureus within the lung alveoli of cystic fibrosis (CF) patients. These infections cause significant lung damage, leading to respiratory failure and death. In an artificial mucin containing medium ASM+, P. aeruginosa forms structures that resemble typical biofilms but are not attached to any surface. We refer to these structures as biofilm like structures (BLS). Using ASM+ in a static microtiter plate culture system, we examined the roles of mucin, extracellular DNA, environmental oxygen (EO2), and quorum sensing (QS) in the development of biofilm-like structures (BLS) by P. aeruginosa; and the effect of EO2 and P. aeruginosa on S. aureus BLS. Results: Under 20% EO2, P. aeruginosa strain PAO1 produced BLS that resemble typical biofilms but are confined to the ASM+ and not attached to the surface. Levels of mucin and extracellular DNA within the ASM+ were optimized to produce robust well developed BLS. At 10% EO2, PAO1 produced thicker, more developed BLS, while under 0% EO2, BLS production was diminished. In contrast, the S. aureus strain AH133 produced well-developed BLS only under 20% EO2. In PAO1, loss of the QS system genes rhlI and rhlR affected the formation of BLS in ASM+ in terms of both structure and architecture. Whether co-inoculated into ASM+ with AH133, or added to established AH133 BLS, PAO1 eliminated AH133 within 48--56 h. Conclusions: The thick, viscous ASM+, which contains mucin and extracellular DNA levels similar to those found in the CF lung, supports the formation of biofilm-like structures similar to the aggregates described within CF airways. Alterations in environmental conditions or in the QS genes of P. aeruginosa, as occurs naturally during the progression of CF lung infection, affect the architecture and quantitative structural features of these BLS. Thus, ASM+ provides an in vitro medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.

Beschreibung: Background: Two important plant pathogenic bacteria Acidovorax oryzae and Acidovorax citrulli are closely related and often not easy to be differentiated from each other, which often resulted in a false identification between them based on traditional methods such as carbon source utilization profile, fatty acid methyl esters, and ELISA detection tests. MALDI-TOF MS and Fourier transform infrared (FTIR) spectra have recently been successfully applied in bacterial identification and classification, which provide an alternate method for differentiating the two species. Results: Characterization and comparison of the 10 A. oryzae strains and 10 A. citrulli strains were performed based on traditional bacteriological methods, MALDI-TOF MS, and FTIR spectroscopy. Our results showed that the identity of the two closely related plant pathogenic bacteria A. oryzae and A. citrulli was able to be confirmed by both pathogenicity tests and species-specific PCR, but the two species were difficult to be differentiated based on Biolog and FAME profile as well as 16S rRNA sequence analysis. However, there were significant differences in MALDI-TOF MS and FTIR spectra between the two species of Acidovorax. MALDI-TOF MS revealed that 22 and 18 peaks were specific to A. oryzae and A. citrulli, respectively, while FTIR spectra of the two species of Acidovorax have the specific peaks at 1738, 1311, 1128, 1078, 989 cm-1 and at 1337, 968, 933, 916, 786 cm-1, respectively. Conclusions: This study indicated that MALDI-TOF MS and FTIR spectra may give a new strategy for rapid bacterial identification and differentiation of the two closely related species of Acidovorax.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Nicolas Sprynski, Christine Felix, David O’Callaghan, Annette C. Vergunst Complementation for virulence of a non-polar virB5 mutant in Brucella suis 1330 was not possible using a pBBR-based plasmid but was with low copy vector pGL10. Presence of the pBBR-based replicon in wildtype B. suis had a dominant negative effect, leading to complete attenuation in J774 macrophages. This was due to pleiotropic effects on VirB protein expression due to multiple copies of the virB promoter region and over expression of VirB5. Functional complementation of mutants in individual components of multiprotein complexes such as bacterial secretion systems, are often problematic; this study highlights the importance of using a low copy vector. Highlights ► A Brucella suis virB5 mutant is attenuated in J774 macrophages. ► VirB5 over expression and/or multiple virB promoter copies attenuate WT B. suis. ► VirB5 over expression and/or multiple virB promoter copies affect other VirB proteins. ► Expressing virB5 from a low copy number plasmid fully complements a virB5 mutant.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Kenta Arai, Fumio Kumakura, Michio Iwaoka Redox-coupled folding pathways of bovine pancreatic ribonuclease A (RNase A) with four intramolecular disulfide (SS) bonds comprise three phases: (I) SS formation to generate partially oxidized intermediate ensembles with no rigid folded structure; (II) SS rearrangement from the three SS intermediate ensemble (3S) to the des intermediates having three native SS linkages; (III) final oxidation of the last native SS linkage to generate native RNase A. We previously demonstrated that DHS ox , a water-soluble selenoxide reagent for rapid and quantitative SS formation, allows clear separation of the three folding phases. In this study, the main conformational folding phase (phase II) has been extensively analyzed at pH 8.0 under a wide range of temperatures (5–45 °C), and thermodynamic and kinetic parameters for the four des intermediates were determined. The free-energy differences (Δ G ) as a function of temperature suggested that the each SS linkage has different thermodynamic and kinetic roles in stability of the native structure. On the other hand, comparison of the rate constants and the activation energies for 3S → des with those reported for the conformational folding of SS-intact RNase A suggested that unfolded des species (desU) having three native SS linkages but not yet being folded are involved in very small amounts (〈1%) in the 3S intermediate ensemble and the desU species would gain the native-like structures via X-Pro isomerization like SS-intact RNase A. It was revealed that DHS ox is useful for kinetic and thermodynamic analysis of the conformational folding process on the oxidative folding pathways of SS-reduced proteins. Graphical abstract Graphical abstract Highlights ► Three phases of the oxidative folding of RNase A were temporally separated using DHS ox . ► Thermodynamic and kinetic parameters of the four des intermediates were obtained using DHS ox . ► Each SS linkage in the native RNase A has different thermodynamic and kinetic roles. ► Unfolded des species (desU) gain native-like structures via X-Pro isomerization. ► All four des species were isolated and allowed to fold to N under aerobic conditions.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Patrick J. Halvey, Daniel C. Liebler, Robbert J.C. Slebos Agents to induce readthrough of premature termination codons (PTCs) are useful research tools and potential therapeutics. Reporters used to detect PTC readthrough are gene-specific and thus are not suited to for general assessment of readthrough activity or in cases where PTC-inactivated genes are unknown. Here we describe a GFP-based reporter construct pMHG-W57 ∗ which is capable of detecting dose-dependent drug-induced PTC readthrough both by fluorescence microscopy and flow cytometry. pMHG-W57 ∗ may be used as a general indicator of PTC readthrough in living cells and obviates the need for gene-specific recoding sequences in reporter constructs. Highlights ► Current reporter systems for translational readthrough are gene-specific and cannot be used in living cells. ► We developed a readthrough reporter based on green fluorescent protein. ► The reporter is gene-independent and can be used to sort living cells based on fluorescence.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Nan Zhang, Martin Buck The widely distributed bacterial σ 54 -dependent transcription regulates pathogenicity and numerous adaptive responses in diverse bacteria. Formation of the σ 54 -dependent open promoter complex is a multi-step process driven by AAA + ATPases. Non-hydrolysable nucleotide analogues are particularly suitable for studying such complexity by capturing various intermediate states along the energy coupling pathway. Here we report a novel ATP analogue, ADP–MgF 3 − , which traps an AAA + ATPase with its target σ 54 . The MgF 3 − -dependent complex is highly homogeneous and functional assays suggest it may represent an early transcription intermediate state valuable for structural studies. Highlights ► Novel ATP analogue ADP–MgF 3 − traps an AAA + ATPase with its target σ 54 . ► MgF 3 − -dependent complex forms a highly homogenous population. ► MgF 3 − -dependent complex represents an early transcription intermediate state.

Beschreibung: Background: Staphylococcus aureus is major human and animal pathogen. Plasmids often carry resistance genes and virulence genes that can disseminate through S. aureus populations by horizontal gene transfer (HGT) mechanisms. Sequences of S. aureus plasmids in the public domain and data from multi-strain microarrays were analysed to investigate (i) the distribution of resistance genes and virulence genes on S. aureus plasmids, and (ii) the distribution of plasmids between S. aureus lineages. Results: A total of 21 plasmid rep gene families, of which 13 were novel to this study, were characterised using a previously proposed classification system. 243 sequenced plasmids were assigned to 39 plasmid groups that each possessed a unique combination of rep genes. We show some resistance genes (including ermC and cat) and virulence genes (including entA, entG, entJ, entP) were associated with specific plasmid groups suggesting there are genetic pressures preventing recombination of these genes into novel plasmid groups. Whole genome microarray analysis revealed that plasmid rep, resistance and virulence genes were associated with S. aureus lineages, suggesting restriction-modification (RM) barriers to HGT of plasmids between strains exist. Conjugation transfer (tra) complex genes were rare. Conclusion: This study argues that genetic pressures are restraining the spread of resistance and virulence genes amongst S. aureus plasmids, and amongst S. aureus populations, delaying the emergence of fully virulent and resistant strains.

Beschreibung: Background: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. Results: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. Conclusions: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.

Beschreibung: Background: The pbr resistance operon from Cupriavidus metallidurans CH34 plasmid pMOL30 confersresistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is aMerR family activator. In other metal sensing MerR family regulators, such as MerR, CueR,and ZntR the cognate regulator binds to a promoter with an unusually long spacer betweenthe 35 and 10 sequences, and activates transcription of resistance genes as a consequenceof binding the appropriate metal. Cysteine residues in these regulators are essential for metalion coordination and activation of expression from their cognate promoter. In this study weinvestigated the interaction of PbrR with the promoter for the structural pbr resistance genes,PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, andeffects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutatedto serine. Results: Gel retardation and footprinting assays using purified PbrR show that it binds to, and protectsfrom DNase I digestion, the PpbrA promoter, which has a 19 bp spacer between its 35 and10 sites. Using beta-galactosidase assays in C. metallidurans, we show that when PpbrA ischanged to an 18 bp spacer, there is an increase in transcriptional activation both in thepresence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in thefully-induced wild-type promoter. Changes to the 10 sequence of PpbrA from TTAAAT tothe consensus E. coli 10 sequence (TATAAT) increased transcriptional activation fromPpbrA, whilst changing the 10 sequence to that of the Tn501 mer promoter (TAAGGT) alsoincreased the transcriptional response, but only in the presence of Pb(II). Individual PbrRmutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutantC132S/C134S, were tested for Pb(II) response from PpbrA, using beta-galactosidase assays inC. metallidurans. The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defectivein Pb(II)-induced activation of PpbrA. Conclusions: These data show that the metal-dependent activation of PbrR occurs by a similar mechanismto that of MerR, but that metal ion coordination is through cysteines which differ from thoseseen in other MerR family regulators, and that the DNA sequence of the 10 promoter affectsexpression levels of the lead resistance genes.

Beschreibung: Background: Brucellosis is a worldwide disease of mammals caused by Alphaproteobacteria in the genus

Beschreibung: Background: A major outbreak of bloody diarrhea associated with Shiga toxin-producing Escherichia coli O104:H4 occurred early in 2011, to which an unusual number of hemolytic uremic syndrome cases were linked. Due to limited information regarding pathogenesis and/or virulence properties of this particular serotype, we investigated the contribution of the aerobactin iron transport system during in vitro and in vivo conditions. Results: A bioluminescent reporter construct was used to perform real-time monitoring of E. coli O104:H4 in a mouse model of infection. We verified that our reporter strain maintained characteristics and growth kinetics that were similar to those of the wild-type E. coli strain. We found that the intestinal cecum of ICR (CD-1) mice was colonized by O104:H4, with bacteria persisting for up to 7 days after intragastric inoculation. MALDI-TOF analysis of heat-extracted proteins was performed to identify putative surface-exposed virulence determinants. A protein with a high similarity to the aerobactin iron receptor was identified and further demonstrated to be up-regulated in E. coli O104:H4 when grown on MacConkey agar or during iron-depleted conditions. Because the aerobactin iron acquisition system is a key virulence factor in Enterobacteriaceae, an isogenic aerobactin receptor (iutA) mutant was created and its intestinal fitness assessed in the murine model. We demonstrated that the aerobactin mutant was out-competed by the wild-type E. coli O104:H4 during in vivo competition experiments, and the mutant was unable to persist in the cecum. Conclusion: Our findings demonstrate that bioluminescent imaging is a useful tool to monitor E. coli O104:H4 colonization properties, and the murine model can become a rapid way to evaluate bacterial factors associated with fitness and/or colonization during E. coli O104:H4 infections.

Beschreibung: Background: The routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non-invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs). Results: Headspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole). Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml]. Conclusions: The results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.

Beschreibung: Background: The parasitic nematode Spirocerca lupi (Spirurida: Thelaziidae), the canine esophagealworm, is the causative agent of spirocercosis, a disease causing morbidity and mortality indogs. Spirocerca lupi has a complex life cycle, involving an obligatory coleopteranintermediate host (vector), an optional paratenic host, and a definitive canid host. Thediagnosis of spirocercosis is challenging, especially in the early disease stages, when adultworms and clinical signs are absent. Thus, alternative approaches are needed to promote earlydiagnosis. The interaction between nematodes and their bacterial symbionts has recentlybecome a focus of novel treatment regimens for other helminthic diseases. Results: Using 16S rDNA-based molecular methods, here we found a novel bacterial symbiont in S.lupi that is closely related to Comamonas species (Brukholderiales: Comamonadaceae) of thebeta-proteobacteria. Its DNA was detected in eggs, larvae and adult stages of S. lupi. Usingfluorescent in situ hybridization technique, we localized Comamonas sp. to the gut epithelialcells of the nematode larvae. Specific PCR enabled the detection of this symbiont's DNA inblood obtained from dogs diagnosed with spirocercosis. Conclusions: The discovery of a new Comamonas sp. in S. lupi increase the complexity of the interactionsamong the organisms involved in this system, and may open innovative approaches fordiagnosis and control of spirocercosis in dogs.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio Hanna-Kirsti S. Leiros, Anita-Elin Fedøy, Ingar Leiros, Ida Helene Steen Isocitrate dehydrogenase (IDH) catalyses the oxidative NAD(P) + -dependent decarboxylation of isocitrate into α-ketoglutarate and CO 2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH ( Ct IDH), a native open apo Ct IDH to 2.35 Å and a quaternary complex of Ct IDH with NADP + , isocitrate and Mg 2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH ( Dp IDH) was also resolved to 1.93 Å. Ct IDH and Dp IDH showed similar global thermal stabilities with melting temperatures of 67.9°C and 66.9°C, respectively. Ct IDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. Ct IDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to Dp IDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in Dp IDH, were absent in Ct IDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251’ and Arg255 ( Ct IDH). These interactions lock the large domain to the small domain and direct NADP + into the correct orientation, which together are important for NADP + selectivity. Graphical abstract Graphical abstract Highlights ► Focus is on a thermophilic (CtIDH) and cold active (DpIDH) isocitrate dehydrogenase. ► Biochemical characterization and three new IDH crystal structures are presented. ► CtIDH has many ionic interactions and hydrogen bonds, which explain the T m of 68°C. ► Prokaryotic IDH subfamily II seems to have a unique locking mechanism. ► The large domain is locked to the small domain and NADP + is also directed correctly.

Beschreibung: Background: Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. Results: Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100 % (200 isolates) for the MTC and 91.4 % (161 isolates) for the NTM. Among the discordant results (8.6 %) for the NTM, one isolate of Mycobacterial species and the an isolate of M. flavescens were found as new sub-types in hsp65 PRA. Conclusions: This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16S rDNA sequencing.

Beschreibung: Background: When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize threeactive [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymescatalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered astandard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H(Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2evolution by intact cells. Hydrogen oxidation activity can be assayed for all threehydrogenases using benzyl viologen (BV; Eo' = -360 mV) as an artificial electron acceptor;however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assaycould differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable tobe visualized on such native gels. This study identifies conditions allowing differentiation ofall three enzymes using simple in-gel zymographic assays. Results: Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 hasbeen described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential forvisualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-Henzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo' = -80 mV) it wasdemonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, whileneither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogendependentreduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 thathad a supernumerary cysteine residue at position 19 of the small subunit substituted forglycine. This finding suggests that tolerance toward oxygen is not the main determinant thatgoverns electron donation to more redox-positive electron acceptors such as NBT. Conclusions: The utilization of particular electron acceptors at different hydrogen concentrations and redoxpotentials correlates with the known physiological functions of the respective hydrogenase.The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenasesprovides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex.

Beschreibung: Background: PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg2+ and Mn2+. Results: Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD. Conclusions: Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB.Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.

Beschreibung: Background: The activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. While the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. The diversity and dynamics of the Archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes. Results: The Archaea community was specialized and dominated by Methanosaeta-like species. During a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. Water temperature appeared to be the process parameter that affected the community composition the most. Several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. The Archaea were estimated to make up 1.6-% of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes. Conclusions: The results presented here show that Archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include Archaea in future studies of microbial communities in activated sludge.

Beschreibung: Background: It has been well established that the Galpha subunit of the heterotrimeric G-protein in the wheatpathogen Stagonospora nodorum is required for a variety of phenotypes includingpathogenicity, melanisation and asexual differentiation. The roles though of the Ggamma and Gbetasubunits though were unclear. The objective of this study was to identify and understand therole of these subunits and assess their requirement for pathogenicity and development. Results: G-protein Ggamma and Gbeta subunits, named Gga1 and Gba1 respectively, were identified in theStagonospora nodorum genome by comparative analysis with known fungal orthologues. Areverse genetics technique was used to study the role of these and revealed that the mutantstrains displayed altered in vitro growth including a differential response to a variety ofexogenous carbon sources. Pathogenicity assays showed that Stagonospora nodorum strainslacking Gba1 were essentially non-pathogenic whilst Gga1-impaired strains displayedsignificantly slower growth in planta. Subsequent sporulation assays showed that like thepreviously described Galpha subunit mutants, both Gba1 and Gga1 were required for asexual sporulation with neither mutant strain being able to differentiate either pycnidia norpycnidiospores under normal growth conditions. Continued incubation at 4degreesC was found tocomplement the mutation in each of the G-protein subunits with nearly wild-type levels ofpycnidia recovered. Conclusion: This study provides further evidence on the significance of cAMP-dependent signaltransduction for many aspects of fungal development and pathogenicity. The observation thatcold temperatures can complement the G-protein sporulation defect now provides an idealtool by which asexual differentiation can now be dissected.

Beschreibung: Background: Because Candida albicans is resistant to several antifungal antibiotics, there is a need toidentify other less toxic natural products, particularly antimicrobial proteins, peptides orbacteriocin like inhibitory substances. An attempt has been made to purify and characterisean anti-Candida compound produced by Enterococcus faecalis. Results: An anti-Candida protein (ACP) produced by E. faecalis active against 8 C. albicans strainswas characterised and partially purified. The ACP showed a broad-spectrum activity againstmultidrug resistant C. albicans MTCC 183, MTCC 7315, MTCC 3958, NCIM 3557, NCIM3471 and DI. It was completely inactivated by treatment with proteinase K and partially bypronase E.The ACP retained biological stability after heat-treatment at 90 degreesC for 20 min, maintainedactivity over a pH range 6-10, and remained active after treatment with alpha-amylase, lipase,organic solvents, and detergents. The antimicrobial activity of the E. faecalis strain was foundexclusively in the extracellular filtrate produced in the late logarithmic growth phase. Thehighest activity (1600 AU mL-1) against C. albicans MTCC 183 was recorded at 48 h ofincubation, and activity decreased thereafter. The peptide showed very lowhaemagglutination and hemolytic activities against human red blood cells. The antimicrobialsubstance was purified by salt-fractionation and chromatography.Partially purified ACP had a molecular weight of approximately 43 KDa in tricine-PAGEanalysis. The 12 amino acid N terminal sequence was obtained by Edman degradation. Thepeptide was de novo sequenced by ESI-MS, and the deduced combined sequence whencompared to other bacteriocins and antimicrobial peptide had no significant sequencesimilarity. Conclusions: The inhibitory activity of the test strain is due to the synthesis of an antimicrobial protein. Toour knowledge, this is the first report on the isolation of a promising non-haemolytic anti-Candida protein from E. faecalis that might be used to treat candidiasis especially inimmunocompromised patients.

Beschreibung: Background: Mycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry. In mycoplasmas lipoproteins are abundant on the membrane surface and play a critical role in interactions with the host, but tools for exploring their molecular biology are limited. Results: In this study we examined whether the alkaline phosphatase gene (phoA) from Escherichia coli could be used as a reporter in mycoplasmas. The promoter region from the gene for elongation factor Tu (ltuf) and the signal and acylation sequences from the vlhA1.1 gene, both from Mycoplasma gallisepticum, together with the coding region of phoA, were assembled in the transposon-containing plasmid pISM2062.2 (pTAP) to enable expression of alkaline phosphatase (AP) as a recombinant lipoprotein. The transposon was used to transform M. gallisepticum strain S6. As a control, a plasmid containing a similar construct, but lacking the signal and acylation sequences, was also produced (pTP) and also introduced into M. gallisepticum. Using a colorimetric substrate for detection of alkaline phosphatase activity, it was possible to detect transformed M. gallisepticum. The level of transcription of phoA in organisms transformed with pTP was lower than in those transformed with pTAP, and alkaline phosphatase was not detected by immunoblotting or enzymatic assays in pTP transformants, eventhough alkaline phosphatase expression could be readily detected by both assays in pTAP transformants. Alkaline phosphatase was shown to be located in the hydrophobic fraction of transformed mycoplasmas following Triton X-114 partitioning and in the membrane fraction after differential fractionation. Trypsin proteolysis confirmed its surface exposure. The inclusion of the VlhA lipoprotein signal sequence in pTAP enabled translocation of PhoA and acylation of the amino terminal cysteine moiety, as confirmed by the effect of treatment with globomycin and radiolabelling studies with [14C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis. Conclusion: This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.

Beschreibung: Background: Enterococci are among the leading causes of hospital-acquired infections in the United Statesand Europe, with Enterococcus faecalis and Enterococcus faecium being the two mostcommon species isolated from enterococcal infections. In the last decade, the proportion ofenterococcal infections caused by E. faecium has steadily increased compared to otherEnterococcus species. Although the underlying mechanism for the gradual replacement of E.faecalis by E. faecium in the hospital environment is not yet understood, many studies usinggenotyping and phylogenetic analysis have shown the emergence of a globally dispersedpolyclonal subclusters of E. faecium strains in clinical environments. Systematic study of themolecular epidemiology and pathogenesis of E. faecium has been hindered by the lack ofclosed, complete E. faecium genomes that can be used as references. Results: In this study, we report the complete genome sequence of the E. faecium strain TX16, alsoknown as DO, which belongs to multilocus sequence type (ST) 18, and was the first E.faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E.faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA)strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, groupin the same clade (referred to as the HA clade) and are evolutionally considerably moreclosely related to each other by phylogenetic and gene content similarity analyses than toisolates in the community-associated (CA) clade with approximately a 3-4% averagenucleotide sequence difference between the two clades at the core genome level. Our studyalso revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HAcladestrains. Mobile elements such as IS16 and transposons were also found almostexclusively in HA strains, as previously reported. Conclusions: Our findings along with other studies show that HA clonal lineages harbor specific geneticelements as well as sequence differences in the core genome which may confer selectionadvantages over the more heterogeneous enterococcal CA E. faecium isolates. Which of thesedifferences are important for the success of specific E. faecium HA lineages in the hospitalenvironment remain(s) to be determined.

Beschreibung: Background: Klebsiella pneumoniae can be found in environmental habitats as well as in hospital settings where it is commonly associated with nosocomial infections. One of the factors that contribute to virulence is its capacity to form biofilms on diverse biotic and abiotic surfaces. The second messenger Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a ubiquitous signal in bacteria that controls biofilm formation as well as several other cellular processes. The cellular levels of this messenger are controlled by c-di-GMP synthesis and degradation catalyzed by diguanylate cyclase (DGC) and phophodiesterase (PDE) enzymes, respectively. Many bacteria contain multiple copies of these proteins with diverse organizational structure that highlight the complex regulatory mechanisms of this signaling network. This work was undertaken to identify DGCs and PDEs and analyze the domain structure of these proteins in K. pneumoniae.FindingsA search for conserved GGDEF and EAL domains in three sequenced K. pneumoniae genomes showed that there were multiple copies of GGDEF and EAL containing proteins. Both single domain and hybrid GGDEF proteins were identified: 21 in K. pneumoniae Kp342, 18 in K. pneumoniae MGH 78578 and 17 in K. pneumoniae NTUH-K2044. The majority had only the GGDEF domain, most with the GGEEF motif, and hybrid proteins containing both GGDEF and EAL domains were also found. The I site for allosteric control was identified only in single GGDEF domain proteins and not in hybrid proteins. EAL-only proteins, containing either intact or degenerate domains, were also identified: 15 in Kp342, 15 in MGH 78578 and 10 in NTUH-K2044. Several input sensory domains and transmembrane segments were identified, which together indicate complex regulatory circuits that in many cases can be membrane associated. Conclusions: The comparative analysis of proteins containing GGDEF/EAL domains in K. pneumoniae showed that most copies were shared among the three strains and that some were unique to a particular strain. The multiplicity of these proteins and the diversity of structural characteristics suggest that the c-di-GMP network in this enteric bacterium is highly complex and reflects the importance of having diverse mechanisms to control cellular processes in environments as diverse as soils or plants and clinical settings.KeywordsKlebsiella pneumoniae, biofilm, diguanylate cyclase, c-di-GMP

Beschreibung: Background: Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. Results: Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100 % (200 isolates) for the MTC and 91.4 % (161 isolates) for the NTM. Among the discordant results (8.6 %) for the NTM, one isolate of Mycobacterial species and the an isolate of M. flavescens were found as new sub-types in hsp65 PRA. Conclusions: This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16S rDNA sequencing.

Beschreibung: Background: Deoxyhypusine synthase (DHS) catalyzes the first step in hypusine biosynthesis of eukaryotic initiation factor 5A (eIF-5A) in Plasmodium falciparum. Target evaluation of parasitic DHS has recently been performed with CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease. CNI-1493 prevented infected mice from experimental cerebral malaria by a decrease in eIF-5A and serum TNF-levels, implicating a link between cytokine signaling and the hypusine pathway. Therefore we addressed the question whether either DHS itself or eIF-5A is required for the outcome of severe malaria. In a first set of experiments we performed an in vitro knockdown of the eIF-5A and DHS proteins by RNA interference (RNAi) in 293T cells. Secondly, transfection of siRNA constructs into murine Plasmodium schizonts was performed which, in turn, were used for infection. Results: 293T cells treated with DHS- and eIF-5A specific siRNAs or control siRNAs were analyzed by RT-PCR to determine endogenous dhs -and eIF-5A mRNA levels. The expressed DHS-shRNA and EIF-5A-shRNA clearly downregulated the corresponding transcript in these cells. Interestingly, mice infected with transgenic schizonts expressing either the eIF-5A or dhs shRNA showed an elevated parasitemia within the first two days post infection which then decreased intermittently. These results were obtained without drug selection. Blood samples, which were taken from the infected mice at day 5 post infection with either the expressed EIF-5A-shRNA or the DHS-shRNA were analyzed by RT-PCR and Western blot techniques, demonstrating the absence of either the hypusinated form of eIF-5A or DHS. Conclusion: Infection of NMRI mice with schizonts from the lethal P. berghei ANKA wildtype strain transgenic for eIF-5A-specific-shRNA or DHS-specific shRNA resulted in low parasitemia 2-9 days post infection before animals succumbed to hyperparasitemia similar to infections with the related but non-lethal phenotype P. berghei strain NK65. RT-PCR and Western blot experiments performed with blood from the transfected erythrocytic stages demonstrated that both genes are important for the proliferation of the parasite. Moreover, these experiments clearly demonstrate that the hypusine pathway in Plasmodium is linked to human iNos induction.

Beschreibung: Background: Aflatoxins are highly carcinogenic compounds produced by Aspergillus species in seeds with high lipid and protein contents. It has been known for over 30 years that peptone is not conducive for AF productions, with reasons unknown. Results: In this study, we showed that when Aspergillus flavus was grown in glucose-containing media, higher initial spore density or higher concentration of peptone promoted both mycelium growth and AF productions, while in peptone-containing media, higher initial spore densities or higher peptone concentrations inhibited AF biosynthesis. Spent medium experiments showed that the inhibited AF productions were regulated in a cell-autonomous manner. Further experiments showed that increased concentrations of peptone inhibited the low density-dependent AF productions but promoted mycelium growth. Expression analyses showed that both regulatory and AF biosynthesis genes were repressed in mycelia cultured with the high initial spore density. Metabolomic studies revealed that, in addition to inhibited AF biosynthesis, mycelia cultured in peptone media with the higher initial spore density showed suppressed fatty acid biosynthesis, while tricarboxylic acid cycle and pentose phosphate pathways were enhanced. Conclusions: We demonstrated that Aspergillus flavus grown in media with peptone as the sole carbon source was able to sense the population density and the peptone concentration to switch between rapid growths and AF productions. Such a highly regulated metabolic switch between rapid growth and AF production may offer Aspergillus flavus a competition advantage in natural ecosystems.

Beschreibung: Background: In this study, we present evidence that proteins encoded by the Locus of EnterocyteEffacement (LEE), considered critical for Escherichia coli O157 (O157) adherence tofollicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do notappear to contribute to O157 adherence to squamous epithelial (RSE) cells also constitutingthis primary site of O157 colonization in cattle. Results: Antisera targeting intimin-gamma, the primary O157 adhesin, and other essential LEE proteinsfailed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, aculture medium that enhances expression of LEE proteins. In addition, RSE adherence of aDMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with itswild-type parent O157 strain grown in the same media. These adherence patterns were incomplete contrast to that observed with HEp-2 cells (the adherence to which is mediated byintimin-gamma), assayed under same conditions. This suggested that proteins other than intimin-gammathat contribute to adherence to RSE cells are expressed by this pathogen during growth inDMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157(DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684proteins including several components of the cattle and human O157 immunome, orthologsof adhesins, hypothetical secreted and outer membrane proteins, in addition to the knownvirulence and LEE proteins. Bioinformatics-based analysis of the components of the O157DMEM proteome revealed several new O157-specific proteins with adhesin potential. Conclusion: Proteins other than LEE and intimin-gamma proteins are involved in O157 adherence to RSE cellsat the bovine RAJ. Such proteins, with adhesin potential, are expressed by this humanpathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSEadherence should provide both valuable insights into the O157-RSE interactions and newtargets for more efficacious anti-adhesion O157 vaccines.

Beschreibung: Background: Dietary supplementation with zinc has been shown to reduce the duration and severity of diarrhoealdisease caused by Enteropathogenic Escherichia coli, common in infants in developing countries. Initially thistherapeutic benefit was attributed to the correction of zinc deficiency in malnourished individuals, but recentlyevidence has emerged that zinc significantly impacts the pathogens themselves: zinc concentrations achievableby oral supplementation can reduce the expression of key virulence-related genes in EPEC and related organisms. Results: Here, we investigate three possible mechanisms for such zinc-induced changes in expression of EPECvirulence: direct interaction of zinc with regulators of LEE operons; genetic interaction of LEE operons withknown regulators of zinc homeostasis; and finally, downregulation of LEE transcription associated withactivation of the ¿E envelope stress response by zinc. We find evidence only for the latter mechanism, includingzinc-induced down-regulation of Type III secretion in EPEC similar to that caused by ammonium metavanadate,another known inducer of the ¿E stress response. Conclusions: We conclude therefore that envelope stress is a major mechanism by which zinc attenuates thevirulence of EPEC and related pathogens.

Beschreibung: Background: Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrhealsyndrome may result from the secretion of various virulence factors including hemolysin BLand nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulatedby the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr isa member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so farbeen studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genesis thus to obtain and characterize holoFnr. Results: Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UVvisibleand EPR spectroscopic analyses together with the chemical estimation of the ironcontent indicated that Fnr binds one [4Fe-4 S]2+ cluster per monomer. Atmospheric O2 causesdisassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- andapoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has ahigher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnrinteract with ResD and PlcR to form a ternary complex. Conclusions: Overall, this work shows that incorporation of the [4Fe-4 S]2+ cluster is not required for DNAbinding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of aternary complex with ResD and PlcR. This points to some new unusual properties of Fnr thatmay have physiological relevance in the redox regulation of enterotoxin gene regulation.

Beschreibung: Publication year: 2011 Source: FEBS Open Bio, Volume 1 Tarlan Mamedov, Vidadi Yusibov Green algae have a great potential as biofactories for the production of proteins. Chlamydomonas reinhardtii , a representative of eukaryotic microalgae, has been extensively used as a model organism to study light-induced gene expression, chloroplast biogenesis, photosynthesis, light perception, cell–cell recognition, and cell cycle control. However, little is known about the glycosylation machinery and N-linked glycan structures of green algae. In this study, we performed mass spectrometry analysis of N-linked oligosaccharides released from total extracts of Chlamydomonas reinhardtii and demonstrated that C. reinhardtii algae possess glycoproteins with mammalian-like sialylated N-linked oligosaccharides. These findings suggest that C. reinhardtii may be an attractive system for expression of target proteins.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Annica Vlad-Fiegen, Anette Langerak, Sonja Eberth, Oliver Müller The Wnt pathway regulates cell proliferation, mobility and differentiation. Among the many Wnt target genes is CCND1 which codes for cyclin D1. Cyclin D1, in complex with cdk4 and cdk6, regulates G1/S phase transition during cell cycle. Independently of CDK, cyclin D1 also regulates the migration of macrophages. Here we analyzed the effects of cyclin D1 on the migration of cancer cell lines using the transwell migration and scratch assays. We also tested the effect of cyclin D1 and β-catenin on E-cadherin-mediated cell–cell contacts. Our results show that the Wnt pathway promotes cellular migration via its target gene cyclin D1. Moreover we show that cyclin D1 influences the actin cytoskeleton and destabilizes adherens junctions. Highlights ► We analyzed the effects of cyclin D1 on the migration of cancer cell lines. ► The Wnt pathway promotes cellular migration via its target gene cyclin D1. ► Cyclin D1 rearranges the actin cytoskeleton and destabilizes adherens junctions.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Hongchang Shen, Yan Fang, Wei Dong, Xueru Mu, Qi Liu, Jiajun Du The insulin like growth factor receptor subtype 1(IGF-1R) plays an important role in cancers transformation and progression. The aim is to investigate the effects of sunitinib on IGF-1R cell signaling transduction, especially on receptor phosphorylation and ubiquitination. In HEK293 cells, IGF-1R signaling pathways are activated in response to IGF-1, which induces obvious phosphorylations of receptor tyrosine and Akt, ERK. However, the phosphorylations of receptor tyrosine, Akt and ERK were significant inhibited by sunitinib. We found that both IGF-1 and sunitinib obviously down regulated the IGF-1R expression. For analysis the ubiquitination, HEK293 cells were simulated with 100 ng/ml IGF-1 or 10 nM sunitinib for 10 min after serum starvation for 24 h. Both IGF-1 and sunitinib could obviously induce the IGF-1R ubiquitination at 10 min compared with control (only serum free, no stimulation), indicating IGF-1 and sunitinib down-regulate the IGF-1R by increasing the receptor degradation through ubiquitination dependent proteasome pathway. We also found that MDM2 combined to IGF-1R in response to sunitinib stimulation. To confirm it, HEK293 cells were transfected with human HA-MDM2 (+MDM2) or siRNA to MDM2 (−MDM2). Following 24 h serum starvation, cells were stimulated with 10 nM sunitinib for 10 min. In over-expressed MDM2 cells, IGF-1R was more ubiquitinated than that in mock-transfected cells (control), and no ubiquitination in −MDM2 cells. These results mean that sunitinib mediates ubiquitination of IGF-1R dependent on MDM2. In summary, sunitinib could block signaling transduction and mediate degradation of IGF-1R.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Leonardo Acuña, Gianluca Picariello, Fernando Sesma, Roberto D. Morero, Augusto Bellomio Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries. Graphical abstract Graphical abstract Highlights ► A chimerical gene encoding a hybrid bacteriocin–microcin was generated by PCR. ► Ent35–MccV is a hybrid bacteriocin containing enterocin CRL35 and microcin V. ► Ent35–MccV is active against Gram-positive and Gram-negative bacteria. ► Enterohemorrhagic Escherichia coli and Listeria monocytogenes are sensitive to Ent35–MccV.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mike J.F. Broderick, Andrey Bobkov, Steve J. Winder Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F-actin have revealed a number of different conformations with calponin homology domains in ‘open’ and ‘closed’ positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F-actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F-actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino-terminal utrophin actin binding domain is in an open conformation in solution and when bound to F-actin. Highlights ► Utrophin actin binding domain crystallises as an open dimer but is monomeric in solution. ► We introduced cysteines in the CH domains to lock the monomer closed. ► Utrophin bound to F-actin with reduced affinity in the closed conformation. ► Differential scanning calorimetry confirmed the open conformation of utrophin on actin.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Vinay Kumar, Gagan D. Gupta Crystals of native Drosophila melanogaster translin diffracted to 7 Å resolution. Reductive methylation of the protein improved crystal quality. The native and methylated proteins showed similar profiles in size-exclusion chromatography analyses but the methylated protein displayed reduced DNA-binding activity. Crystals of the methylated protein diffracted to 4.2 Å resolution at BM14 of the ESRF synchrotron. Crystals with 49% solvent content belonged to monoclinic space group P 2 1 with eight protomers in the asymmetric unit. Only 2% of low-resolution structures with similar low percentage solvent content were found in the PDB. The crystal structure, solved by molecular replacement method, refined to R work ( R free ) of 0.24 (0.29) with excellent stereochemistry. The crystal structure clearly shows that drosophila protein exists as an octamer, and not as a decamer as expected from gel-filtration elution profiles. The similar octameric quaternary fold in translin orthologs and in translin–TRAX complexes suggests an up-down dimer as the basic structural subunit of translin-like proteins. The drosophila oligomer displays asymmetric assembly and increased radius of gyration that accounts for the observed differences between the elution profiles of human and drosophila proteins on gel-filtration columns. This study demonstrates clearly that low-resolution X-ray structure can be useful in understanding complex biological oligomers. Highlights ► Methylation yielded diffraction-quality crystals of Drosophila translin. ► The crystal structure revealed asymmetric octameric assembly of Drosophila translin. ► Up-down dimer is the basic structural subunit of translin-like proteins. ► The radius of gyration of the Drosophila oligomer is larger than that of human translin. ► Low-resolution X-ray structures facilitate understanding of biological complexes.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mamoru Hagihara, Ayaka Takei, Takeshi Ishii, Fumio Hayashi, Kenji Kubota, Kaori Wakamatsu, Nobukazu Nameki Choline- O -sulfate (2-(trimethylammonio)ethyl sulfate, COS) is a naturally occurring osmolyte that is synthesized by plants, lichens, algae, fungi, and several bacterial species. We examined the inhibitory effects of COS on amyloid formation of the human islet amyloid polypeptide (hIAPP or amylin) using a thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy and transmission electron microscopy. The results showed that COS suppresses a conformational change of hIAPP from a random coil to a β-sheet structure, resulting in the inhibition of amyloid formation. Comparisons with various structural analogs including carnitine, acetylcholine and non-detergent sulfobetaines (NDSBs) using the ThT fluorescence assay showed that COS is the most effective inhibitor of hIAPP amyloid formation, suggesting that the sulfate group, which is unique to COS, significantly contributes to the inhibition. Highlight ► Choline- O -sulfate (COS), an osmolyte, inhibits amyloid formation of hIAPP. ► COS is the most effective inhibitor of amyloid formation among structural analogs. ► The sulfate group appears to be involved in the inhibition.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mark J. Holness, Gulrez Zariwala, Celia G. Walker, Mary C. Sugden We studied adipocytes from 8-week-old control rat offspring (CON) or rat offspring subjected to maternal low (8%) protein (MLP) feeding during pregnancy/lactation, a procedure predisposing to obesity. Acute exposure to isoproterenol or adenosine enhanced PDK4 and PPARγ mRNA gene expression in CON and MLP adipocytes. Enhanced adipocyte Pdk4 expression correlated with increased PPARγ expression. Higher levels of PDK4 and PPARγ were observed in MLP adipocytes. SCD1 is a PPARγ target. Isoproterenol enhanced adipocyte PDK4 and SCD1 gene expression in parallel. This could reflect augmented PPARγ expression together with enhanced lipolytic stimulation to supply endogenous PPARγ ligands, allowing enhanced adipocyte PDK4 and SCD1 expression via PPARγ activation. In contrast, the effect of adenosine to increase PDK4 expression is independent of stimulation of lipolysis and, as SCD1 expression was unaffected by adenosine, unlikely to reflect PPARγ activation. Increased adipocyte expression of both PDK4 and SCD1 in the MLP model could participate as components of a “thrifty” phenotype, favouring the development of obesity. Highlights ► An early-life dietary intervention increasing the risk of obesity enhances adipocyte Pdk4 expression. ► Adenosine increases adipocyte PDK4 expression. ► An early-life dietary intervention augments PPARγ expression and enhances lipolytic stimulation. ► Enhanced lipolysis could supply PPARγ ligands enhancing adipocyte PDK4 via PPARγ activation.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Carol A. Chrestensen, Jonathan L. McMurry, John C. Salerno Endothelial nitric oxide synthase (eNOS) contains a motif similar to recognition sequences in known MAPK binding partners. In optical biosensing experiments, eNOS bound p38 and ERK with ∼100 nM affinity and complex kinetics. Binding is diffusion-limited ( k on ∼ .15 × 10 6 M −1 s −1 ). Neuronal NOS also bound p38 but exhibited much slower and weaker binding. p38-eNOS binding was inhibited by calmodulin. Evidence for a ternary complex was found when eNOS bound p38 was exposed to CaM, increasing the apparent dissociation rate. These observations strongly suggest a direct role for MAPK in regulation of NOS with implications for signaling pathways including angiogenesis and control of vascular tone. Highlights ► MAP kinases bind to a pentabasic sequence in the eNOS autoinhibitory insertion. ► MAP kinases bind poorly or not at all to nNOS. ► MAP kinases interact weakly with CaM binding, increasing CaM release. ► The MAPK binding site is adjacent to the phosphorylation sites of other kinases.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mariana Tamazato Longhi, Nathalie Cella Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation. Highlights ► Three endogenous maspin tyrosine phosphoforms were identified in non-transformed mammary epithelial cells. ► Tyrosine phosphatase inhibition results in rapid increase in maspin levels. ► Tyrosine phosphatase inhibition results in maspin accumulation in the cytoplasm.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Hiromitsu Araki, Christoph Knapp, Peter Tsai, Cristin Print Most “omics” experiments require comprehensive interpretation of the biological meaning of gene lists. To address this requirement, a number of gene set analysis (GSA) tools have been developed. Although the biological value of GSA is strictly limited by the breadth of the gene sets used, very few methods exist for simultaneously analysing multiple publically available gene set databases. Therefore, we constructed GeneSetDB ( http://genesetdb.auckland.ac.nz/haeremai.html ), a comprehensive meta-database, which integrates 26 public databases containing diverse biological information with a particular focus on human disease and pharmacology. GeneSetDB enables users to search for gene sets containing a gene identifier or keyword, generate their own gene sets, or statistically test for enrichment of an uploaded gene list across all gene sets, and visualise gene set enrichment and overlap using a clustered heat map. Highlights ► Gene Set Analysis (GSA) can reveal biological meaning from “omics” experiments. ► It is currently difficult to apply GSA to the range of available gene set databases. ► We constructed GeneSetDB, a comprehensive meta-database for gene set analysis. ► GeneSetDB integrates 26 databases containing different types of information. ► GeneSetDB lets biologists visualise the gene sets represented in their “omics” results.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio Ryo Furukawa, Yuma Yamada, Yuichi Matsushima, Yu-ichi Goto, Hideyoshi Harashima Mitochondrial transcription factor A (TFAM) packages mitochondrial DNA (mtDNA) and plays a critical role in mtDNA maintenance, replication and transcription. We report herein on the effects of DNA packaged with TFAM on the transcription process. Our initial findings indicated that the transcription of the TFAM/DNA complex was activated, when the complex was formed at an optimal ratio. We also found that TFAM has a significant advantage over protamine, a nuclear DNA packaging protein, from the viewpoint of transcription efficiency. This result indicates that TFAM can be useful packaging protein for exogenous DNA to achieve mitochondrial transgene expression. Graphical abstract Graphical abstract Highlights ► The base-pair interval at which mitochondrial transcription factor A (TFAM) binds to DNA is critical for transcription. ► The TFAM/DNA complex was transcriptionally active only when the packaging of DNA was optimal. ► The inhibition of the transcription of a TFAM/DNA complex at high ratios appears to result from the overpackaging of DNA by TFAM. ► Our findings indicated that the manner in which DNA is packaged with TFAM has an impact on transcription activation.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio Yossi Laviv, Helen Toledano, Shalom Michowiz, Olga Dratviman-Storobinsky, Yuval Turm, Suzana Fichman-Horn, Ella Kagnovski, Nitza Goldenberg-Cohen Fifty-two samples of pediatric low-grade glioma (48 primary, 4 recurrent) were analyzed for BRAF copy number variation (digital PCR analysis, CopyCaller) and point mutations of BRAF V600E, and exon 5 Q209 in GNAQ , and GNA11 , using the MALDI-TOF mass spectrometer with validation by direct sequencing. An increased BRAF copy number was found in 18/47 primary samples tested; 15 of them (83.3%) were pilocytic astrocytomas. A BRAF mutation was found in 3/48 primary tumors, all with a normal BRAF copy number and no GNAQ mutation. One sample had a GNAQ209 mutation (Q209P626) with a normal BRAF gene ; none of the tumors had a GNA11Q209 mutation. Recurrent or progressive tumors, analyzed in four patients, had the same molecular genotype as their primary. Increased BRAF copy number and activating BRAF mutations may be involved in the development of low-grade glioma via overactivation of the Ras/Raf pathway. This is the first report of a mutation in GNAQ209 in pediatric low-grade glioma. Understanding the molecular mechanisms underlying glioma initiation and growth may assist in the development of targeted therapies. Highlights ► We investigated the occurrence of BRAF , GNAQ , and GNA11 mutations in pediatric low-grade glioma. ► We found an increase in the copy number of BRAF. ► BRAF and GNAQ mutations are mutually exclusive. ► Understanding the molecular basis of low-grade glioma may increase treatment options.

Beschreibung: Publication year: 2011 Source: FEBS Open Bio, Volume 1 Mariana Giró, Romina D. Ceccoli, Hugo O. Poli, Néstor Carrillo, Anabella F. Lodeyro Oxidative stress in plants causes ferredoxin down-regulation and NADP + shortage, over-reduction of the photosynthetic electron transport chain, electron leakage to oxygen and generation of reactive oxygen species (ROS). Expression of cyanobacterial flavodoxin in tobacco chloroplasts compensates for ferredoxin decline and restores electron delivery to productive routes, resulting in enhanced stress tolerance. We have designed an in vivo system to optimize flavodoxin reduction and NADP + regeneration under stress using a version of cyanobacterial ferredoxin–NADP + reductase without the thylakoid-binding domain. Co-expression of the two soluble flavoproteins in the chloroplast stroma resulted in lines displaying maximal tolerance to redox-cycling oxidants, lower damage and decreased ROS accumulation. The results underscore the importance of chloroplast redox homeostasis in plants exposed to adverse conditions, and provide a tool to improve crop tolerance toward environmental hardships. Graphical abstract Graphical abstract Highlights ► Expression of cyanobacterial flavodoxin in tobacco plants confers increased tolerance to oxidative stress. ► Expression of ferredoxin–NADP + reductase in tobacco does not provide oxidative stress tolerance. ► The presence of ferredoxin–NADP + reductase significantly increases oxidative stress tolerance conferred by flavodoxin in transgenic plants. ► Stress tolerance in plants depends on chloroplast redox homeostasis.

Beschreibung: Publication year: 2011 Source: FEBS Open Bio, Volume 1 Sabrina Siamer, Oriane Patrit, Mathilde Fagard, Naïma Belgareh-Touzé, Marie-Anne Barny Erwinia amylovora is responsible for fire blight, a necrotic disease of apples and pears. E. amylovora relies on a type III secretion system (T3SS) to induce disease on host plants. DspA/E belongs to the AvrE family of type III effector. Effectors of the AvrE family are injected via the T3SS in plant cell and are important to promote bacterial growth following infection and to suppress plant defense responses. Their mode of action in the plant cells is unknown. Here we study the physiological effects induced by dsp A/E expression in the yeast Saccharomyces cerevisiae . Expression of dspA/E in the yeast inhibits cell growth. This growth inhibition is associated with perturbations of the actin cytoskeleton and endocytosis.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Yoshihiro Kawamura, Shigeru Takasaki, Masashi Mizokami The recommended treatment for patients with chronic hepatitis C, pegylated interferon α (PEG-IFN-α) plus rebavirin (RBV), does not provide a sustained virologic response in all patients, especially those with hepatitis C virus (HCV) genotype 1. It is therefore important to predict whether or not a new patient with HCV genotype 1 will be cured by the recommended treatment. We propose a prediction method for a new patient using a decision tree learning model based on SNPs evaluated in a genome-wide association study. By the decision tree learning for 142 Japanese patients with HCV genotype 1 (78 with null virologic response and 64 with virologic response), we can predict with high probability (93%) whether or not a new patient with HCV will be helped by the recommended treatment. Highlights ► We modeled drug responses using decision tree learning based on SNPs in a genome-wide association study. ► We can predict the drug responses of a new patient with HCV genotype 1. ► Responsiveness to pegylated interferon α (PEG-IFN-α) plus rebavirin (RBV) treatment was predicted. ► We can predict with 93% probability whether a new patient with HCV genotype 1 will be helped by drug treatment.

Beschreibung: Background: Plant root exudates have been shown to play an important role in mediating interactions between plant growth-promoting rhizobacteria (PGPR) and their host plants. Most investigations were performed on Gram-negative rhizobacteria, while much less is known about Gram-positive rhizobacteria. To elucidate early responses of PGPR to root exudates, we investigated changes in the transcriptome of a Gram-positive PGPR to plant root exudates. Results: Bacillus amyloliquefaciens FZB42 is a well-studied Gram-positive PGPR. To obtain a comprehensive overview of FZB42 gene expression in response to maize root exudates, microarray experiments were performed. A total of 302 genes representing 8.2% of the FZB42 transcriptome showed significantly altered expression levels in the presence of root exudates. The majority of the genes (261) was up-regulated after incubation of FZB42 with root exudates, whereas only 41 genes were down-regulated. Several groups of the genes which were strongly induced by the root exudates are involved in metabolic pathways relating to nutrient utilization, bacterial chemotaxis and motility, and non-ribosomal synthesis of antimicrobial peptides and polyketides. Conclusions: Here we present a transcriptome analysis of the root-colonizing bacterium Bacillus amyloliquefaciens FZB42 in response to maize root exudates. The 302 genes identified as being differentially transcribed are proposed to be involved in interactions of Gram-positive bacteria with plants.

Beschreibung: Background: The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulenceplasmid. To identify possible genetic determinants of pS88 virulence, we examined thetranscriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes,and 35 ORFs of unknown function. Results: Quantification of plasmidic transcripts was obtained by quantitative real-time reversetranscription of extracted RNA, normalized on three housekeeping genes. The transcriptomeof E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtainedduring growth in Luria Bertani broth, with and without iron depletion. We also analyzed thetranscriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection.The transcriptome obtained after ex vivo growth in serum and urine was very similar to thoseobtained in iron-depleted LB broth. Genes encoding iron acquisition systems were stronglyupregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function andphysically linked to aerobactin and salmochelin loci, respectively, were also highly expressedin iron-depleted conditions and may correspond to ancillary iron acquisition genes. FourORFs were induced ex vivo, independently of the iron concentration. Other putative virulencegenes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similarpattern of induction but at much higher levels. Conclusion: We identify new pS88 genes potentially involved in the growth of E. coli meningitis strainS88 in human serum and urine.

Beschreibung: Background: The production of Streptococcus pyogenes exoproteins, many of which contribute tovirulence, is regulated in response to nutrient availability. CodY is a transcriptional regulatorthat controls gene expression in response to amino acid availability. The purpose of this studywas to identify differences in the expression of streptococcal exoproteins associated withdeletion of the codY gene. Results: We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant thepost-exponential phase of growth. We used both one and two-dimensional gel electrophoresisto separate exoproteins. Proteins that were significantly different in abundance upon repeatedanalysis were identified with tandem mass spectrometry. The production of the secretedcysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putativeadhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from thecodY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encodednuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were lessabundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assaysshowed greater DNase activity in culture supernatants isolated in the post-exponential phaseof growth from the codY mutant strain compared to the wild-type strain. Becauseextracellular nucleases and proteases can influence biofilm formation, we also measured theability of the strains to form biofilms during growth with both rich medium (Todd Hewittyeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomasscompared to the wild-type strain. Conclusions: Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenesexoproteins, including DNases, a protease, and hylauronidase, which together may alleviatestarvation by promoting dissemination of the pathogen to nutrient rich environments and byhydrolysis of host macromolecules.

Beschreibung: Publication year: 2012 Source: FEBS Open Bio Karolina Stefanowicz, Nausicaä Lannoo, Paul Proost, Els J.M. Van Damme The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N - and O -glycans containing N -acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly- N- acetyllactosamine ([Galβ1-4GlcNAc] n ) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides. Highlights ► Recombinant expression of a new type of Arabidopsis F-box protein. ► Arabidopsis F-box-Nictaba protein is a functional lectin. ► Arabidopsis F-box-Nictaba recognizes N-acetyllactosamine and Lewis structures.

Beschreibung: Background: Microbial anaerobic digestion (AD) is used as a waste treatment process to degrade complex organic compounds into methane. The archaeal and bacterial taxa involved in AD are well known, whereas composition of the fungal community in the process has been less studied. The present study aimed to reveal the composition of archaeal, bacterial and fungal communities in response to increasing organic loading in mesophilic and thermophilic AD processes by applying 454 amplicon sequencing technology. Furthermore, a DNA microarray method was evaluated in order to develop a tool for monitoring the microbiological status of AD. Results: The 454 sequencing showed that the diversity and number of bacterial taxa decreased with increasing organic load, while archaeal i.e. methanogenic taxa remained more constant. The number and diversity of fungal taxa increased during the process and varied less in composition with process temperature than bacterial and archaeal taxa, even though the fungal diversity increased with temperature as well. Evaluation of the microarray using AD sample DNA showed correlation of signal intensities with sequence read numbers of corresponding target groups. The sensitivity of the test was found to be about 1%. Conclusions: The fungal community survives in anoxic conditions and grows with increasing organic loading, suggesting that Fungi may contribute to the digestion by metabolising organic nutrients for bacterial and methanogenic groups. The microarray proof of principle tests suggest that the method has the potential for semiquantitative detection of target microbial groups given that comprehensive sequence data is available for probe design.

Beschreibung: Background: Uropathogenic strains of Escherichia coli cause symptomatic infections whereas asymptomatic bacteriuria (ABU) strains are well adapted for growth in the human urinary tract, where they establish long-term bacteriuria. Human urine is a very complex growth medium that could be perceived by certain bacteria as a stressful environment. To investigate a possible imbalance between endogenous oxidative response and antioxidant mechanisms, lipid oxidative damage estimated as thiobarbituric acid reactive substances (TBARS) content was evaluated in twenty-one E. coli belonging to various pathovars and phylogenetic groups. Antioxidant defense mechanisms were also analysed. Results: During exponential growth in urine, TBARS level differs between strains, without correlation with the ability to grow in urine which was similarly limited for commensal, ABU and uropathogenic strains. In addition, no correlation between TBARS level and the phylogroup or pathogenic group is apparent. The growth of ABU strain 83972 was associated with a high level of TBARS and more active antioxidant defenses that reduce the imbalance. Conclusions: Our results indicate that growth capacity in urine is not a property of ABU strains. However, E. coli isolates respond very differently to this stressful environment. In strain ABU 83972, on one hand, the increased level of endogenous reactive oxygen species may be responsible for adaptive mutations. On the other hand, a more active antioxidant defense system could increase the capacity to colonize the bladder.

Beschreibung: Background: Listeria monocytogenes can cause invasive diseases in humans and farm animals and is frequently isolated from dairy products and poultry. Listeriosis is uncommon in China but L. monocytogenes has been isolated from foods and food processing environments in China. However little is known of genetic diversity of Chinese L. monocytogenes isolates and their relationships with global isolates. Results: Two hundred and twelve isolates of L. monocytogenes from food sources from 12 provinces/cities in China were analysed by serotyping, Pulsed Field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST). The predominant serotypes are 1/2a, 1/2b and 1/2c accounting for 90.1% of the isolations. PFGE divided the isolates into 61 pulse types (PTs). Twenty nine PTs were represented by more than one isolates with PT GX6A16.0004 containing the most number of isolates. MLST differentiated the isolates into 36 STs, among which 15 were novel. The most common 3 STs were ST9 (29.1%), ST8 (10.7%) and ST87 (9.2%), accounting for 49.0% of the isolates. Conclusions: STs prevalent in other parts of the world are also prevalent in China including 7 STs (ST1-ST3, ST5, ST6, ST8, ST9) which caused maternal fetal infections or outbreaks, suggesting that these STs potentially can also cause severe human infections or outbreaks in China. Surveillance of these STs will provide important information for prevention of listeriosis. This study also enhances our understanding of genetic diversity of L. monocytogenes in China.

Beschreibung: Background: Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production. Results: Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility. Conclusions: Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such a gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane.

Beschreibung: Background: Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically¿¿¿104-fold higher than the B. pseudomallei and B. mallei LD50in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. Results: Madagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was 105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect¿s open circulatory system in vivo. Conclusions: The results demonstrate that MH cockroaches are an attractive alternative to mammals to study host-pathogen interactions and may allow the identification of new Burkholderia virulence determinants. The importance of T6SS-1 as a virulence factor in MH cockroaches and rodents suggests that the primary role of this secretion system is to target evasion of the innate immune system.

Beschreibung: Background: Concrete corrosion of wastewater collection systems is a significant cause of deterioration and premature collapse. Failure to adequately address the deteriorating infrastructure networks threatens our environment, public health, and safety. Analysis of whole-metagenome pyrosequencing data and 16S rRNA gene clone libraries was used to determine microbial composition and functional genes associated with biomass harvested from crown (top) and invert (bottom) sections of a corroded wastewater pipe. Results: Taxonomic and functional analysis demonstrated that approximately 90% of the total diversity was associated with the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The top (TP) and bottom pipe (BP) communities were different in composition, with some of the differences attributed to the abundance of sulfide-oxidizing and sulfate-reducing bacteria. Additionally, human fecal bacteria were more abundant in the BP communities. Among the functional categories, proteins involved in sulfur and nitrogen metabolism showed the most significant differences between biofilms. There was also an enrichment of genes associated with heavy metal resistance, virulence (protein secretion systems) and stress response in the TP biofilm, while a higher number of genes related to motility and chemotaxis were identified in the BP biofilm. Both biofilms contain a high number of genes associated with resistance to antibiotics and toxic compounds subsystems. Conclusions: The functional potential of wastewater biofilms was highly diverse with level of COG diversity similar to that described for soil. On the basis of the metagenomic data, some factors that may contribute to niche differentiation were pH, aerobic conditions and availability of substrate, such as nitrogen and sulfur. The results from this study will help us better understand the genetic network and functional capability of microbial members of wastewater concrete biofilms.

Beschreibung: Background: Intragenomic recombination between babA and babB mediates antigenic variations and may help H. pylori colonization. This study determined whether variable genotypes of babA and babB correlate to different clinical disease outcomes, and can distribute over the different gastric niches. Results: This study enrolled 92 clinical strains (45 from peptic ulcer, 27 from gastritis, and 20 from gastric cancer) to detect whether the babA and babB are at locus A or B by PCR reactions using the primers designed from the upstream and variable region of the babA and babB genes. Four genotypes of babA and babB (A B, AB B, A AB, AB AB) were found. The distribution of the 4 genotypes in 92 clinical strains was significantly different among patients with different gastric diseases (p 〈 0.05). The isolates from gastric cancer patients had a higher rate of AB AB genotype than those from non-cancer patients 40.0% vs. 9.7%, p 〈 0.05). The AB AB genotype was associated with a higher intensity of ntestinal metaplasia (p 〈 0.05), but did not correlate with a higher inflammation and colonization density in gastric histology (p 〉 0.05). Besides, the study enrolled 19 patients to verify whether variable genotypes of babAB existed in the different gastric niches. Among the patients infected with more than one babAB genotypes over antrum and corpus, there were higher rate of genotypes as A B or AB AB in isolates from antrum than in those from corpus (75.0 % vs. 16.7%, p

Beschreibung: Background: Drug resistance displays a problem for the therapy of Mycobacterium tuberculosis infections. For molecular resistance testing, it is essential to have precise knowledge on genomic variations involved in resistance development. However, data from high-incidence settings are only sparely available. Therefore we performed a systematic approach and analyzed a total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone for mutations in katG, rpoB, rrs, rpsL, gidB, embB, pncA and where applicable in inhA and ahpC. Of the strains investigated 50 were either mono- or poly-resistant to isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide or MDR and 47 fully susceptible strains served as controls. Results: The majority of isoniazid and rifampin resistant strains had mutations in katG315 (71.9%) and rpoB531 (50%). However, rpoB mutations in codons 511, 516 and 533 were also detected in five rifampin susceptible strains. MIC determinations revealed low-level rifampin resistance for those strains. Thus, the sensitivity and specificity of sequencing of katG for detection of drug resistance were 86.7% and 100% and for sequencing of rpoB 100% and 93.8%, respectively. Strikingly, none of the streptomycin resistant strains had mutations in rrs, but 47.5% harboured mutations in rpsL. Further changes were detected in gidB. Among ethambutol resistant strains 46.7% had mutations at embB306. Pyrazinamide resistant strains displayed a variety of mutations throughout pncA. The specificities of sequencing of rpsL, embB and pncA for resistance detection were high (96-100%), whereas sensitivities were lower (48.8%, 73.3%, 70%). Conclusions: Our study reveals a good correlation between data from molecular and phenotypic resistance testing in this high-incidence setting. However, the fact that particular mutations in rpoB are not linked to high-level resistance is challenging and demonstrates that careful interpretation of molecular resistance assays is mandatory. In addition, certain variations, especially in gidB, appear to be phylogenetically informative polymorphisms rather than markers for drug resistance.

Beschreibung: Background: Rhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28degreesC and 35degreesC. Results: Two-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses. Conclusions: Our data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species

Beschreibung: Background: The vaginal microbiome plays an important role in urogenital health. Quantitative real time Polymerase Chain Reaction (qPCR) assays for the most prevalent vaginal Lactobacillus species and bacterial vaginosis species G. vaginalis and A. vaginae exist, but qPCR information regarding variation over time is still very limited. We set up qPCR assays for a selection of seven species and defined the temporal variation over three menstrual cycles in a healthy Caucasian population with a normal Nugent score. We also explored differences in qPCR data between these healthy women and an 'at risk' clinic population of Caucasian, African and Asian women with and without bacterial vaginosis (BV), as defined by the Nugent score. Results: Temporal stability of the Lactobacillus species counts was high with L. crispatus counts of 10*8 copies/mL and L. vaginalis counts of 10*6 copies/mL. We identified 2 types of 'normal flora' and one 'BV type flora' with latent class analysis on the combined data of all women. The first group was particularly common in women with a normal Nugent score and was characterized by a high frequency of L. crispatus, L. iners, L. jensenii, and L. vaginalis and a correspondingly low frequency of L. gasseri and A. vaginae. The second group was characterized by the predominance of L. gasseri and L. vaginalis and was found most commonly in healthy Caucasian women. The third group was commonest in women with a high Nugent score but was also seen in a subset of African and Asian women with a low Nugent score and was characterized by the absence of Lactobacillus species (except for L. iners) but the presence of G. vaginalis and A. vaginae. Conclusions: We have shown that the quantification of specific bacteria by qPCR contributes to a better description of the non-BV vaginal microbiome, but we also demonstrated that differences in populations such as risk and ethnicity also have to be taken into account. We believe that our selection of indicator organisms represents a feasible strategy for the assessment of the vaginal microbiome and could be useful for monitoring the microbiome in safety trials of vaginal products.

Beschreibung: Background: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. Results: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.750.78 Mbp genomes and UUR serovars were 0.840.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. Conclusions: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The 4 overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that 6 ureaplasmas exist as quasispecies rather than as stable serovars in their native environment. Therefore, differentialpathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.

Beschreibung: Background: Metal ions are important micronutrients in cellular metabolism, but excess ions that cause toxic reactive oxygen species are harmful to cells. In bacteria, Fur family proteins such as Fur, Zur and PerR manage the iron and zinc uptake and oxidative stress responses,respectively. The single Fur-like protein (annotated as PerR) in Streptococcus suis has been demonstrated to be involved in zinc and iron uptake in previous studies, but the reports on oxidative stress response and gene regulation are limited. Results: In the present study, the perR gene deletion mutant [increment]perR was constructed in Streptococcus suis serotype 2 strain SC-19, and the mutant strain [increment]perR exhibited less sensitivity to H2O2 stress compared to the wild-type. The dpr and metQIN were found to be upregulated in the [increment]perR strain compared with SC-19. Electrophoretic mobility shift assays showed that the promoters of dpr and metQIN could be bound by the PerR protein. These results suggest that dpr and metQIN are members of the PerR regulon of S. suis. dpr encodes a Dps-like peroxide resistance protein, and the dpr knockout strains ([increment]dpr and [increment]dpr[increment]perR) were highly sensitive to H2O2. MetQIN is a methionine transporter, and the increased utilization of methionine in the [increment]perR strain indirectly affected the peroxide resistance. Using a promoter-EGFP genefusion reporting system, we found that the PerR regulon was induced by H2O2, and the induction was modulated by metal ions. Finally, we found that the pathogenicity of the perRmutant was attenuated and easily cleared by mice. Conclusions: These data strongly suggest that the Fur-like protein PerR directly regulates dpr and metQIN and plays a crucial role in oxidative stress response in S. suis.

Beschreibung: Background: Recent studies have identified in Mycobacterium avium subsp. paratuberculosis (MAP), already known as a pathogen in ruminants, a potential zoonotic agent of some autoimmune diseases in humans. Therefore, considering the possible risk for public health, it is necessary a thorough understanding of MAP's gene expression during infection of human host as well as the identification of its immunogenic and/or virulence factors for the development of appropriate diagnostic and therapeutic tools. Results: In order to characterize MAP's transcriptome during macrophage infection, we analyzed for the first time the whole gene expression of a human derived strain of MAP in simulated intraphagosomal conditions and after intracellular infection of the human macrophage cell line THP-1 by using the DNA-microarray technology. Results showed that MAP shifts its transcriptome to an adaptive metabolism for an anoxic environment and nutrient starvation. It up-regulates several response factors to oxidative stress or intracellular conditions and allows, in terms of transcription, a passive surface peptidoglycan spoliation within the macrophage along with an intensification of the anabolic activity for lipidic membrane structures. Conclusions: These results indicate a possible interactive system between MAP and its host cell based on the internal mimicry unlike other intracellular pathogens, bringing new hypothesis in the virulence and pathogenicity of MAP and its importance in human health.

Beschreibung: Background: In the fission yeast Schizosaccharomyces pombe, the phx1+ (pombe homeobox) gene was initially isolated as a multi-copy suppressor of lysine auxotrophy caused by depletion of copper/zinc-containing superoxide dismutase (CuZn-SOD). Overproduction of Phx1 increased the synthesis of homocitrate synthase, the first enzyme in lysine biosynthetic pathway, which is labile to oxidative stress. Phx1 has a well conserved DNA-binding domain called homeodomain at the N-terminal region and is predicted to be a transcription factor in S. pombe. However, its role has not been revealed in further detail. Here we examined its expression pattern and the phenotype of its null mutant to get clues on its function. Results: Fluorescence from the Phx1-GFP expressed from a chromosomal fusion gene demonstrated that it is localized primarily in the nucleus, and is distinctly visible during the stationary phase. When we replaced the N-terminal homeobox domain of Phx1 with the DNA binding domain of Pap1, a well-characterized transcription factor, the chimeric protein caused the elevation of transcripts from Pap1-dependent genes such as ctt1+ and trr1+, suggesting that Phx1 possesses transcriptional activating activity when bound to DNA. The amount of phx1+ transcripts sharply increased as cells entered the stationary phase and was maintained at high level throughout the stationary phase. Nutrient shift down to low nitrogen or carbon sources caused phx1+ induction during the exponential phase, suggesting that cells need Phx1 for maintenance function during nutrient starvation. The delta-phx1 null mutant showed decreased viability in long-term culture, whereas overproduction of Phx1 increased viability. Decrease in long-term survival was also observed for delta-phx1 under N- or C-starved conditions. In addition, delta-phx1 mutant was more sensitive to various oxidants and heat shock. When we examined sporulation of the delta-phx1/delta-phx1 diploid strain, significant decrease in the formation of meiotic spores was observed. Conclusions: Phx1 is a transcriptional regulator whose synthesis is elevated during stationary phase and by nutrient starvation in S. pombe. It supports long-term survival and stress tolerance against oxidation and heat, and plays a key role in the formation of meiotic spores.

Beschreibung: Background: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population

Beschreibung: Background: Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causingthe mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with variousplants of economic interest in a non pathogenic manner. Results: A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrcgenes encoding for the type three secretion system (T3SS) proteins. To investigate thecontribution of T3SS to the plant-bacterial interaction process we generated mutant strains ofH. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause themottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains alsodid not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonizationexperiments showed that mutations in hrpE and hrcN genes reduced the capacity of H.rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involvedin the endophytic colonization. Conclusions: Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development ofthe mottled stripe disease and endophytic colonization of rice.

Beschreibung: Background: Playing a strategic role in the host immune function, the intestinal microbiota has beenrecently hypothesized to be involved in the etiology of atopy. In order to investigate thegastrointestinal microbial ecology of atopic disease, here we performed a pilot comparativemolecular analysis of the faecal microbiota in atopic children and healthy controls. Results: Nineteen atopic children and 12 healthy controls aged 4-14 years were enrolled. Stools werecollected and the faecal microbiota was characterized by means of the already developedphylogenetic microarray platform, HTF-Microbi.Array, and quantitative PCR. The intestinalmicrobiota of atopic children showed a significant depletion in members of the Clostridiumcluster IV, Faecalibacterium prausnitzii, Akkermansia muciniphila and a correspondingincrease of the relative abundance of Enterobacteriaceae. Conclusion: Depleted in key immunomodulatory symbionts, the atopy-associated microbiota canrepresent an inflammogenic microbial consortium which can contribute to the severity of thedisease. Our data open the way to the therapeutic manipulation of the intestinal microbiota inthe treatment of atopy by means of pharmaceutical probiotics.

Beschreibung: Background: Tuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom. Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different regions of Italy to validate the method under different environmental conditions. Results: The primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma production during the three years of the study was correlated with the concentration of T. magnatum DNA. Conclusions: Taken together, these results suggest that the specific real-time PCR assay perfected in this study could be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières.

Beschreibung: Background: The mucus layer covering the human intestinal epithelium forms a dynamic surface for host-microbial interactions. In addition to the environmental factors affecting the intestinal equilibrium, such as diet, it is well established that the microbiota composition is individually driven, but the host factors determining the composition have remained unresolved. Results: In this study, we show that ABO blood group is involved in differences in relative proportion and overall profiles of intestinal microbiota. Specifically, the microbiota from the individuals harbouring the B antigen (secretor B and AB) differed from the non-B antigen groups and also showed higher diversity of the Eubacterium rectale-Clostridium coccoides (EREC) and Clostridium leptum (CLEPT) -groups in comparison with other blood groups. Conclusions: Our novel finding indicates that the ABO blood group is one of the genetically determined host factors modulating the composition of the human intestinal microbiota, thus enabling new applications in the field of personalized nutrition and medicine.

Beschreibung: Background: Several strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains. Through PCR interrogation of integration hotspots, chromosome walking, island-tagging and fosmid-based marker rescue we captured and sequenced KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116, a bloodstream isolate from a patient with pneumonia and neutropenic sepsis. Results: The 14.0 kb KpGI-5 island exhibited a genome-anomalous G+C content, possessed near-perfect 46 bp direct repeats, encoded a gamma1 chaperone/usher fimbrial cluster (fim2) and harboured seven other predicted genes of unknown function. Transcriptional analysis demonstrated expression of three fim2 genes, and suggested that the fim2A-fim2K cluster comprised an operon. As fimbrial systems are frequently implicated in pathogenesis, we examined the role of fim2 by analysing KR2107, a streptomycin-resistant derivative of KR116, and three isogenic mutants (Deltafim, Deltafim2 and DeltafimDeltafim2) using biofilm assays, human cell adhesion assays and pair-wise competition-based murine models of intestinal colonization, lung infection and ascending urinary tract infection. Although no statistically significant role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence: KR2107 (most virulent), KR2107[increment]fim2, KR2107[increment]fim and KR2107[increment]fim[increment]fim2 (least virulent). Thus, despite lack of statistical evidence there was a suggestion that fim and fim2 contribute additively to virulence in these murine infection models. However, further studies would be necessary to substantiate this hypothesis. Conclusion: Although fim2 was present in 13% of Klebsiella spp. strains investigated, no obvious in vitro or in vivo role for the locus was identified, although there were subtle hints of involvement in urovirulence and bacterial dissemination from the respiratory tract. Based on our findings and on parallels with other fimbrial systems, we propose that fim2 has the potential to contribute beneficially to pathogenesis and/or environmental persistence of Klebsiella strains, at least under specific yet-to-be identified conditions.

Beschreibung: After publication of this work [Grabowska et al. BMC Microbiol 2011, 11:166.], it came to our attention that the grant numbers in the Acknowledgements section were incorrect. This work was supported by two grants from Polish Ministry of Science and Higher Education (No. N303 341835 and N401 183 31/3968) and by intramural grant of University of Warsaw (BW 19126).

Beschreibung: Background: Similar to Gram-negative bacteria, the outer membrane (OM) of the pathogenic spirochete, Borrelia burgdorferi, contains integral OM-spanning proteins (OMPs), as well as membrane-anchored lipoproteins. Although the mechanism of OMP biogenesis is still not well-understood, recent studies have indicated that a heterooligomeric OM protein complex, known as BAM (beta-barrel assembly machine) is required for proper assembly of OMPs into the bacterial OM. We previously identified and characterized the essential beta-barrel OMP component of this complex in B. burgdorferi, which we determined to be a functional BamA ortholog. Results: In the current study, we report on the identification of two additional protein components of the B. burgdorferi BAM complex, which were identified as putative lipoproteins encoded by ORFs BB0324 and BB0028. Biochemical assays with a BamA-depleted B. burgdorferi strain indicate that BB0324 and BB0028 do not readily interact with the BAM complex without the presence of BamA, suggesting that the individual B. burgdorferi BAM components may associate only when forming a functional BAM complex. Cellular localization assays indicate that BB0324 and BB0028 are OM-associated subsurface lipoproteins, and in silico analyses indicate that BB0324 is a putative BamD ortholog. Conclusions: The combined data suggest that the BAM complex of B. burgdorferi contains unique protein constituents which differ from those found in other proteobacterial BAM complexes. The novel findings now allow for the B. burgdorferi BAM complex to be further studied as a model system to better our understanding of spirochetal OM biogenesis in general.

Beschreibung: RationalePulmonary Alveolar Proteinosis (PAP) patients exhibit an acquired deficiency of biologically active granulocyte-macrophage colony stimulating factor (GM-CSF) attributable to GM-CSF specific autoantibodies. PAP alveolar macrophages are foamy, lipid-filled cells with impaired surfactant clearance and markedly reduced expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) and the PPARgamma-regulated ATP binding cassette (ABC) lipid transporter, ABCG1. An open label proof of concept Phase II clinical trial was conducted in PAP patients using rituximab, a chimeric murine-human monoclonal antibody directed against B lymphocyte specific antigen CD20. Rituximab treatment decreased anti-GM-CSF antibody levels in bronchoalveolar lavage (BAL) fluid, and 7/9 patients completing the trial demonstrated clinical improvement as measured by arterial blood oxygenation.ObjectivesThis study sought to determine whether rituximab therapy would restore lipid metabolism in PAP alveolar macrophages. Methods: BAL samples were collected from patients pre- and 6-months post-rituximab infusion for evaluation of mRNA and lipid changes. Results: Mean PPARgamma and ABCG1 mRNA expression increased 2.8 and 5.3-fold respectively (p [less than or equal to] 0.05) after treatment. Lysosomal phospholipase A2 (LPLA2) (a key enzyme in surfactant degradation) mRNA expression was severely deficient in PAP patients pre-treatment but increased 2.8-fold post-treatment. In supplemental animal studies, LPLA2 deficiency was verified in GM-CSF KO mice but was not present in macrophage-specific PPARgamma KO mice compared to wild-type controls. Oil Red O intensity of PAP alveolar macrophages decreased after treatment, indicating reduced intracellular lipid while extracellular free cholesterol increased in BAL fluid. Furthermore, total protein and Surfactant protein A were significantly decreased in the BAL fluid post therapy. Conclusions: Reduction in GM-CSF autoantibodies by rituximab therapy improves alveolar macrophage lipid metabolism by increasing lipid transport and surfactant catabolism. Mechanisms may involve GM-CSF stimulation of alveolar macrophage ABCG1 and LPLA2 activities by distinct pathways.

Beschreibung: Background: In recent years, Staphylococcus epidermidis (Se) has become a major nosocomial pathogen and the most common cause of infections of implanted prostheses and other indwelling devices. This is due in part to avid biofilm formation by Se on device surfaces. However, it still remains unknown that how the process of Se biofilm development is associated with relapsed infection in such patients. Results: We have identified clinical Se isolates displaying enhanced biofilm dispersal and self-renewal relative to reference strain. These isolates also exhibit enhanced initial cell attachment, extracellular DNA release, cell autolysis and thicker microcolonies during biofilm development relative to reference strain. Our genetic analyses suggest that these clinical isolates exhibit significant downregulation of RNAIII, the effector molecule of the agr quorum sensing system, and upregulation of the autolysin gene atlE. Isogenic deletion of the agr system in Se 1457 confirmed that agr negatively regulating atlE resulted in enhanced initial cell attachment, extracellular DNA release, cell autolysis and biofilm formation abilities. In contrast, double deletion of agr and atlE significantly abolished these features. Conclusions: Collectively, these data reveal the role of agr system in long-term biofilm development and pathogenesis during Se caused indwelling devices-related relapsed infection.

Beschreibung: Background: Microbes are extensively associated with insects, playing key roles in insect defense,nutrition and reproduction. Most of the associations reported involve Proteobacteria. Despitethe fact that Actinobacteria associated with insects were shown to produce antibiotic barriersagainst pathogens to the hosts or to their food and nutrients, there are few studies focusing ontheir association with insects. Thus, we surveyed the Actinobacteria diversity on a specificregion of the midgut of seven species of stinkbugs (Hemiptera: Pentatomidae) known to carrya diversity of symbiotically-associated Proteobacteria. Results: A total of 34 phylotypes were placed in 11 different Actinobacteria families. Dichelopsmelacanthus held the highest diversity with six actinobacteria families represented by ninephylotypes. Thyanta perditor (n = 7), Edessa meditabunda (n = 5), Loxa deducta (n = 4) andPellea stictica (n = 3) were all associated with three families. Piezodorus guildini (n = 3) andNezara viridula (n = 3) had the lowest diversity, being associated with two(Propionibacteriaceae and Mybacteriaceae) and one (Streptomyceataceae) families,respectively. Corynebacteriaceae and Mycobacteriaceae were the most common familieswith phylotypes from three different insect species each one. Conclusions: Many phylotypes shared a low 16 S rRNA gene similarity with their closest type strains andformed new phyletic lines on the periphery of several genera. This is a strong indicative thatstinkbug caeca can harbor new species of actinobacteria, which might be derived fromspecific associations with the species of stinkbugs studied. Although the well-known role ofactinobacteria as a source of biomolecules, the ecological features of these symbionts on thestinkbugs biology remain unknown.

Beschreibung: Background: Clostridium butyricum has become increasingly important in preventing and treatingintestinal inflammation. In the intestine it may increase the resistance of the gut to pathogeninvasion via inducing the secretion of anti-inflammatory cytokines. Interleukin 10 (IL-10)plays a central role in preventing certain inflammatory diseases by down-regulatinginflammatory cascades. In a previous study, we observed that the level of IL-10 mRNA wasmodulated by C. butyricum. The aim of this study was to investigate whether C. butyricumachieves its beneficial effects through IL-10. Results: We treated HT-29 cells with anti-IL-10 (IL-10 antibody) or siIL-10 (IL-10 small interferingRNA) to disrupt IL-10. In both cases, the effects of C. butyricum-induced NF-kappaB activationand IL-8 expression were enhanced. We also found that neutralization or knockdown of IL-10 could induce apoptosis and necrosis of HT-29 cells treated with C. butyricum comparedwith control cells. Conclusions: These findings show that IL-10 serves an important role in C. butyricum-mediated immuneprotection, and in host recognition of C. butyricum.

Beschreibung: Background: Lamivudine (LAM) is associated with the highest known rate of resistance mutations amongnucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAMcontinues in widespread use, especially in combination therapies. The primary LAMresistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim ofthis study was to characterize Brazilian HBV isolates from acute and chronic cases by directsequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencingmethod capable of detecting single-nucleotide polymorphisms. HBV DNA from serumsamples of 20 individuals with acute HBV infection and 44 with chronic infectionundergoing antiviral therapies containing LAM were analyzed by direct sequencing andpyrosequencing methods. Results: Phylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D andF) for the Brazilian population in both acute and chronic infections. However, withingenotype A isolates, subgenotype A2 was more frequently detected in acute cases than inchronic cases (P = 0.012). As expected, none of the individuals with acute hepatitis B hadLAM-resistant isolates as a dominant virus population, whether detected by direct sequencingor pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20)had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patientsundergoing LAM treatment, YMDD mutants were frequently found as a dominant viruspopulation. In cases where wild-type virus was the dominant population, subpopulations ofYMDD variants were usually found, demonstrating the complexity of HBV quasispecies. Conclusions: YMDD variants were frequently detected as a minor population in acute HBV infection. Theoccurrence of pre-existing variants may lead to a high frequency of resistant mutants duringantiviral therapy in the chronic phase. In chronic infection, detection of YMDD variantsbefore virological or biochemical breakthrough might contribute to making better therapychoices and thus improving treatment outcome.

Beschreibung: Background: Lysostaphin and the catalytic domain of LytM cleave pentaglycine crossbridges ofStaphylococcus aureus peptidoglycan. The bacteriocin lysostaphin is secreted byStaphylococcus simulans biovar staphylolyticus and directed against the cell walls ofcompeting S. aureus. LytM is produced by S. aureus as a latent autolysin and can beactivated in vitro by the removal of an N-terminal domain and occluding region. Results: We compared the efficacies of the lysostaphin and LytM catalytic domains using a newlydeveloped model of chronic S. aureus infected eczema. Lysostaphin was effective, like inother models. In contrast, LytM was not significantly better than control. The differenttreatment outcomes could be correlated with in vitro properties of the proteins, including proteolytic stability, affinity to cell wall components other than peptidoglycan, and sensitivityto the ionic milieu. Conclusions: Although lysostaphin and LytM cleave the same peptide bond in the peptidoglycan, the twoenzymes have very different environmental requirements what is reflected in their contrastingperformance in mouse eczema model.

Beschreibung: Background: Bacteremia due to Salmonella spp. is a life-threatening condition and is commonly associated with immune compromise. A 2009 observational study estimated risk factors for the ten most common non-typhoidal Salmonella (NTS) serovars isolated from Thai patients between 2002-2007. In this study, 60.8% of Salmonella enterica serovar Enteritidis isolates (n = 1517) were recovered from blood specimens and infection with Salmonella serovar Enteritidis was a statistically significant risk factor for bacteremia when compared to other NTS serovars. Based on this information, we characterized a subset of isolates collected in 2008 to determine if specific clones were recovered from blood or stool specimens at a higher rate. Twenty blood isolates and 20 stool isolates were selected for antimicrobial resistance testing (MIC), phage typing, PFGE, and MLVA.ResultEight antibiogrammes, seven MLVA types, 14 XbaI/BlnI PFGE pattern combinations, and 11 phage types were observed indicating considerable diversity among the 40 isolates characterized. Composite analysis based on PFGE and MLVA data revealed 22 genotypes. Seven of the genotypes containing two or more isolates were from both stool and blood specimens originating from various months and zones. Additionally, those genotypes were all further discriminated by phage type and/or antibiogramme. Ninety percent of the isolates were ciprofloxacin resistant. Conclusions: The increased percentage of bloodstream infections as described in the 2009 observational study could not be attributed to a single clone. Future efforts should focus on assessing the immune status of bacteriaemic patients and identifying prevention and control measures, including attribution studies characterizing non-clinical (animal, food, and environmental) isolates.

Beschreibung: Background: Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. Results: The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. Conclusions: Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or "disease specialized" while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters.

Beschreibung: Background: Hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by Salmonella enterica serovar Typhimurium (S. Typhimurium) upon internalization by phagocytic cells. In order to survive, bacteria must sense these signals and modulate gene expression. Growing evidence indicates that the ArcAB two component system plays a role in the resistance to reactive oxygen species. We investigated the influx of H2O2 and HOCl through OmpW and the role of ArcAB in modulating its expression after exposure to both toxic compounds in S. Typhimurium. Results: H2O2 and HOCl influx was determined both in vitro and in vivo. A S. Typhimurium ompW mutant strain ([increment] ompW) exposed to sub-lethal levels of H2O2 and HOCl showed a decreased influx of both compounds as compared to a wild type strain. Further evidence of H2O2 and HOCl diffusion through OmpW was obtained by using reconstituted proteoliposomes. We hypothesized that ompW expression should be negatively regulated upon exposure to H2O2 and HOCl to better exclude these compounds from the cell. As expected, qRT-PCR showed a negative regulation in a wild type strain treated with sub-lethal concentrations of these compounds. A bioinformatic analysis in search for potential negative regulators predicted the presence of three ArcA binding sites at the ompW promoter region. By electrophoretic mobility shift assay (EMSA) and using transcriptional fusions we demonstrated an interaction between ArcA and one site at the ompW promoter region. Moreover, qRT-PCR showed that the negative regulation observed in the wild type strain was lost in an arcA and in arcB mutant strains. Conclusions: OmpW allows the influx of H2O2 and HOCl and is negatively regulated by ArcA by direct interaction with the ompW promoter region upon exposure to both toxic compounds.

Beschreibung: Background: Diseases from Staphylococcus aureus are a major problem in Indian hospitals and recent studies point to infiltration of community associated methicillin resistant S. aureus (CA-MRSA) into hospitals. Although CA-MRSA are genetically different from nosocomial MRSA, the distinction between the two groups is blurring as CA-MRSA are showing multidrug resistance and are endemic in many hospitals. Our survey of samples collected from Indian hospitals between 2004 and 2006 had shown mainly hospital associated methicillin resistant Staphylococcus aureus (HA-MRSA) carrying staphylococcal cassette chromosome mec (SCCmec) type III and IIIA. But S. aureus isolates collected from 2007 onwards from community and hospital settings in India have shown SCCmec type IV and V cassettes while several variations of type IV SCCmec cassettes from IVa to IVj have been found in other parts of the world. In the present study, we have collected nasal swabs from rural and urban healthy carriers and pus, blood etc from in patients from hospitals to study the distribution of SCCmec types and sequence types (ST) in the community and hospital environment. We performed molecular characterization of all the isolates to determine their lineage and microarray of select isolates from each sequence type to analyze their toxins, virulence and immune-evasion factors. Results: Molecular analyses of 68 S. aureus isolates from in and around Bengaluru and three other Indian cities have been carried out. Our strains fall into fifteen ST with all major clonal complexes (CC) present along with some minor ones. The dominant MRSA clones are ST22 and ST772 among healthy carriers and patients. We are reporting two methicillin sensitive S. aureus (MSSA) isolates belonging to ST291 (related to ST398 which is live stock associated), ST1208 (CC8), and ST672 as emerging MRSA clones in this study for the first time. Sixty nine percent of isolates carry Panton- Valentine Leucocidin genes (PVL) along with many other toxins. There is more diversity of ST among methicillin sensitive S. aureus than resistant ones. Microarray analysis of isolates belonging to different ST for the first time gives an insight into major toxins, virulence factors, adhesion and immune evasion factors present among the isolates in various parts of India. Conclusions: S. aureus isolates reported in this study belong to a highly diverse group of ST and CC and we are reporting several new ST which have not been reported earlier along with factors influencing virulence and host pathogen interactions.