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  • Synthetic Biology and Assembly Cloning  (70)
  • Environmental Microbiology  (51)
  • Pathogens & Pathogenicity  (51)
  • Oxford University Press  (172)
  • American Chemical Society
  • Cell Press
  • Copernicus
  • Public Library of Science (PLoS)
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  • 1
    Publication Date: 2016-07-15
    Description: Invertebrate animal species that can withstand temperatures as high as 37°C, the human body temperature, are limited. In the present study, we utilized the two-spotted cricket, Gryllus bimaculatus , which lives in tropical and subtropical regions, as an animal model of human pathogenic bacterial infection. Injection of Pseudomonas aeruginosa or Staphylococcus aureus into the hemolymph killed crickets. Injected P. aeruginosa or S. aureus proliferated in the hemolymph until the cricket died. The ability of these pathogenic bacteria to kill the crickets was blocked by the administration of antibiotics. S. aureus gene-knockout mutants of virulence factors, including cvfA, agr and srtA , exhibited decreased killing ability compared with the parent strain. The dose at which 50% of crickets were killed by P. aeruginosa or S. aureus was not decreased at 37°C compared with that at 27°C. Injection of Listeria monocytogenes , which upregulates toxin expression at 37°C, killed crickets, and the dose at which 50% of crickets were killed was decreased at 37°C compared with that at 27°C. These findings suggest that the two-spotted cricket is a useful model animal for evaluating the virulence properties of various human pathogenic bacteria at variable temperature including 37°C.
    Keywords: Pathogens & Pathogenicity
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 2
    Publication Date: 2016-07-20
    Description: Four antibiotics (pamamycin, oligomycin A, oligomycin B and echinosporin) were isolated and characterized from the fermentation broth of the marine Streptomyces strains B8496 and B8739. Bioassays revealed that each of these compounds impaired motility and caused subsequent lysis of P. viticola zoospores in a dose- and time-dependent manner. Pamamycin displayed the strongest motility inhibitory and lytic activities (IC 50 0.1 μg mL –1 ) followed by oligomycin B (IC 50 0.15 and 0.2 μg mL –1 ) and oligomycin F (IC 50 0.3 and 0.5 μg mL –1 ). Oligomycin A and echinosporin also showed motility inhibitory activities against the zoospores with IC 50 values of 3.0 and 10.0 μg mL –1 , respectively. This is the first report of motility inhibitory and lytic activities of these antibiotics against zoospores of a phytopathogenic peronosporomycete. Structures of all the isolated compounds were determined based on detailed spectroscopic analysis.
    Keywords: Environmental Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 3
    Publication Date: 2016-07-31
    Description: In sulfidic environments, microbes oxidize reduced sulfur compounds via several pathways. We used metagenomics to investigate sulfur metabolic pathways from microbial mat communities in two subterranean sulfidic streams in Lower Kane Cave, WY, USA and from Glenwood Hot Springs, CO, USA. Both unassembled and targeted recA gene assembly analyses revealed that these streams were dominated by Epsilonproteobacteria and Gammaproteobacteria , including groups related to Sulfurovum , Sulfurospirillum , Thiothrix and an epsilonproteobacterial group with no close cultured relatives. Genes encoding sulfide:quinone oxidoreductase (SQR) were abundant at all sites, but the specific SQR type and the taxonomic affiliation of each type differed between sites. The abundance of thiosulfate oxidation pathway genes (Sox) was not consistent between sites, although overall they were less abundant than SQR genes. Furthermore, the Sox pathway appeared to be incomplete in all samples. This work reveals both variations in sulfur metabolism within and between taxonomic groups found in these systems, and the presence of novel epsilonproteobacterial groups.
    Keywords: Environmental Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 4
    Publication Date: 2016-07-31
    Description: Pseudomonas aeruginosa is an opportunistic pathogen with high resistance to a wide variety of antimicrobials. The multidrug resistance pump MexAB-OprM promotes the efflux of various antibiotics, mostly when mutations accumulate in the transcriptional regulators MexR, NalC and NalD, thereby causing MexAB-OprM overexpression. In this work, a characterization of 50 P. aeruginosa isolates obtained from Brazilian agricultural soils to determine the reasons of their resistance to aztreonam was done. The majority of the isolates showed higher aztreonam resistance than wild-type strain by MIC method. DNA sequence analysis of mexR , nalC and nalD genes from 13 of these isolates showed the amino acid substitution in NalC for all tested isolates, just one mutation was detected in MexR and none in NalD. Furthermore, an increase in the level of mexA expression by real-time RT-PCR analysis in eight isolates harboring mutations in NalC was found. Although there was not a relationship between MIC of aztreonam and the level of mexA expression, on the other hand, the results presented here suggest that novel mutations in NalC, including Arg 97 -Gly and Ala 186 -Thr, are related to MexAB-OprM overexpression causing aztreonam resistance in P. aeruginosa environmental isolates.
    Keywords: Environmental Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 5
    Publication Date: 2016-07-31
    Description: The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila . Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans . The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts.
    Keywords: Pathogens & Pathogenicity
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 6
    Publication Date: 2016-07-31
    Description: Sedge-dominated wetlands on the Qinghai–Tibetan Plateau are methane emission centers. Methanotrophs at these sites play a role in reducing methane emissions, but relatively little is known about the composition of active methanotrophs in these wetlands. Here, we used DNA stable isotope probing to identify the key active aerobic methanotrophs in three sedge-dominated wetlands on the plateau. We found that Methylocystis species were active in two peatlands, Hongyuan and Dangxiong. Methylobacter species were found to be active only in Dangxiong peat. Hongyuan peat had the highest methane oxidation rate, and cross-feeding of carbon from methanotrophs to methylotrophic Hyphomicrobium species was observed. Owing to a low methane oxidation rate during the incubation, the labeling of methanotrophs in Maduo wetland samples was not detected. Our results indicate that there are large differences in the activity of methanotrophs in the wetlands of this region.
    Keywords: Environmental Microbiology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 7
    Publication Date: 2016-08-05
    Description: Here we present the generation and function of two sets of bacterial plasmids that harbor fluorescent genes encoding either blue, cyan, yellow or red fluorescent proteins. In the first set, protein expression is controlled by the strong and constitutive nptII promoter whereas in the second set, the strong tac promoter was chosen that underlies LacI q regulation. Furthermore, the plasmids are mobilizable, contain Tn 7 transposons and a temperature-sensitive origin of replication. Using Escherichia coli S17-1 as donor strain, the plasmids allow fast and convenient Tn 7 -transposon delivery into many enterobacterial hosts, such as the here-used E. coli O157:H7. This procedure omits the need of preparing competent recipient cells and antibiotic resistances are only transiently conferred to the recipients. As the fluorescence proteins show little to no overlap in fluorescence emission, the constructs are well suited for the study of multicolored synthetic bacterial communities during biofilm production or in host colonization studies, e.g. of plant surfaces. Furthermore, tac promoter-reporter constructs allow the generation of so-called reproductive success reporters, which allow to estimate past doublings of bacterial individuals after introduction into environments, emphasizing the role of individual cells during colonization.
    Keywords: Environmental Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 8
    Publication Date: 2016-08-05
    Description: Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli , enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli , hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC.
    Keywords: Pathogens & Pathogenicity
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 9
    Publication Date: 2016-07-28
    Description: Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 10
    Publication Date: 2016-06-23
    Description: Spa -typing and microarray techniques were used to study epidemiological changes in methicillin-resistant Staphylococcus aureus (MRSA) in South-East Austria. The population structure of 327 MRSA isolated between 2002 and 2012 was investigated. MRSA was assigned to 58 different spa types and 14 different MLST CC (multilocus sequence type clonal complexes); in particular, between 2007 and 2012, an increasing diversity in MRSA clones could be observed. The most abundant clonal complex was CC5. On the respective SCC mec cassettes, the CC5 isolates differed clearly within this decade and CC5/SCC mec I, the South German MRSA, predominant in 2002, was replaced by CC5/SCC mec II, the Rhine-Hesse MRSA in 2012. Whereas in many European countries MLST CC22-MRSA (EMRSA 15, the Barnim epidemic MRSA) is predominant, this clone occurred in Austria nearly 10 years later than in neighbouring countries. CC45, the Berlin EMRSA, epidemic in Germany, was only sporadically found in South-East Austria. The Irish ST8-MRSA-II represented by spa -type t190 was frequently found in 2002 and 2007, but disappeared in 2012. Our results demonstrate clonal replacement of MRSA clones within the last years in Austria. Ongoing surveillance is warranted for detection of changes within the MRSA population.
    Keywords: Environmental Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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