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  • Articles  (116)
  • Environmental Microbiology  (51)
  • Biotechnology & Synthetic Biology  (41)
  • Synthetic Biology and Assembly Cloning  (24)
  • Oxford University Press  (116)
  • 2015-2019  (116)
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  • Articles  (116)
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  • Oxford University Press  (116)
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  • 1
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    Oligomycins and pamamycin homologs impair motility and induce lysis of zoospores of the grapevine downy mildew pathogen, Plasmopara viticola (2016)
    Oxford University Press
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    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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  • 2
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    Diverse sulfur metabolisms from two subterranean sulfidic spring systems (2016)
    Oxford University Press
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    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
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  • 3
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    Mutations in NalC induce MexAB-OprM overexpression resulting in high level of aztreonam resistance in environmental isolates of Pseudomonas aeruginosa (2016)
    Oxford University Press
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    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
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  • 4
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    Identification of active aerobic methanotrophs in plateau wetlands using DNA stable isotope probing (2016)
    Oxford University Press
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    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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  • 5
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    MiniTn7-transposon delivery vectors for inducible or constitutive fluorescent protein expression in Enterobacteriaceae (2016)
    Oxford University Press
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    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
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  • 6
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    Post-translational control of genetic circuits using Potyvirus proteases (2016)
    Oxford University Press
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    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
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 7
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    Increase of genetic diversity and clonal replacement of epidemic methicillin-resistant Staphylococcus aureus strains in South-East Austria (2016)
    Oxford University Press
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    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
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  • 8
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    Effects of dietary fibre source on microbiota composition in the large intestine of suckling piglets (2016)
    Oxford University Press
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    Publication Date: 2016-06-23
    Description: This study aimed to investigate the effects of dietary fibre sources on the gut microbiota in suckling piglets, and to test the hypothesis that a moderate increase of dietary fibre may affect the gut microbiota during the suckling period. Suckling piglets were fed different fibre-containing diets or a control diet from postnatal day 7 to 22. Digesta samples from cecum, proximal colon and distal colon were used for Pig Intestinal Tract Chip analysis. The data showed that the effects of fibre-containing diet on the gut microbiota differed in the fibre source and gut location. The alfalfa diet increased Clostridium cluster XIVb and Sporobacter termitidis in the cecum compared to the pure cellulose diet. Compared to the control diet, the alfalfa diet also increased Coprococcus eutactus in the distal colon, while the pure cellulose diet decreased Eubacterium pyruvativorans in the cecum. The pure cellulose diet increased Prevotella ruminicola compared to the wheat bran diet. Interestingly, the alfalfa group had the lowest abundance of the potential pathogen Streptococcus suis in the cecum and distal colon. These results indicated that a moderate increase in dietary fibres affected the microbial composition in suckling piglets, and that the alfalfa inclusion produced some beneficial effects on the microbial communities.
    Keywords: Environmental Microbiology
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  • 9
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    Microbial detoxification in the gut of a specialist avian herbivore, the Greater Sage-Grouse (2016)
    Oxford University Press
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    Publication Date: 2016-06-23
    Description: One function of the gut microbiota gaining recent attention, especially in herbivorous mammals and insects, is the metabolism of plant secondary metabolites (PSMs). We investigated whether this function exists within the gut communities of a specialist avian herbivore. We sequenced the cecal metagenome of the Greater Sage-Grouse ( Centrocercus urophasianus ), which specializes on chemically defended sagebrush ( Artemisia spp.). We predicted that the cecal metagenome of the sage-grouse would be enriched in genes associated with the metabolism of PSMs when compared to the metagenome of the domestic chicken. We found that representation of microbial genes associated with ‘xenobiotic degradation and metabolism’ was 3-fold higher in the sage-grouse cecal metagenomes when compared to that of the domestic chicken. Further, we identified a complete metabolic pathway for the degradation of phenol to pyruvate, which was not detected in the metagenomes of the domestic chicken, bovine rumen or 14 species of mammalian herbivores. Evidence of monoterpene degradation (a major class of PSMs in sagebrush) was less definitive, although we did detect genes for several enzymes associated with this process. Overall, our results suggest that the gut microbiota of specialist avian herbivores plays a similar role to the microbiota of mammalian and insect herbivores in degrading PSMs.
    Keywords: Environmental Microbiology
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  • 10
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    Endosymbiotic bacteria in honey bees: Arsenophonus spp. are not transmitted transovarially (2016)
    Oxford University Press
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    Publication Date: 2016-06-23
    Description: Intracellular endosymbiotic bacteria are common and can play a crucial role for insect pathology. Therefore, such bacteria could be a potential key to our understanding of major losses of Western honey bees ( Apis mellifera ) colonies. However, the transmission and potential effects of endosymbiotic bacteria in A. mellifera and other Apis spp. are poorly understood. Here, we explore the prevalence and transmission of the genera Arsenophonus , Wolbachia , Spiroplasma and Rickettsia in Apis spp. Colonies of A. mellifera ( N = 33, with 20 eggs from worker brood cells and 100 adult workers each) as well as mated honey bee queens of A. cerana , A. dorsata and A. florea ( N = 12 each) were screened using PCR. While Wolbachia , Spiroplasma and Rickettsia were not detected, Arsenophonus spp. were found in 24.2% of A. mellifera colonies and respective queens as well as in queens of A. dorsata (8.3%) and A. florea (8.3%), but not in A. cerana . The absence of Arsenophonus spp. from reproductive organs of A. mellifera queens and surface-sterilized eggs does not support transovarial vertical transmission. Instead, horizontal transmission is most likely.
    Keywords: Environmental Microbiology
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  • 11
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    Widespread ability of fungi to drive quinone redox cycling for biodegradation (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: Wood-rotting fungi possess remarkably diverse extracellular oxidation mechanisms, including enzymes, such as laccase and peroxidases, and Fenton chemistry. The ability to biologically drive Fenton chemistry by the redox cycling of quinones has previously been reported to be present in both ecologically diverging main groups of wood-rotting basidiomycetes. Therefore, we investigated whether it is even more widespread among fungal organisms. Screening of a diverse selection of a total of 18 ascomycetes and basidiomycetes for reduction of the model compound 2,6-dimethoxy benzoquinone revealed that all investigated strains were capable of reducing it to its corresponding hydroquinone. In a second step, depolymerization of the synthetic polymer polystyrene sulfonate was used as a proxy for quinone-dependent Fenton-based biodegradation capabilities. A diverse subset of the strains, including environmentally ubiquitous molds, white-rot fungi, as well as peatland and aquatic isolates, caused substantial depolymerization indicative for the effective employment of quinone redox cycling as biodegradation tool. Our results may also open up new paths to utilize diverse fungi for the bioremediation of recalcitrant organic pollutants.
    Keywords: Environmental Microbiology
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  • 12
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    Endophytic fungi associated with Sudanese medicinal plants show cytotoxic and antibiotic potential (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: In this study, we isolated 15 endophytic fungi from five Sudanese medicinal plants. Each fungal endophytic strain was identified by sequencing of internal transcribed spacer (ITS) regions of rDNA. Ethyl acetate extracts were prepared from each endophyte cultivated in vitro and tested for their respective antibacterial activities and antiproliferative activities against human cancer cells. Antibacterial screening was carried out against two bacterial strains: Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus , by the broth dilution method. Cell viability was evaluated by the MTT procedure after exposure of MCF7 breast cancer cells and HT29 or HCT116 human colon adenocarcinoma cells to each endophytic extract. Of interest, Byssochlamys spectabilis isolated from Euphorbia prostata showed cytotoxicity (IC 50 = 1.51 ± 0.2 μg mL –1 ) against MCF7 cells, but had a low effect against HT29 or HCT116 cells (IC 50 〉 20 μg mL –1 ). Cladosporium cladosporioides 2, isolated from Vernonia amygdalina leaves, showed antiproliferative activities against MCF7 cells (IC 50 = 10.5 ± 1.5 μg mL –1 ) only. On the other hand, B. spectabilis and Alternaria sp. extract had antibacterial activities against the S. aureus strain. The findings of this work revealed that endophytic fungi associated with medicinal plants from Sudan could be considered as an attractive source of new therapeutic compounds.
    Keywords: Biotechnology & Synthetic Biology
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  • 13
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    Properties and biotechnological applications of ice-binding proteins in bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: Ice-binding proteins (IBPs), such as antifreeze proteins (AFPs) and ice-nucleating proteins (INPs), have been described in diverse cold-adapted organisms, and their potential applications in biotechnology have been recognized in various fields. Currently, both IBPs are being applied to biotechnological processes, primarily in medicine and the food industry. However, our knowledge regarding the diversity of bacterial IBPs is limited; few studies have purified and characterized AFPs and INPs from bacteria. Phenotypically verified IBPs have been described in members belonging to Gammaproteobacteria, Actinobacteria and Flavobacteriia classes, whereas putative IBPs have been found in Gammaproteobacteria, Alphaproteobacteria and Bacilli classes. Thus, the main goal of this minireview is to summarize the current information on bacterial IBPs and their application in biotechnology, emphasizing the potential application in less explored fields such as agriculture. Investigations have suggested the use of INP-producing bacteria antagonists and AFPs-producing bacteria (or their AFPs) as a very attractive strategy to prevent frost damages in crops. UniProt database analyses of reported IBPs (phenotypically verified) and putative IBPs also show the limited information available on bacterial IBPs and indicate that major studies are required.
    Keywords: Environmental Microbiology
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  • 14
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    Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1 (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA , cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C–50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of K cat and K cat / K m for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research.
    Keywords: Environmental Microbiology
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  • 15
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    A case in support of implementing innovative bio-processes in the metal mining industry (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: The metal mining industry faces many large challenges in future years, among which is the increasing need to process low-grade ores as accessible higher grade ores become depleted. This is against a backdrop of increasing global demands for base and precious metals, and rare earth elements. Typically about 99% of solid material hauled to, and ground at, the land surface currently ends up as waste (rock dumps and mineral tailings). Exposure of these to air and water frequently leads to the formation of acidic, metal-contaminated run-off waters, referred to as acid mine drainage, which constitutes a severe threat to the environment. Formation of acid drainage is a natural phenomenon involving various species of lithotrophic (literally ‘rock-eating’) bacteria and archaea, which oxidize reduced forms of iron and/or sulfur. However, other microorganisms that reduce inorganic sulfur compounds can essentially reverse this process. These microorganisms can be applied on industrial scale to precipitate metals from industrial mineral leachates and acid mine drainage streams, resulting in a net improvement in metal recovery, while minimizing the amounts of leachable metals to the tailings storage dams. Here, we advocate that more extensive exploitation of microorganisms in metal mining operations could be an important way to green up the industry, reducing environmental risks and improving the efficiency and the economy of metal recovery.
    Keywords: Environmental Microbiology
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  • 16
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    Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast (2015)
    Oxford University Press
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    Publication Date: 2015-05-03
    Description: Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5–2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 17
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    A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications (2015)
    Oxford University Press
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    Publication Date: 2015-04-21
    Description: RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP -transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 18
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    Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley (2016)
    Oxford University Press
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    Publication Date: 2016-01-09
    Description: Proteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a ‘DNA pulley’ for position-resolved nano-mechanical measurements of protein-DNA interactions. A molecule of DNA is tethered by one end to a glass surface, and by the other end to a magnetic bead. The DNA is stretched horizontally by a magnet, and a nanoscale knife made of silicon nitride is manipulated to contact, bend and scan along the DNA. The mechanical profile of the DNA at the contact with the knife is probed via nanometer-precision optical tracking of the magnetic bead. This system enables detection of protein bumps on the DNA and localization of their binding sites. We study theoretically the technical requirements to detect mechanical heterogeneities in the DNA itself.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 19
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    Dealing with the genetic load in bacterial synthetic biology circuits: convergences with the Ohm's law (2016)
    Oxford University Press
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    Publication Date: 2016-01-09
    Description: Synthetic biology seeks to envision living cells as a matter of engineering. However, increasing evidence suggests that the genetic load imposed by the incorporation of synthetic devices in a living organism introduces a sort of unpredictability in the design process. As a result, individual part characterization is not enough to predict the behavior of designed circuits and thus, a costly trial-error process is eventually required. In this work, we provide a new theoretical framework for the predictive treatment of the genetic load. We mathematically and experimentally demonstrate that dependences among genes follow a quantitatively predictable behavior. Our theory predicts the observed reduction of the expression of a given synthetic gene when an extra genetic load is introduced in the circuit. The theory also explains that such dependence qualitatively differs when the extra load is added either by transcriptional or translational modifications. We finally show that the limitation of the cellular resources for gene expression leads to a mathematical formulation that converges to an expression analogous to the Ohm's law for electric circuits. Similitudes and divergences with this law are outlined. Our work provides a suitable framework with predictive character for the design process of complex genetic devices in synthetic biology.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 20
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    Nitric oxide scavengers differentially inhibit ammonia oxidation in ammonia-oxidizing archaea and bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-04-01
    Description: Differential inhibitors are important for measuring the relative contributions of microbial groups, such as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), to biogeochemical processes in environmental samples. In particular, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) represents a nitric oxide scavenger used for the specific inhibition of AOA, implicating nitric oxide as an intermediate of thaumarchaeotal ammonia oxidation. This study investigated four alternative nitric oxide scavengers for their ability to differentially inhibit AOA and AOB in comparison to PTIO. Caffeic acid, curcumin, methylene blue hydrate and trolox were tested on Nitrosopumilus maritimus , two unpublished AOA representatives (AOA-6f and AOA-G6) as well as the AOB representative Nitrosomonas europaea . All four scavengers inhibited ammonia oxidation by AOA at lower concentrations than for AOB. In particular, differential inhibition of AOA and AOB by caffeic acid (100 μM) and methylene blue hydrate (3 μM) was comparable to carboxy-PTIO (100 μM) in pure and enrichment culture incubations. However, when added to aquarium sponge biofilm microcosms, both scavengers were unable to inhibit ammonia oxidation consistently, likely due to degradation of the inhibitors themselves. This study provides evidence that a variety of nitric oxide scavengers result in differential inhibition of ammonia oxidation in AOA and AOB, and provides support to the proposed role of nitric oxide as a key intermediate in the thaumarchaeotal ammonia oxidation pathway.
    Keywords: Environmental Microbiology
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    De novo biosynthesis of resveratrol by site-specific integration of heterologous genes in Escherichia coli (2016)
    Oxford University Press
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    Publication Date: 2016-04-08
    Description: Resveratrol is a well-known triphenolic natural product present in red wine. For its contribution to human health, the demand for resveratrol as a food and nutrition supplement has increased significantly. In recent years, the rapid development of synthetic biology has promoted extensive work to increase the production of resveratrol in microbes. However, supplementation of expensive phenylpropanoic precursors was required in current engineered strains. Here, we first utilized the site-specific integration strategy to produce resveratrol in Escherichia coli . The genes tal , 4cl and sts were site-specific integrated into the loci of genes tyrR and trpED in the chromosome of E. coli BW25113 (DE3). The final strain was capable of producing 4.612 mg L –1 of resveratrol from glucose.
    Keywords: Biotechnology & Synthetic Biology
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    Overexpression of two stress-responsive, small, non-coding RNAs, 6S and tmRNA, imparts butanol tolerance in Clostridium acetobutylicum (2016)
    Oxford University Press
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    Publication Date: 2016-04-08
    Description: While extensively studied in several model organisms, the role of small, non-coding RNAs in the stress response remains largely unexplored in Clostridium organisms. About 100 years after the first industrial Acetone–Butanol–Ethanol fermentation process, based on the Weizmann Clostridium acetobutylicum strain, strain tolerance to butanol remains a crucial factor limiting the economics of the process. Several studies have examined the response of this organism to metabolite stress, and several genes have been engaged to impart enhanced tolerance, but no sRNAs have yet been directly engaged in this task. We show that the two stress-responsive sRNAs, 6S and tmRNA, upon overexpression impart tolerance to butanol as assessed by viability assays under process-relevant conditions. 6S overexpression enhances cell densities as well as butanol titres. We discuss the likely mechanisms that these two sRNAs might engage in this tolerance phenotype. Our data support the continued exploration of sRNAs as a basis for engineering enhanced tolerance and enhanced solvent production, especially because sRNA-based strategies impose a minimal metabolic burden on the cells.
    Keywords: Biotechnology & Synthetic Biology
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    Neofiscalin A and fiscalin C are potential novel indole alkaloid alternatives for the treatment of multidrug-resistant Gram-positive bacterial infections (2016)
    Oxford University Press
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    Publication Date: 2016-07-02
    Description: Ten indole alkaloids were obtained from the marine sponge-associated fungus Neosartorya siamensis KUFA 0017. We studied the antimicrobial properties of these and of three other compounds previously isolated from the soil fungus N. siamensis KUFC 6349. Only neofiscalin A showed antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE); with a minimum inhibitory concentration (MIC) of 8 μg mL –1 against both strains. Another compound, fiscalin C, presented synergistic activity against MRSA when combined with oxacillin, although alone showed no antibacterial effect. Moreover, neofiscalin A, when present at sub-MICs, hampered the ability of both MRSA and VRE strains to form a biofilm. Additionally, the biofilm inhibitory concentration values of neofiscalin A against the MRSA and VRE isolates were 96 and 80 μg mL –1 , respectively. At a concentration of 200 μg mL –1 , neofiscalin A was able to reduce the metabolic activity of the biofilms by ~50%. One important fact is that our results also showed that neofiscalin A had no cytotoxicity against a human brain capillary endothelial cell line.
    Keywords: Biotechnology & Synthetic Biology
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    A novel isolate and widespread abundance of the candidate alphaproteobacterial order (Ellin 329), in southern Appalachian peatlands (2016)
    Oxford University Press
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    Publication Date: 2016-07-02
    Description: Peatlands of all latitudes play an integral role in global climate change by serving as a carbon sink and a primary source of atmospheric methane; however, the microbial ecology of mid-latitude peatlands is vastly understudied. Herein, next generation Illumina amplicon sequencing of small subunit rRNA genes was utilized to elucidate the microbial communities in three southern Appalachian peatlands. In contrast to northern peatlands, Proteobacteria dominated over Acidobacteria in all three sites. An average of 11 bacterial phyla was detected at relative abundance values 〉1%, with three candidate divisions (OP3, WS3 and NC10) represented, indicating high phylogenetic diversity. Physiological traits of isolates within the candidate alphaproteobacterial order, Ellin 329, obtained here and in previous studies indicate that bacteria of this order may be involved in hydrolysis of poly-, di- and monosaccharides. Community analyses indicate that Ellin 329 is the third most abundant order and is most abundant near the surface layers where plant litter decomposition should be primarily occurring. In sum, members of Ellin 329 likely play important roles in organic matter decomposition, in southern Appalachian peatlands and should be investigated further in other peatlands and ecosystem types.
    Keywords: Environmental Microbiology
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    Marine phage genomics: the tip of the iceberg (2016)
    Oxford University Press
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    Publication Date: 2016-07-02
    Description: Marine viruses are the most abundant biological entity in the oceans, the majority of which infect bacteria and are known as bacteriophages. Yet, the bulk of bacteriophages form part of the vast uncultured dark matter of the microbial biosphere. In spite of the paucity of cultured marine bacteriophages, it is known that marine bacteriophages have major impacts on microbial population structure and the biogeochemical cycling of key elements. Despite the ecological relevance of marine bacteriophages, there are relatively few isolates with complete genome sequences. This minireview focuses on knowledge gathered from these genomes put in the context of viral metagenomic data and highlights key advances in the field, particularly focusing on genome structure and auxiliary metabolic genes.
    Keywords: Environmental Microbiology
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    Plants of the fynbos biome harbour host species-specific bacterial communities (2016)
    Oxford University Press
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    Publication Date: 2016-07-02
    Description: The fynbos biome in South Africa is globally recognised as a plant biodiversity hotspot. However, very little is known about the bacterial communities associated with fynbos plants, despite interactions between primary producers and bacteria having an impact on the physiology of both partners and shaping ecosystem diversity. This study reports on the structure, phylogenetic composition and potential roles of the endophytic bacterial communities located in the stems of three fynbos plants ( Erepsia anceps , Phaenocoma prolifera and Leucadendron laureolum ). Using Illumina MiSeq 16S rRNA sequencing we found that different subpopulations of Deinococcus-Thermus, Alphaproteobacteria, Acidobacteria and Firmicutes dominated the endophytic bacterial communities. Alphaproteobacteria and Actinobacteria were prevalent in P. prolifera , whereas Deinococcus-Thermus dominated in L. laureolum , revealing species-specific host–bacteria associations. Although a high degree of variability in the endophytic bacterial communities within hosts was observed, we also detected a core microbiome across the stems of the three plant species, which accounted for 72% of the sequences. Altogether, it seems that both deterministic and stochastic processes shaped microbial communities. Endophytic bacterial communities harboured putative plant growth-promoting bacteria, thus having the potential to influence host health and growth.
    Keywords: Environmental Microbiology
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    The unconventional secretion of PepN is independent of a functional autophagy machinery in the filamentous fungus Aspergillus niger (2016)
    Oxford University Press
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    Publication Date: 2016-07-02
    Description: During unconventional protein secretion (UPS), proteins do not pass through the classical endoplasmic reticulum (ER)–Golgi-dependent pathway, but are transported to the cell membrane via alternative routes. One type of UPS is dependent on several autophagy-related (Atg) proteins in yeast and mammalian cells, but mechanisms for unconventional secretion are largely unknown for filamentous fungi. In this study, we investigated whether the autophagy machinery is used for UPS in the filamentous fungus Aspergillus niger . An aspartic protease, which we called PepN, was identified as being likely to be secreted unconventionally, as this protein is highly abundant in culture filtrates during carbon starvation while it lacks a conventional N-terminal secretion sequence. We analysed the presence of PepN in the culture filtrates of carbon starved wild-type, atg1 and atg8 deletion mutant strains by Western blot analysis and by secretome analysis using nanoLC-ESI-MS/MS (wild-type and atg8 deletion mutant). Besides the presence of carbohydrate-active enzymes and other types of proteases, PepN was abundantly found in culture filtrates of both wild-type and atg deletion strains, indicating that the secretion of PepN is independent of the autophagy machinery in A. niger and hence most likely occurs via a different mechanism.
    Keywords: Biotechnology & Synthetic Biology
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    Blue light-mediated transcriptional activation and repression of gene expression in bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes.
    Keywords: Synthetic Biology and Assembly Cloning
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    Millimeter scale electron conduction through exoelectrogenic mixed species biofilms (2016)
    Oxford University Press
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    Publication Date: 2016-07-03
    Description: The functioning of many natural and engineered environments is dependent on long distance electron transfer mediated through electrical currents. These currents have been observed in exoelectrogenic biofilms and it has been proposed that microbial biofilms can mediate electron transfer via electrical currents on the centimeter scale. However, direct evidence to confirm this hypothesis has not been demonstrated and the longest known electrical transfer distance for single species exoelectrogenic biofilms is limited to 100 μm. In the present study, biofilms were developed on electrodes with electrically non-conductive gaps from 50 μm to 1 mm and the in situ conductance of biofilms was evaluated over time. Results demonstrated that the exoelectrogenic mixed species biofilms in the present study possess the ability to transfer electrons through electrical currents over a distance of up to 1 mm, 10 times further than previously observed. Results indicate the possibility of interspecies interactions playing an important role in the spatial development of exoelectrogenic biofilms, suggesting that these biological networks might remain conductive even at longer distance. These findings have significant implications in regards to future optimization of microbial electrochemical systems.
    Keywords: Environmental Microbiology
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    CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated ‘CasHRA ( Cas 9-facilitated H omologous R ecombination A ssembly)’ that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo ; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 ( M inimal G enome of Escherichia coli ) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions.
    Keywords: Synthetic Biology and Assembly Cloning
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    Effects of MreB paralogs on poly-{gamma}-glutamic acid synthesis and cell morphology in Bacillus amyloliquefaciens (2016)
    Oxford University Press
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    Publication Date: 2016-08-27
    Description: Actin-like MreB paralogs play important roles in cell shape maintenance, cell wall synthesis and the regulation of the D,L-endopeptidases, CwlO and LytE. The gram-positive bacteria, Bacillus amyloliquefaciens LL3, is a poly--glutamic acid (-PGA) producing strain that contains three MreB paralogs: MreB, Mbl and MreBH. In B. amyloliquefaciens , CwlO and LytE can degrade -PGA. In this study, we aimed to test the hypothesis that modulating transcript levels of MreB paralogs would alter the synthesis and degradation of -PGA. The results showed that overexpression or inhibition of MreB, Mbl or MreBH had distinct effects on cell morphology and the molecular weight of the -PGA products. In fermentation medium, cells of mreB inhibition mutant were 50.2% longer than LL3, and the -PGA titer increased by 55.7%. However, changing the expression level of mbl showed only slight effects on the morphology, -PGA molecular weight and titer. In the mreBH inhibition mutant, -PGA production and its molecular weight increased by 56.7% and 19.4%, respectively. These results confirmed our hypothesis that suppressing the expression of MreB paralogs might reduce -PGA degradation, and that improving the cell size could strengthen -PGA synthesis. This is the first report of enhanced -PGA production via suppression of actin-like MreB paralogs.
    Keywords: Biotechnology & Synthetic Biology
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    Simultaneous detection of somatic and F-specific coliphages in different settings by Escherichia coli strain CB390 (2016)
    Oxford University Press
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    Publication Date: 2016-08-28
    Description: Bacteriophages are increasingly being used as water quality indicators. Two groups of phages infecting Escherichia coli , somatic and F-specific coliphages, are being considered as indicators of fecal and viral contamination for several types of water around the world. However, some uncertainties remain regarding which coliphages to assess. Recently, E. coli strain CB390 has been reported to be suitable for simultaneous detection of both groups, which seems to be more informative than determining only one of the groups. Here, a significant number of samples from different settings, mostly those where F-specific phages have been reported to outnumber somatic coliphages, are analyzed for somatic coliphages, F-specific RNA phages by standardized methods and coliphages detected by host strain CB390. The results presented here confirm that the numbers of phages counted using CB390 are equivalent to the sum of the somatic and F-specific coliphages counted independently in all settings. Hence the usefulness of this strain for simultaneous detection of somatic and F-specific coliphages is confirmed. Also, sets of data on the presence of coliphages in reclaimed and groundwater are reported.
    Keywords: Environmental Microbiology
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    Robust, tunable genetic memory from protein sequestration combined with positive feedback (2015)
    Oxford University Press
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    Publication Date: 2015-10-15
    Description: Natural regulatory networks contain many interacting components that allow for fine-tuning of switching and memory properties. Building simple bistable switches, synthetic biologists have learned the design principles of complex natural regulatory networks. However, most switches constructed so far are so simple (e.g. comprising two regulators) that they are functional only within a limited parameter range. Here, we report the construction of robust, tunable bistable switches in Escherichia coli using three heterologous protein regulators (ExsADC) that are sequestered into an inactive complex through a partner swapping mechanism. On the basis of mathematical modeling, we accurately predict and experimentally verify that the hysteretic region can be fine-tuned by controlling the interactions of the ExsADC regulatory cascade using the third member ExsC as a tuning knob. Additionally, we confirm that a dual-positive feedback switch can markedly increase the hysteretic region, compared to its single-positive feedback counterpart. The dual-positive feedback switch displays bistability over a 10 6 -fold range of inducer concentrations, to our knowledge, the largest range reported so far. This work demonstrates the successful interlocking of sequestration-based ultrasensitivity and positive feedback, a design principle that can be applied to the construction of robust, tunable, and predictable genetic programs to achieve increasingly sophisticated biological behaviors.
    Keywords: Synthetic Biology and Assembly Cloning
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    Industrial production of acetone and butanol by fermentation--100 years later (2016)
    Oxford University Press
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    Publication Date: 2016-06-08
    Description: Microbial production of acetone and butanol was one of the first large-scale industrial fermentation processes of global importance. During the first part of the 20th century, it was indeed the second largest fermentation process, superseded in importance only by the ethanol fermentation. After a rapid decline after the 1950s, acetone-butanol-ethanol (ABE) fermentation has recently gained renewed interest in the context of biorefinery approaches for the production of fuels and chemicals from renewable resources. The availability of new methods and knowledge opens many new doors for industrial microbiology, and a comprehensive view on this process is worthwhile due to the new interest. This thematic issue of FEMS Microbiology Letters, dedicated to the 100th anniversary of the first industrial exploitation of Chaim Weizmann's ABE fermentation process, covers the main aspects of old and new developments, thereby outlining a model development in biotechnology. All major aspects of industrial microbiology are exemplified by this single process. This includes new technologies, such as the latest developments in metabolic engineering, the exploitation of biodiversity and discoveries of new regulatory systems such as for microbial stress tolerance, as well as technological aspects, such as bio- and down-stream processing.
    Keywords: Biotechnology & Synthetic Biology
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    A new lactobacilli in vivo expression system for the production and delivery of heterologous proteins at mucosal surfaces (2016)
    Oxford University Press
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    Publication Date: 2016-06-02
    Description: Food-grade lactic acid bacteria, such as lactobacilli, represent good candidates for the development of mucosal vectors. Indeed, they are generally recognized as safe microorganisms and some strains display beneficial effects (probiotics). In this study, we described a new lactobacilli in vivo expression (LIVE) system for the production and delivery of therapeutic molecules at mucosal surfaces. The versatility and functionality of this system was successfully validated in several lactobacilli species; furthermore, we assessed in vivo LIVE system in two different mouse models of human pathologies: (i) a model of therapy against intestinal inflammation (inflammatory bowel diseases) and (ii) a model of vaccination against dental caries. We demonstrated that Lactobacillus gasseri expressing the anti-inflammatory cytokine IL-10 under LIVE system efficiently delivered the recombinant protein at mucosal surfaces and display anti-inflammatory effects. In the vaccination model against caries, LIVE system allowed the heterologous expression of Streptococcus mutans antigen GbpB by L. gasseri , leading to a stimulation of the host immune response.
    Keywords: Biotechnology & Synthetic Biology
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    Bioreactors and in situ product recovery techniques for acetone-butanol-ethanol fermentation (2016)
    Oxford University Press
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    Publication Date: 2016-06-02
    Description: The microbial fermentation process is one of the sustainable and environment-friendly ways to produce 1-butanol and other bio-based chemicals. The success of the fermentation process greatly relies on the choice of bioreactors and the separation methods. In this review, the history and the performance of bioreactors for the acetone–butanol–ethanol (ABE) fermentation is discussed. The subject is then focused on in situ product recovery (ISPR) techniques, particularly for the integrated extraction-gas stripping. The usefulness of this promising hybrid ISPR device is acknowledged by its incorporation with batch, fed-batch and continuous processes to improve the performance of ABE fermentation.
    Keywords: Biotechnology & Synthetic Biology
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    Uptake and effect of rare earth elements on gene expression in Methylosinus trichosporium OB3b (2016)
    Oxford University Press
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    Publication Date: 2016-06-04
    Description: It is well known that Methylosinus trichosporium OB3b has two forms of methane monooxygenase (MMO) responsible for the initial conversion of methane to methanol, a cytoplasmic (soluble) methane monooxygenase and a membrane-associated (particulate) methane monooxygenase, and that copper strongly regulates expression of these alternative forms of MMO. More recently, it has been discovered that M. trichosporium OB3b has multiple types of the methanol dehydrogenase (MeDH), i.e. the Mxa-type MeDH (Mxa-MeDH) and Xox-type MeDH (Xox-MeDH), and the expression of these two forms is regulated by the availability of the rare earth element (REE), cerium. Here, we extend these studies and show that lanthanum, praseodymium, neodymium and samarium also regulate expression of alternative forms of MeDH. The effect of these REEs on MeDH expression, however, was only observed in the absence of copper. Further, a mutant of M. trichosporium OB3b, where the Mxa-MeDH was knocked out, was able to grow in the presence of lanthanum, praseodymium and neodymium, but was not able to grow in the presence of samarium. Collectively, these data suggest that multiple levels of gene regulation by metals exist in M. trichosporium OB3b, but that copper overrides the effect of other metals by an as yet unknown mechanism.
    Keywords: Environmental Microbiology
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    Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches (2016)
    Oxford University Press
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    Publication Date: 2016-06-03
    Description: We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong 70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.
    Keywords: Synthetic Biology and Assembly Cloning
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    Microbial impact on polysulfide dynamics in the environment (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: Polysulfides (S x 2– ) are sulfide oxidation intermediates that are important for a variety of environmentally relevant processes including pyrite formation, organic matter sulfidization, isotope exchange among reduced sulfur species, and metal chelation. In addition to their chemical reactivity, laboratory experiments with microbial cultures and enzymes indicate both indirect and direct roles for microorganisms in affecting polysulfide chemistry in natural environments through production and consumption. As polysulfides have been detected in a wide array of natural systems ranging from microbial mats to hydrothermal vents, constraining their biogeochemical cycling has broad impacts. However, many questions remain regarding the processes responsible for polysulfide dynamics in these environments and the precise role that microorganisms play in these processes. This review provides a summary of laboratory experiments investigating the role of polysulfides in microbial metabolism, and observations of polysulfides in the environment in order to provide further insight into and highlight open questions about this significant component of the sulfur cycle.
    Keywords: Environmental Microbiology
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    Solid-phase cloning for high-throughput assembly of single and multiple DNA parts (2015)
    Oxford University Press
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    Publication Date: 2015-04-21
    Description: We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of 〉60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of 〉150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
    Keywords: Synthetic Biology and Assembly Cloning
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    Metabolic modeling of clostridia: current developments and applications (2016)
    Oxford University Press
    Details
    Publication Date: 2016-01-29
    Description: Anaerobic Clostridium spp. is an important bioproduction microbial genus that can produce solvents and utilize a broad spectrum of substrates including cellulose and syngas. Genome-scale metabolic (GSM) models are increasingly being put forth for various clostridial strains to explore their respective metabolic capabilities and suitability for various bioconversions. In this study, we have selected representative GSM models for six different clostridia ( Clostridium acetobutylicum , C. beijerinckii , C. butyricum , C. cellulolyticum , C. ljungdahlii and C. thermocellum ) and performed a detailed model comparison contrasting their metabolic repertoire. We also discuss various applications of these GSM models to guide metabolic engineering interventions as well as assessing cellular physiology.
    Keywords: Biotechnology & Synthetic Biology
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  • 42
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    Mercury resistance transposons in Bacilli strains from different geographical regions (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: A total of 65 spore-forming mercury-resistant bacteria were isolated from natural environments worldwide in order to understand the acquisition of additional genes by and dissemination of mercury resistance transposons across related Bacilli genera by horizontal gene movement. PCR amplification using a single primer complementary to the inverted repeat sequence of Tn MERI1 -like transposons showed that 12 of 65 isolates had a transposon-like structure. There were four types of amplified fragments: Tn 5084 , Tn 5085 , Tn d MER3 (a newly identified deleted transposon-like fragment) and Tn 6294 (a newly identified transposon). Tn d MER3 is a 3.5-kb sequence that carries a merRETPA operon with no merB or transposase genes. It is related to the mer operon of Bacillus licheniformis strain FA6-12 from Russia. DNA homology analysis shows that Tn 6294 is an 8.5-kb sequence that is possibly derived from Tn d MER3 by integration of a Tn MERI1 -type transposase and resolvase genes and in addition the merR2 and merB1 genes. Bacteria harboring Tn 6294 exhibited broad-spectrum mercury resistance to organomercurial compounds, although Tn 6294 had only merB1 and did not have the merB2 and merB3 sequences for organomercurial lyases found in Tn 5084 of B. cereus strain RC607. Strains with Tn 6294 encode mercuric reductase (MerA) of less than 600 amino acids in length with a single N-terminal mercury-binding domain, whereas MerA encoded by strains MB1 and RC607 has two tandem domains. Thus, Tn d MER3 and Tn 6294 are shorter prototypes for Tn MERI1 -like transposons. Identification of Tn 6294 in Bacillus sp. from Taiwan and in Paenibacillus sp. from Antarctica indicates the wide horizontal dissemination of Tn MERI1 -like transposons across bacterial species and geographical barriers.
    Keywords: Environmental Microbiology
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  • 43
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    Synthesis and cell-free cloning of DNA libraries using programmable microfluidics (2016)
    Oxford University Press
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    Publication Date: 2016-03-01
    Description: Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 44
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    A gene network engineering platform for lactic acid bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-03-01
    Description: Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 45
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    Acetone-butanol-ethanol fermentation of corn stover: current production methods, economic viability and commercial use (2016)
    Oxford University Press
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    Publication Date: 2016-03-02
    Description: Biobutanol is a next-generation liquid biofuel with properties akin to those of gasoline. There is a widespread effort to commercialize biobutanol production from agricultural residues, such as corn stover, which do not compete with human and animal foods. This pursuit is backed by extensive government mandates to expand alternative energy sources. This review provides an overview of research on biobutanol production using corn stover feedstock. Structural composition, pretreatment, sugar yield (following pretreatment and hydrolysis) and generation of lignocellulose-derived microbial inhibitory compounds (LDMICs) from corn stover are discussed. The review also discusses different Clostridium species and strains employed for biobutanol production from corn stover-derived sugars with respect to solvent yields, tolerance to LDMICs and in situ solvent recovery (integrated fermentation). Further, the economics of cellulosic biobutanol production are highlighted and compared to corn starch-derived ethanol and gasoline. As discussed herein, the economic competitiveness of biobutanol production from corn stover largely depends on feedstock processing and fermentation process design.
    Keywords: Biotechnology & Synthetic Biology
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  • 46
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    Fungal denitrification: Bipolaris sorokiniana exclusively denitrifies inorganic nitrogen in the presence and absence of oxygen (2016)
    Oxford University Press
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    Publication Date: 2016-02-07
    Description: Fungi may play an important role in the production of the greenhouse gas nitrous oxide (N 2 O). Bipolaris sorokiniana is a ubiquitous saprobe found in soils worldwide, yet denitrification by this fungal strain has not previously been reported. We aimed to test if B. sorokiniana would produce N 2 O and CO 2 in the presence of organic and inorganic forms of nitrogen (N) under microaerobic and anaerobic conditions. Nitrogen source (organic-N, inorganic-N, no-N control) significantly affected N 2 O and CO 2 production both in the presence and absence of oxygen, which contrasts with bacterial denitrification. Inorganic N addition increased denitrification of N 2 O (from 0 to 0.3 μg N 2 0-N h –1  g –1 biomass) and reduced respiration of CO 2 (from 0.1 to 0.02 mg CO 2 h –1  g –1 biomass). Isotope analyses indicated that nitrite, rather than ammonium or glutamine, was transformed to N 2 O. Results suggest the source of N may play a larger role in fungal N 2 O production than oxygen status.
    Keywords: Environmental Microbiology
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  • 47
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    A Pseudomonas putida double mutant deficient in butanol assimilation: a promising step for engineering a biological biofuel production platform (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn 5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn 5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant.
    Keywords: Biotechnology & Synthetic Biology
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  • 48
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    Frontiers in microbial 1-butanol and isobutanol production (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: The heavy dependence on petroleum-derived fuel has raised concerns about energy sustainability and climate change, which have prompted researchers to explore fuel production from renewable sources. 1-Butanol and isobutanol are promising biofuels that have favorable properties and can also serve as solvents or chemical feedstocks. Microbial production of these alcohols provides great opportunities to access a wide spectrum of renewable resources. In recent years, research has improved the native 1-butanol production and has engineered isobutanol production in various organisms to explore metabolic diversity and a broad range of substrates. This review focuses on progress in metabolic engineering for the production of these two compounds using various resources.
    Keywords: Biotechnology & Synthetic Biology
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  • 49
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    Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba ), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera.
    Keywords: Environmental Microbiology
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  • 50
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    Recent progress in biobutanol tolerance in microbial systems with an emphasis on Clostridium (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: Biobased production of butanol promises a more sustainable route for industrial production. However, butanol toxicity remains a barrier for achieving high product titers. Investigation into butanol stress has shed some light on its modes of toxicity. Unfortunately, there still remain significant shortfalls in our understanding of the complex interactions of butanol with cells. To address this knowledge gap, a diverse range of tools have been employed to gain a better understanding of the adverse effects of butanol on the cell. These findings have lead to the identification of possible molecular mechanisms associated with butanol tolerance, which can be harnessed for future strain development efforts. This review focuses on recent efforts to address the toxicity of butanol in microbial producers and offers some perspectives on the future direction of this research sector.
    Keywords: Biotechnology & Synthetic Biology
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  • 51
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    Synergistic effect of calcium and zinc on glucose/xylose utilization and butanol tolerance of Clostridium acetobutylicum (2016)
    Oxford University Press
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    Publication Date: 2016-02-25
    Description: Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L –1 were achieved from 69.3 g L –1 glucose with butanol/ABE productivities of 0.40 and 0.65 g L –1 h –1 compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L –1 h –1 obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L –1 butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum .
    Keywords: Biotechnology & Synthetic Biology
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    Pseudomonas putida KT2440 markerless gene deletion using a combination of {lambda} Red recombineering and Cre/loxP site-specific recombination (2016)
    Oxford University Press
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    Publication Date: 2016-02-07
    Description: Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/P mekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida KT2440.
    Keywords: Biotechnology & Synthetic Biology
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  • 53
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    Evaluation of viability, metabolic activity and spore quantity in clostridial cultures during ABE fermentation (2016)
    Oxford University Press
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    Publication Date: 2016-03-02
    Description: Flow cytometry, in combination with fluorescent staining, was used to evaluate population heterogeneity in acetone-butanol–ethanol fermentation that was carried out with type strain Clostridium beijerinckii NCIMB 8052 and non-type C. pasteurianum NRRL B-598. A combination of propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), PI plus Syto-9 and bis-oxonol (BOX) alone were employed to distinguish between active and damaged cells together with simultaneous detection of spores. These strategies provided valuable information on the physiological state of clostridia. CFDA and PI staining gave the best separation of four distinct subpopulations of enzymatically active cells, doubly stained cells, damaged cells and spores. Proportional representation of cells in particular sub-regions correlated with growth characteristics, fermentation parameters such as substrate consumption and product formation in both species under different cultivation conditions.
    Keywords: Biotechnology & Synthetic Biology
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    Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor (2015)
    Oxford University Press
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    Publication Date: 2015-12-02
    Description: Optimizing bio-production involves strain and process improvements performed as discrete steps. However, environment impacts genotype and a strain that is optimal under one set of conditions may not be under different conditions. We present a methodology to simultaneously vary genetic and process factors, so that both can be guided by design of experiments (DOE). Advances in DNA assembly and gene insulation facilitate this approach by accelerating multi-gene pathway construction and the statistical interpretation of screening data. This is applied to a 6-aminocaproic acid (6-ACA) pathway in Escherichia coli consisting of six heterologous enzymes. A 32-member fraction factorial library is designed that simultaneously perturbs expression and media composition. This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly). Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease. Therefore, if genotype and media were varied separately this would lead to a suboptimal combination. This is applied to the design of a strain and media composition that increases 6-ACA from 9 to 48 mg/l in a single optimization step. This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.
    Keywords: Synthetic Biology and Assembly Cloning
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    Simultaneous saccharification and fermentation of dilute alkaline-pretreated corn stover for enhanced butanol production by Clostridium saccharobutylicum DSM 13864 (2016)
    Oxford University Press
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    Publication Date: 2016-02-03
    Description: Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF.
    Keywords: Biotechnology & Synthetic Biology
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    Solid and liquid media for isolating and cultivating acidophilic and acid-tolerant sulfate-reducing bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-04-24
    Description: Growth media have been developed to facilitate the enrichment and isolation of acidophilic and acid-tolerant sulfate-reducing bacteria (aSRB) from environmental and industrial samples, and to allow their cultivation in vitro . The main features of the ‘standard’ solid and liquid devised media are as follows: (i) use of glycerol rather than an aliphatic acid as electron donor; (ii) inclusion of stoichiometric concentrations of zinc ions to both buffer pH and to convert potentially harmful hydrogen sulphide produced by the aSRB to insoluble zinc sulphide; (iii) inclusion of Acidocella aromatica (an heterotrophic acidophile that does not metabolize glycerol or yeast extract) in the gel underlayer of double layered (overlay) solid media, to remove acetic acid produced by aSRB that incompletely oxidize glycerol and also aliphatic acids (mostly pyruvic) released by acid hydrolysis of the gelling agent used (agarose). Colonies of aSRB are readily distinguished from those of other anaerobes due to their deposition and accumulation of metal sulphide precipitates. Data presented illustrate the effectiveness of the overlay solid media described for isolating aSRB from acidic anaerobic sediments and low pH sulfidogenic bioreactors.
    Keywords: Environmental Microbiology
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    Construction and characterization of three protein-targeting expression system in Lactobacillus casei (2016)
    Oxford University Press
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    Publication Date: 2016-03-13
    Description: We previously reported that the β-1,4-Mannanase ( manB ) gene from Bacillus pumilus functions as a good reporter gene in Lactobacillus casei . Two vectors were constructed. One carries the signal peptide of secretion protein Usp45 (SP Usp45 ) from Lactococcus lactis (pELSH), and the other carries the full-length S-layer protein, SlpA, from L. acidophilus (pELWH). In this work, another vector, pELSPH, was constructed to include the signal peptide of protein SlpA (SP SlpA ), and the capacity of all three vectors to drive expression of the manB gene in L. casei was evaluated. The results showed that SP Usp45 is functionally recognized and processed by the L. casei secretion machinery. The SP Usp45 -mediated secretion efficiency was ~87%, and SP SlpA drove the export of secreted ManB with ~80% efficiency. SP SlpA secretion was highly efficient, and expressed SlpA was anchored to the cell wall by an unknown secretion mechanism. Full-length SlpA drove the cell wall-anchored expression of an SlpA-ManB fusion protein but at a much lower level than that of protein SlpA.
    Keywords: Biotechnology & Synthetic Biology
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    A rapid and sensitive colorimetric measurement of antibiotic efficacy against Escherichia coli in vitro (2016)
    Oxford University Press
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    Publication Date: 2016-03-13
    Description: A common dye of prussian blue (PB) as an indicator was used to develop a colorimetric method for detecting the efficacy of the antibiotics in vitro. Considering the electronic production capacity of microbial respiration, ferricyanide was employed in transferring electrons from target microorganism of Escherichia coli ( E. coli ) to produce ferrocyanide. Subsequently, ferrocyanide reacted with ferric ions to form PB. In view of relationship between the PB yield and the bacterial activity, the efficacy of the antibiotics on E. coli was directly detected at 700 nm of PB absorption. When the 5% activity of antibiotics on 20 isolates of E. coli was quantified as 5% efficacy, the applied concentrations of eight antibiotics, such as cefepime, ceftriaxone sodium, cefoperazone sodium, piperacillin sodium, amoxicillin, gentamicin, amikacin and levofloxacin were 2, 2, 4, 4, 10, 4, 8 and 8 μg mL –1 , respectively. To compare with minimum inhibitory concentration results obtained by Clinical and Laboratory Standards Institute broth macrodilution method, the results of PB methods showed good agreements except with gentamicin. Paired t- test result ( P ) also showed that difference between two methods was statistically significant ( P = 0.006).
    Keywords: Environmental Microbiology
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    Hetero-oligomeric MspA pores in Mycobacterium smegmatis (2016)
    Oxford University Press
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    Publication Date: 2016-03-13
    Description: The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.
    Keywords: Biotechnology & Synthetic Biology
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    Multiplex pairwise assembly of array-derived DNA oligonucleotides (2016)
    Oxford University Press
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    Publication Date: 2016-03-19
    Description: While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.
    Keywords: Synthetic Biology and Assembly Cloning
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    Exposure of Metarhizium acridum mycelium to light induces tolerance to UV-B radiation (2016)
    Oxford University Press
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    Publication Date: 2016-03-04
    Description: Metarhizium acridum is an entomopathogenic fungus commonly used as a bioinsecticide. The conidium is the fungal stage normally employed as field inoculum in biological control programs and must survive under field conditions such as high ultraviolet-B (UV-B) exposure. Light, which is an important stimulus for many fungi, has been shown to induce the production of M. robertsii conidia with increased stress tolerance. Here we show that a two-hour exposure to white or blue/UV-A light of fast-growing mycelium induces tolerance to subsequent UV-B irradiation. Red light, however, does not have the same effect. In addition, we established that this induction can take place with as little as 1 min of white-light exposure. This brief illumination scheme could be relevant in future studies of M. acridum photobiology and for the production of UV-B resistant mycelium used in mycelium-based formulations for biological control.
    Keywords: Environmental Microbiology
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    Photorhabdus luminescens LN2 requires rpoS for nematicidal activity and nematode development (2016)
    Oxford University Press
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    Publication Date: 2016-03-04
    Description: Photorhabdus (Enterobacteriaceae) bacteria are pathogenic to insects and mutualistic with entomopathogenic Heterorhabditis nematodes . Photorhabdus luminescens subsp. akhurstii LN2, associated with Heterorhabditis indica LN2, shows nematicidal activity against H. bacteriophora H06 infective juveniles (IJs). In the present study, an rpoS mutant of P. luminescens LN2 was generated through allelic exchange to examine the effects of rpoS deletion on the nematicidal activity and nematode development. The results showed that P. luminescens LN2 required rpoS for nematicidal activity against H06 nematodes, normal IJ recovery and development of H. indica LN2, however, not for the bacterial colonization in LN2 and H06 IJs. This provides cues for further understanding the role of rpoS in the mutualistic association between entomopathogenic nematodes and their symbionts.
    Keywords: Environmental Microbiology
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    Butanol formation from gaseous substrates (2016)
    Oxford University Press
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    Publication Date: 2016-03-04
    Description: Mostly, butanol is formed as a product by saccharolytic anaerobes, employing the so-called ABE fermentation (for acetone–butanol–ethanol). However, this alcohol can also be produced from gaseous substrates such as syn(thesis) gas (major components are carbon monoxide and hydrogen) by autotrophic acetogens. In view of economic considerations, a biotechnological process based on cheap and abundant gases such as CO and CO 2 as a carbon source is preferable to more expensive sugar or starch fermentation. In addition, any conflict for use of substrates that can also serve as human nutrition is avoided. Natural formation of butanol has been found with, e.g. Clostridium carboxidivorans , while metabolic engineering for butanol production was successful using, e.g. C. ljungdahlii . Production of butanol from CO 2 under photoautotrophic conditions was also possible by recombinant DNA construction of a respective cyanobacterial Synechococcus sp. PCC 7942 strain.
    Keywords: Biotechnology & Synthetic Biology
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    Diversity and distribution of catechol 2, 3-dioxygenase genes in surface sediments of the Bohai Sea (2016)
    Oxford University Press
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    Publication Date: 2016-05-05
    Description: Catechol 2, 3-dioxygenase (C23O) is the key enzyme for aerobic aromatic degradation. Based on clone libraries and quantitative real-time polymerase chain reaction, we characterized diversity and distribution patterns of C23O genes in surface sediments of the Bohai Sea. The results showed that sediments of the Bohai Sea were dominated by genes related to C23O subfamily I.2.A. The samples from wastewater discharge area (DG) and aquaculture farm (KL) showed distinct composition of C23O genes when compared to the samples from Bohai Bay (BH), and total organic carbon was a crucial determinant accounted for the composition variation. C6BH12-38 and C2BH2-35 displayed the highest gene copies and highest ratios to the 16S rRNA genes in KL, and they might prefer biologically labile aromatic hydrocarbons via aquaculture inputs. Meanwhile, C7BH3-48 showed the highest gene copies and highest ratios to the 16S rRNA genes in DG, and this could be selective effect of organic loadings from wastewater discharge. An evident increase in C6BH12-38 and C7BH3-48 gene copies and reduction in diversity of C23O genes in DG and KL indicated composition perturbations of C23O genes and potential loss in functional redundancy. We suggest that ecological habitat and trophic specificity could shape the distribution of C23O genes in the Bohai Sea sediments.
    Keywords: Environmental Microbiology
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    Lichen-forming fungus Caloplaca flavoruscens inhibits transcription factors and chromatin remodeling system in fungi (2016)
    Oxford University Press
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    Publication Date: 2016-05-20
    Description: Lichen-forming fungi and extracts derived from them have been used as alternative medicine sources for millennia and recently there has been a renewed interest in their known bioactive properties for anticancer agents, cosmetics and antibiotics. Although lichen-forming fungus-derived compounds are biologically and commercially valuable, few studies have been performed to determine their modes of action. This study used chemical-genetic and chemogenomic high-throughput analyses to gain insight into the modes of action of Caloplaca flavoruscens extracts. High-throughput screening of 575 lichen extracts was performed and 39 extracts were identified which inhibited yeast growth. A C. flavoruscens extract was selected as a promising antifungal and was subjected to genome-wide haploinsufficiency profiling and homozygous profiling assays. These screens revealed that yeast deletion strains lacking Rsc8, Pro1 and Toa2 were sensitive to three concentrations (IC 25.5 , IC 25 and IC 50 , respectively) of C. flavoruscens extract. Gene-enrichment analysis of the data showed that C. flavoruscens extracts appear to perturb transcription and chromatin remodeling.
    Keywords: Biotechnology & Synthetic Biology
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    Cytotoxic damage of soybean agglutinin on intestinal epithelial cells of broiler chicks: in vitro protection by Bifidobacterium infantis CRL1395 (2016)
    Oxford University Press
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    Publication Date: 2016-05-20
    Description: Plant lectins, which are proteins/glycoproteins present in a wide range of vegetables, fruits, cereals and beans, are resistant to digestive enzymes and food cooking temperatures. They bind reversibly to specific glycosidic residues expressed on the membrane of intestinal epithelial cells (IEC) and cause anti-nutritional effects in humans and animals. Soybean lectin (SBA) has been detected in poultry diets, and its ability to bind to the intestinal epithelium has been reported. The development of new methods for removing SBA from feeds or to prevent interaction with the intestinal mucosa is of interest. In this study, the in vitro cytotoxicity of SBA on IEC of chicks was demonstrated for the first time. The LD 50 , assessed after 2 h exposure of IEC to SBA, was 6.13 μg mL –1 . The ability of Bifidobacterium infantis CRL1395 to bind SBA on the bacterial envelope was confirmed, and prevention of IEC cytotoxicity by lectin removal was demonstrated. Safety of B. infantis CRL1395, resistance to gastrointestinal stress and adhesion were also determined. It was concluded that the early administration of B. infantis CRL1395 to chicks would effectively reduce the toxicity of SBA. Besides, it would favour the colonization of the gut with a beneficial microbiota.
    Keywords: Biotechnology & Synthetic Biology
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    Impact of pnpR, a LysR-type regulator-encoding gene, on the cellular processes of Pseudomonas putida DLL-E4 (2016)
    Oxford University Press
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    Publication Date: 2016-05-20
    Description: LysR-type transcriptional regulators (LTTRs) regulate various cellular processes in bacteria. pnpR is an LTTR-encoding gene involved in the regulation of hydroquinone (HQ) degradation, and its effects on the cellular processes of Pseudomonas putida DLL-E4 were investigated at the physiological, biochemical and molecular levels. Reverse transcription polymerase chain reaction revealed that pnpR positively regulated its own expression and that of the pnpC1C2DECX1X2 operon; additionally, pnpR partially regulated the expression of pnpA when P. putida was grown on para -nitrophenol (PNP) or HQ. Strains DLL-E4 and DLL- pnpR exhibited similar cellular morphologies and growth rates. Transcriptome analysis revealed that pnpR regulated the expression of genes in addition to those involved in PNP degradation. A total of 20 genes were upregulated and 19 genes were downregulated by at least 2-fold in strain DLL- pnpR relative to strain DLL-E4. Bioinformatic analysis revealed putative PnpR-binding sites located in the upstream regions of genes involved in PNP degradation, carbon catabolite repression and other cellular processes. The utilization of L-aspartic acid, L-histidine, L-pyroglutamic acid, L-serine, -aminobutyric acid, D,L-lactic acid, D-saccharic acid, succinic acid and L-alaninamide was increased at least 1.3-fold in strain DLL- pnpR as shown by BIOLOG assays, indicating that pnpR plays a potential negative regulation role in the utilization of carbon sources.
    Keywords: Environmental Microbiology
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    Catalytic upgrading of butyric acid towards fine chemicals and biofuels (2016)
    Oxford University Press
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    Publication Date: 2016-04-08
    Description: Fermentation-based production of butyric acid is robust and efficient. Modern catalytic technologies make it possible to convert butyric acid to important fine chemicals and biofuels. Here, current chemocatalytic and biocatalytic conversion methods are reviewed with a focus on upgrading butyric acid to 1-butanol or butyl-butyrate. Supported Ruthenium- and Platinum-based catalyst and lipase exhibit important activities which can pave the way for more sustainable process concepts for the production of green fuels and chemicals.
    Keywords: Biotechnology & Synthetic Biology
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    Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum (2016)
    Oxford University Press
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    Publication Date: 2016-04-15
    Description: Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner -based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP- based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A , thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum .
    Keywords: Biotechnology & Synthetic Biology
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    Keeping it simple: lessons from the golden era of antibiotic discovery (2016)
    Oxford University Press
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    Publication Date: 2016-04-15
    Description: Bacteria are becoming increasingly resistant to currently used antibiotics. At the same time, little progress has been made in discovering new antibacterial drugs to combat resistant organisms. History teaches us that ‘high tech’ target-based complex methods are not synonymous with success and a return to simple, systematic screening of natural products against bacteria from traditional and novel resources holds our greatest hope of success.
    Keywords: Biotechnology & Synthetic Biology
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    n-butanol: challenges and solutions for shifting natural metabolic pathways into a viable microbial production (2016)
    Oxford University Press
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    Publication Date: 2016-04-15
    Description: The economic upturn of the past 200 years would not have been conceivable without fossil resources such as coal and oil. However, the fossil-based economy increasingly reaches its limits and displays contradictions. Bioeconomy, strategically combining economy and ecology willing to make biobased and sustainable growth possible, is promising to make a significant contribution towards solving these issues. In this context, microbial bioconversions are promising to support partially the increasing need for materials and fuels starting from fresh, preferably waste, biomass. Butanol is a very attractive molecule finding applications both as a chemical platform and as a fuel. Today it principally derives from petroleum, but it also represents the final product of microbial catabolic pathways. Because of the need to maximize yield, titer and productivity to make the production competitive and viable, the challenge is to transform a robustly regulated metabolic network into the principal cellular activity. However, this goal can only be accomplished by a profound understanding of the cellular physiology, survival strategy and sensing/signalling cascades. Here, we shortly review on the natural cellular pathways and circumstances that lead to n -butanol accumulation, its physiological consequences that might not match industrial needs and on possible solutions for circumventing these natural constraints.
    Keywords: Biotechnology & Synthetic Biology
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    Downstream process options for the ABE fermentation (2016)
    Oxford University Press
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    Publication Date: 2016-04-20
    Description: Butanol is a very interesting substance both for the chemical industry and as a biofuel. The classical distillation process for the removal of butanol is far too energy demanding, at a factor of 220% of the energy content of butanol. Alternative separation processes studied are hybrid processes of gas-stripping, liquid–liquid extraction and pervaporation with distillation and a novel adsorption/drying/desorption hybrid process. Compared with the energy content of butanol, the resulting energy demand for butanol separation and concentration of optimized hybrid processes is 11%–22% for pervaporation/distillation and 11%–17% for liquid–liquid extraction/distillation. For a novel adsorption/drying/desorption process, the energy demand is 9.4%. But all downstream process options need further proof of industrial applicability.
    Keywords: Biotechnology & Synthetic Biology
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    System-level modeling of acetone-butanol-ethanol fermentation (2016)
    Oxford University Press
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    Publication Date: 2016-04-20
    Description: Acetone–butanol–ethanol (ABE) fermentation is a metabolic process of clostridia that produces bio-based solvents including butanol. It is enabled by an underlying metabolic reaction network and modulated by cellular gene regulation and environmental cues. Mathematical modeling has served as a valuable strategy to facilitate the understanding, characterization and optimization of this process. In this review, we highlight recent advances in system-level, quantitative modeling of ABE fermentation. We begin with an overview of integrative processes underlying the fermentation. Next we survey modeling efforts including early simple models, models with a systematic metabolic description, and those incorporating metabolism through simple gene regulation. Particular focus is given to a recent system-level model that integrates the metabolic reactions, gene regulation and environmental cues. We conclude by discussing the remaining challenges and future directions towards predictive understanding of ABE fermentation.
    Keywords: Biotechnology & Synthetic Biology
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    Methanogen communities in a municipal landfill complex in China (2016)
    Oxford University Press
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    Publication Date: 2016-04-20
    Description: Landfills are significant global sources of atmospheric methane, but little is known about the ecology and community structure of methanogens in these sites. Here, we investigated the methanogen community based on methyl coenzyme M reductase A gene amplicons in the vertical profiles of three different sites at a municipal landfill complex in China. Links between methanogen communities and refuse properties were explored using multivariate analysis. Clone library results showed that most clones (92%) were related to the hydrogenotrophic methanogens, Methanomicrobiales. Almost all of the Methanomicrobiales clones retrieved in this study are members of the genus Methanoculleus . Eight clones were affiliated with the genus Methanofollis . The remaining clones were clustered within the genus Methanosarcina . Terminal restriction fragment length polymorphism profiles showed that the landfill was predominated by 22 taxa, making up 69%–96% of the community. Of these, a single taxon comprised 36%–65% of the communities across all sites and depths. Principal components analysis separated the methanogen community into three groups, irrespective of site or depth. Redundancy analysis suggested that total phosphorus and pH play roles in structuring methanogen communities in landfills.
    Keywords: Environmental Microbiology
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    Cry-like genes, in an uncommon gene configuration, produce a crystal that localizes within the exosporium when expressed in an acrystalliferous strain of Bacillus thuringiensis (2016)
    Oxford University Press
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    Publication Date: 2016-02-10
    Description: Cry proteins are pesticidal toxins produced by the bacterium Bacillus thuringiensis (Bt), which aggregate in sporulating cells to form a crystal. Except in a relatively few cases, these crystals are located outside the exosporium that surrounds the spore. Bt2-56 is a strain of Bt that has the relatively uncommon characteristic of locating its Cry protein-containing crystal within the exosporium, and in association with a long, multifiber filament. With the ultimate goal of both understanding and manipulating the localization of Cry proteins within the exosporium, we sought to identify the genes coding for the exosporium-localized Cry proteins in Bt2-56. Herein we show (i) that five cry-like genes are present in the genome of Bt2-56, (ii) that two pairs of these genes show organizational similarity to a relatively uncommon gene configuration that coexpress a cry gene along with a gene whose product aids crystal formation and (iii) that when one of these two gene pairs (cry21A-cdA) is expressed in an acrystalliferous strain of Bt, crystals are formed that localize within the exosporium. In Bt ssp. finitimus , the only other strain in which crystal localization has been studied, a Cry protein needed expression of two non-cry ORFs in order to localize within the exosporium, indicating that there are some mechanistic differences for crystal localization between Bt ssp. finitimus and Bt2-56.
    Keywords: Biotechnology & Synthetic Biology
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    Production of biobutanol from cellulose hydrolysate by the Escherichia coli co-culture system (2016)
    Oxford University Press
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    Publication Date: 2016-02-12
    Description: The commercialization of the n -butanol bioprocess is largely dependent on the price of feedstocks. Renewable cellulose appears to be an appealing feedstock. The microbial production of n -butanol still remains challenging because of the limited availability of intracellular NADH. To address this issue, an Escherichia coli strain carrying the clostridial CoA-dependent pathway was supplied with heterologous formate dehydrogenase. With the cellulose hydrolysate of rice straw, this single strain produced cellulosic biobutanol with a production yield at 35% of the theoretical and a productivity of 0.093 g L –1  h –1 . In an alternative method, the production involved a co-culture system consisting of two separate strains provided with the full CoA-dependent pathway. This system achieved a production yield and productivity reaching 62.8% of the theoretical and 0.163 g L –1  h –1 , respectively. The result indicates that the E. coli co-culture system is technically promising for the production of cellulosic biobutanol.
    Keywords: Biotechnology & Synthetic Biology
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    First description of 'Chalky back phenomenon in banana prawns (Fenneropenaeus merguiensis) and its possible association with Vibrio and Photobacterium species (2016)
    Oxford University Press
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    Publication Date: 2016-02-12
    Description: Here we report a newly identified ‘Chalky back’ phenomenon in banana prawns ( Fenneropenaeus merguiensis ) farmed in North Queensland, Australia. This was characterized by localized white discoloured segmentation of the cervical groove, moreover, after cooking the prawns exploded, making them unfit for commercial sale. Histological examination revealed breakdown of gut and abdominal muscle tissue in some moribund specimens. We selectively isolated Vibrio spp., which are known prawn pathogens, from healthy and Chalky back specimens. Isolated bacteria were identified, typed and tested for the presence of eight virulence genes (VGs), biofilm formation, adherence and cytotoxicity to fish cells. In all, 32 isolates were recovered and identified as Vibrio harveyi , V. owensii , V. sinaloensis -like, V. campbellii , V. shilonii , Vibrio sp. and Photobacterium damselae using 16S rRNA gene sequencing. All V. harveyi carried VGs coding for haemolysin, tox R and flagella; formed biofilm; and adhered to both cell lines. This was similar to the V. sinaloensis -like strains that were only isolated from Chalky back specimens. Our data suggest that Vibrio spp. may play a role in the pathogenesis of Chalky back. This study is the first report of Chalky back phenomenon in farmed banana prawns that needs to be closely monitored by the industry.
    Keywords: Environmental Microbiology
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    A part toolbox to tune genetic expression in Bacillus subtilis (2016)
    Oxford University Press
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    Publication Date: 2016-09-03
    Description: Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli , such libraries are lacking for the Gram-positive model Bacillus subtilis , a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis . We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ~14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis .
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
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    Interspecific cooperation: enhanced growth, attachment and strain-specific distribution in biofilms through Azospirillum brasilense-Pseudomonas protegens co-cultivation (2016)
    Oxford University Press
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    Publication Date: 2016-10-30
    Description: Plant-growth-promoting bacteria belonging to Azospirillum and Pseudomonas genera are major inhabitants of the rhizosphere. Both are increasingly commercialized as crops inoculants. Interspecific interaction in the rhizosphere is critical for inoculants aptness. The objective of this work was to evaluate Azospirillum and Pseudomonas interaction in mixed biofilms by co-cultivation of the model strains Azospirillum brasilense Sp245 and Pseudomonas protegens CHA0. The results revealed enhanced growth of both strains when co-cultured in static conditions. Moreover, Sp245 biofilm formed in plastic surfaces was increased 2-fold in the presence of CHA0. Confocal microscopy revealed highly structured mixed biofilms showing Sp245 mainly on the bottom and CHA0 towards the biofilm surface. In addition, A. brasilense biofilm was thicker and denser when co-cultured with P. protegens. In a colony–colony interaction assay, Sp245 changed nearby CHA0 producing small colony phenotype, which accounts for a diffusible metabolite mediator; though CHA0 spent medium did not affect Sp245 colony phenotype. Altogether, these results point to a cooperative interaction between A. brasilense Sp245 and P. protegens CHA0 in which both strains increase their static growth and produce structured mixed biofilms with a strain-specific distribution.
    Keywords: Environmental Microbiology
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    Rumen microbiota for wild boreal cervids living in the same habitat (2016)
    Oxford University Press
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    Publication Date: 2016-10-26
    Description: Knowledge about the factors shaping the rumen microbiota in wild animals is limited. Therefore, the aim of this study was to compare the microbiota from the three cervid species moose ( Alces alces , n = 5), red deer ( Cervus elaphus , n = 4) and roe deer ( Capreolus capreolus , n = 12), sharing the same habitat. Using deep 16S rRNA gene sequencing, we found that the largest species moose had the highest number of unique operational taxonomic units. Furthermore, red deer and moose shared more of the microbiota, compared with the smallest species, roe deer, with Firmicutes and Euryarchaeota being significantly overrepresented for the shared microbiota. These differences could not be explained by diet or range. The animals largely shared the same range, and there are no systematic differences in diet. We therefore believe rumen physiology can be one of the main contributing factors to the observed distribution of the rumen microbiota in cervid species.
    Keywords: Environmental Microbiology
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    Preparation and evaluation of MS2 bacteriophage-like particles packaging hepatitis E virus RNA (2016)
    Oxford University Press
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    Publication Date: 2016-11-09
    Description: Hepatitis E virus (HEV) is the pathogen causing hepatitis E (HE). It arouses global public health concern since it is a zoonotic disease. The objective of this letter is to report a cost-effective internal control prepared for monitoring procedures of HEV reverse transcriptase (RT)-PCR detection. A selected conserved HEV RNA fragment was integrated into the downstream of the truncated MS2 bacteriophage genome based on Armored RNA technology. The resulting MS2-HEV gene harbored by the pET-28b-MS2-HEV plasmid was transformed into E. coli BL21(DE3) for expression analysis by SDS-PAGE. The expression products were purified and concentrated by ultrasonication and ultrafiltration separation. The morphology and stability properties of the virus-like particles (VLPs) were evaluated by electron microscopy scanning and nuclease challenges, respectively. SDS-PAGE results showed that the constructed MS2-HEV gene expressed efficiently and the purity of the VLPs was highly consistent with the result in electron microscopy. Stability evaluation results demonstrated that the prepared VLPs exhibited strong resistance to DNase I and RNase A attacks and also performed long-lasting protection of coated HEV RNA for at least 4 months at –20°C. These data revealed that the prepared VLPs meet the basic requirements of use as internal control material in the HEV RNA amplification assay.
    Keywords: Biotechnology & Synthetic Biology
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    Epigenetics knocks on synthetic biology's door (2016)
    Oxford University Press
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    Publication Date: 2016-09-08
    Description: Epigenetics is the study of heritable changes in gene expression without concomitant changes in DNA sequence. Due to its relevance in development, differentiation and human health, epigenetics has recently become an emerging area of science with regard to eukaryotic organisms and has shown enormous potential in synthetic biology. However, significant examples of epigenetic regulation in bacterial synthetic biology have not yet been reported. In the current study, we present the first model of such an epigenetic circuit. We termed the circuit the alternator circuit because parental cells carrying this circuit and their progeny alternate between distinct and heritable cellular fates without undergoing changes in genome sequence. Furthermore, we demonstrated that the alternator circuit exhibits hysteresis because its output depends not only on its present state but also on its previous states.
    Keywords: Biotechnology & Synthetic Biology
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    The endolysin from the Enterococcus faecalis bacteriophage VD13 and conditions stimulating its lytic activity (2016)
    Oxford University Press
    Details
    Publication Date: 2016-10-12
    Description: Bacteriophages produce endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant endolysins can lyse Gram-positive bacteria when added exogenously. As a potential alternative antimicrobial, we cloned and expressed the enterococcal VD13 bacteriophage endolysin. VD13 endolysin has a CHAP catalytic domain with 92% identity with the bacteriophage IME-EF1 endolysin. The predicted size of VD13 endolysin is ~27 kDa as verified by SDS-PAGE. The VD13 endolysin lyses Enterococcus faecalis strains, but not E. faecium or other non-enterococci. VD13 endolysin has activity from pH 4 to pH 8, with peak activity at pH 5, and exhibits greater activity in the presence of calcium. Optimum activity at pH 5 occurs in the absence of NaCl. VD13 endolysin, in ammonium acetate (C 2 H 3 O 2 NH 4 ) calcium chloride (CaCl 2 ) buffer pH 5, is stimulated to higher activity upon heating at temperatures up to 65°C for 30 min, whereas activity is lost upon heating to 42°C, in pH 7 buffer.
    Keywords: Biotechnology & Synthetic Biology
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    The transcriptional factor TtsI is involved in a negative regulation of swimming motility in Mesorhizobium loti MAFF303099 (2016)
    Oxford University Press
    Details
    Publication Date: 2016-10-12
    Description: Mesorhizobium loti MAFF303099 has a functional Type III secretion system (T3SS) that is involved in the determination of competitiveness for legume nodulation. Here we demonstrate that the transcriptional factor TtsI, which positively regulates T3SS genes expression, is involved in a negative regulation of M. loti swimming motility in soft-agar. Conditions that induce T3SS expression affect flagella production. The same conditions also affect promoter activity of M. loti visN gene, a homolog to the positive regulator of flagellar genes that has been described in other rhizobia. Defects in T3SS complex assembly at membranes limited the negative regulation of motility by the expression of TtsI.
    Keywords: Environmental Microbiology
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    Fire regime, not time-since-fire, affects soil fungal community diversity and composition in temperate grasslands (2016)
    Oxford University Press
    Details
    Publication Date: 2016-09-08
    Description: Frequent burning is commonly undertaken to maintain diversity in temperate grasslands of southern Australia. How burning affects below-ground fungal community diversity remains unknown. We show, using a fungal rDNA metabarcoding approach (Illumina MiSeq), that the fungal community composition was influenced by fire regime (frequency) but not time-since-fire. Fungal community composition was resilient to direct fire effects, most likely because grassland fires transfer little heat to the soil. Differences in the fungal community composition due to fire regime was likely due to associated changes that occur in vegetation with recurrent fire, via the break up of obligate symbiotic relationships. However, fire history only partially explains the observed dissimilarity in composition among the soil samples, suggesting a distinctiveness in composition in each grassland site. The importance of considering changes in soil microbe communities when managing vegetation with fire is highlighted.
    Keywords: Environmental Microbiology
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    Cultivation of an immobilized (hyper)thermophilic marine microbial community in a bioreactor (2016)
    Oxford University Press
    Details
    Publication Date: 2016-09-08
    Description: Cultivation in a bioreactor of immobilized deep-sea hydrothermal microbial community was tested in order to assess the stability and reactivity of this new system. A community composed of eight hydrothermal strains was entrapped in a polymer matrix that was used to inoculate a continuous culture in a gas-lift bioreactor. The continuous culture was performed for 41 days at successively 60°C, 55°C, 60°C, 85°C and 60°C, at pH 6.5, in anaerobic condition and constant dilution rate. Oxic stress and pH variations were tested at the beginning of the incubation. Despite these detrimental conditions, three strains including two strict anaerobes were maintained in the bioreactor. High cell concentrations (3 x 10 8 cells mL –1 ) and high ATP contents were measured in both liquid fractions and beads. Cloning-sequencing and qPCR revealed that Bacillus sp. dominated at the early stage, and was later replaced by Thermotoga maritima and Thermococcus sp. Acetate, formate and propionate concentrations varied simultaneously in the liquid fractions. These results demonstrate that these immobilized cells were reactive to culture conditions. They were protected inside the beads during the stress period and released in the liquid fraction when conditions were more favorable. This confirms the advantage of immobilization that highlights the resilience capacity of certain hydrothermal microorganisms after a stress period.
    Keywords: Biotechnology & Synthetic Biology
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  • 87
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    Degradation and emission of carbonyl sulfide, an atmospheric trace gas, by fungi isolated from forest soil (2016)
    Oxford University Press
    Details
    Publication Date: 2016-09-11
    Description: Soil is thought to be important both as a source and a sink of carbonyl sulfide (COS) in the troposphere, but the mechanism affecting COS uptake, especially for fungi, remains uncertain. Fungal isolates that were collected randomly from forest soil showed COS-degrading ability at high frequencies: 38 out of 43 isolates grown on potato dextrose agar showed degradation of 30 ppmv COS within 24 h. Of these isolates, eight degraded 30 ppmv of COS to below the detection limit within 2 h. These isolates also showed an ability to degrade COS included in ambient air (around 500 pptv) and highly concentrated (12 500 ppmv) level, even though the latter is higher than the lethal level for mammals. COS-degrading activity was estimated by using ergosterol as a biomass index for fungi. Trichoderma sp. THIF08 had the highest COS-degrading activity of all the isolates. Interestingly, Umbelopsis/Mortierella spp. THIF09 and THIF13 were unable to degrade 30 ppmv COS within 24 h, and actually emitted COS during the cultivation in ambient air. These results indicate a fungal contribution to the flux of COS between the terrestrial and atmospheric environments.
    Keywords: Environmental Microbiology
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    PCR-based detection of composite transposons and translocatable units from oral metagenomic DNA (2016)
    Oxford University Press
    Details
    Publication Date: 2016-09-17
    Description: A composite transposon is a mobile genetic element consisting of two insertion sequences (ISs) flanking a segment of cargo DNA often containing antibiotic resistance (AR) genes. Composite transposons can move as a discreet unit. There have been recently several reports on a novel mechanism of movement of an IS 26 -based composite transposon through the formation of a translocatable unit (TU), carrying the internal DNA segment of a composite transposon and one copy of a flanking IS. In this study, we determined the presence of composite transposons and TUs in human oral metagenomic DNA using PCR primers from common IS elements. Analysis of resulting amplicons showed four different IS 1216 composite transposons and one IS 257 composite transposon in our metagenomic sample. As our PCR strategy would also detect TUs, PCR was carried out to detect circular TUs predicted to originate from these composite transposons. We confirmed the presence of two novel TUs, one containing an experimentally proven antiseptic resistance gene and another containing a putative universal stress response protein (UspA) encoding gene. This is the first report of a PCR strategy to amplify the DNA segment on composite transposons and TUs in metagenomic DNA. This can be used to identify AR genes associated with a variety of mobile genetic elements from metagenomes.
    Keywords: Environmental Microbiology
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    A simple technique for suppressor detection in Escherichia coli (2016)
    Oxford University Press
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    Publication Date: 2016-10-12
    Description: To study the viability of a gyrA S83 stop mutation found in an Escherichia coli J53 ciprofloxacin-resistant strain (J53 CipR27), a pBR322 derivative was constructed with a TAG mutation in the bla gene knocking out ampicillin resistance. Ampicillin resistance was restored, suggesting that the strain contains tRNA suppressor activity able to suppress the UAG codon gyrA and allow viability. The method was applied to 22 unique clinical E. coli isolates, and all were found to have low-level suppressor activity.
    Keywords: Biotechnology & Synthetic Biology
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  • 90
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    Degradation of carbonyl sulfide by Actinomycetes and detection of clade D of {beta}-class carbonic anhydrase (2016)
    Oxford University Press
    Details
    Publication Date: 2016-10-12
    Description: Carbonyl sulfide (COS) is an atmospheric trace gas and one of the sources of stratospheric aerosol contributing to climate change. Although one of the major sinks of COS is soil, the distribution of COS degradation ability among bacteria remains unclear. Seventeen out of 20 named bacteria belonging to Actinomycetales had COS degradation activity at mole fractions of 30 parts per million by volume (ppmv) COS. Dietzia maris NBRC 15801 T and Mycobacterium sp. THI405 had the activity comparable to a chemolithoautotroph Thiobacillus thioparus THI115 that degrade COS by COS hydrolase for energy production. Among 12 bacteria manifesting rapid degradation at 30 ppmv COS, D. maris NBRC 15801 T and Streptomyces ambofaciens NBRC 12836 T degraded ambient COS (~500 parts per trillion by volume). Geodermatophilus obscurus NBRC 13315 T and Amycolatopsis orientalis NBRC 12806 T increased COS concentrations. Moreover, six of eight COS-degrading bacteria isolated from soils had partial nucleotide sequences similar to that of the gene encoding clade D of β-class carbonic anhydrase, which included COS hydrolase. These results indicate the potential importance of Actinomycetes in the role of soils as sinks of atmospheric COS.
    Keywords: Environmental Microbiology
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    Enterotoxigenic Escherichia coli heat-stable toxin and heat-labile toxin toxoid fusion 3xSTaN12S-dmLT induces neutralizing anti-STa antibodies in subcutaneously immunized mice (2016)
    Oxford University Press
    Details
    Publication Date: 2016-11-17
    Description: Enterotoxigenic Escherichia coli (ETEC) bacteria producing heat-stable toxin (STa) and/or heat-labile toxin (LT) are among top causes of children's diarrhea and travelers’ diarrhea. Currently no vaccines are available for ETEC associated diarrhea. A major challenge in developing ETEC vaccines is the inability to stimulate protective antibodies against the key STa toxin that is potently toxic and also poorly immunogenic. A recent study suggested toxoid fusion 3xSTa N12S -dmLT, which consists of a monomer LT toxoid (LT R192G/L211A ) and three copies of STa toxoid STa N12S , may represent an optimal immunogen inducing neutralizing antibodies against STa toxin [IAI 2014, 82(5):1823-32]. In this study, we immunized mice with this fusion protein following a different parenteral route and using different adjuvants to further characterize immunogenicity of this toxoid fusion. Data from this study showed that 3xSTa N12S -dmLT toxoid fusion induced neutralizing anti-STa antibodies in the mice following subcutaneous immunization, as effectively as in the mice under intraperitoneal route. Data also indicated that double mutant LT (dmLT) can be an effective adjuvant for this toxoid fusion in mice subcutaneous immunization. Results from this study affirmed that toxoid fusion 3xSTa N12S -dmLT induces neutralizing antibodies against STa toxin, suggesting this toxoid fusion is potentially a promising immunogen for ETEC vaccine development.
    Keywords: Biotechnology & Synthetic Biology
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    OmpF of Pectobacterium carotovorum subsp. carotovorum Pcc3 is required for carocin D sensitivity (2016)
    Oxford University Press
    Details
    Publication Date: 2016-12-23
    Description: Carocin D is a bacteriocin produced by Pectobacterium carotovorum subsp. carotovorum Pcc21. Carocin D inhibits the growth of P . carotovorum subsp. carotovorum and closely related strains. Pectobacterium carotovorum subsp. carotovorum is a causative bacterium for soft rot disease and leads to severe economic losses. Bacteriocins recognize and interact with a specific membrane protein of target bacteria as a receptor. To identify the receptor responsible for carocin D recognition, mutants that underwent a phenotypic change from carocin D sensitivity to carocin D insensitivity were screened. Based on Tn 5 insertions, carocin D sensitivity was dependent on expression of the outer membrane protein OmpF. The insensitivity of the mutant (Pcc3MR) to carocin D was complemented with ompF from carocin D-sensitive strains, not from carocin D-resistant strains. The selectivity between sensitive and resistant strains could be attributed to variation in OmpFs in the cell-surface-exposed regions. Based on sequence analysis and complementation assays, it appears that carocin D uses OmpF as a receptor and is translocated by the TonB system. According to previously reported translocation mechanisms of colicins, OmpF works along with the TolA system rather than the TonB system. Therefore, the current findings suggest that carocin D is imported by a unique colicin-like bacteriocin translocation system.
    Keywords: Biotechnology & Synthetic Biology
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    Bacterial biodegradation of neonicotinoid pesticides in soil and water systems (2016)
    Oxford University Press
    Details
    Publication Date: 2016-12-23
    Description: Neonicotinoids are neurotoxic systemic insecticides used in plant protection worldwide. Unfortunately, application of neonicotinoids affects both beneficial and target insects indiscriminately. Being water soluble and persistent, these pesticides are capable of disrupting both food chains and biogeochemical cycles. This review focuses on the biodegradation of neonicotinoids in soil and water systems by the bacterial community. Several bacterial strains have been isolated and identified as capable of transforming neonicotinoids in the presence of an additional carbon source. Environmental parameters have been established for accelerated transformation in some of these strains. Studies have also indicated that enhanced biotransformation of these pesticides can be accomplished by mixed microbial populations under optimised environmental conditions. Substantial research into the identification of neonicotinoid-mineralising bacterial strains and identification of the genes and enzymes responsible for neonicotinoid degradation is still required to complete the understanding of microbial biodegradation pathways, and advance bioremediation efforts.
    Keywords: Environmental Microbiology
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    New and highly active microbial phosphotriesterase sources (2016)
    Oxford University Press
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    Publication Date: 2016-12-29
    Description: Many toxic insecticides used worldwide as well as some chemical warfare agents are phosphotriester derivatives. Therefore, detoxification of organophosphorus compounds has become the subject of many studies and in particular bioremediation, based on the phosphotriesterase catalysed hydrolysis of these compounds, has shown to be an effective and ecological methodology. In order to identify new bacterial phosphotriesterases, a simple and sensitive fluorimetric screening method on solid media was employed that allowed the selection of six strains with phosphotriesterase activity. Since pH and temperature are important parameters for bioremediation of contaminated soils and waters, the influence of these variables on the rate of the enzymatic hydrolysis was assessed. This study afforded notable results, being the most remarkable one the increased activity exhibited by Nocardia asteroides and Streptomyces setonii strains at 50°C, 7 and 30 times higher than at 30°C, respectively. Compared with the results obtained with Brevundimonas diminuta , whose activity is usually considered as reference, an increase of 26 and 75 times is observed, respectively.
    Keywords: Environmental Microbiology
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    High-affinity phosphate-binding protein (PBP) for phosphorous recovery: proof of concept using recombinant Escherichia coli (2016)
    Oxford University Press
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    Publication Date: 2016-10-30
    Description: Phosphorus (P) is a critical, non-renewable nutrient; yet excess discharges can lead to eutrophication and deterioration of water quality. Thus, P removal from water must be coupled with P recovery to achieve sustainable P management. P-specific proteins provide a novel, promising approach to recover P from water. Bacterial phosphate-binding proteins (PBPs) are able to effectively remove phosphate, achieving extremely low levels in water (i.e. 0.015 mg-P L –1 ). A prerequisite of using PBP for P recovery, however, is not only removal, but also controlled P release, which has not yet been reported. Phosphate release using recombinant PBP-expressing Escherichia coli was explored in this study. Escherichia coli was genetically modified to overexpress PBP in the periplasmic space. The impacts of ionic strength, temperature and pH on phosphate release were assessed. PBP-expressed E. coli demonstrated consistently superior ability to adsorb more phosphate from liquid and release more phosphate under controlled conditions relative to negative controls (unexpressed PBP E. coli and E. coli K12). Lower pH (3.8), higher temperature (35ºC) and higher ionic strength (100 mM KCl) facilitated increased phosphate release, providing a maximum of 2.1% P recovery within 3 h. This study provides proof of concept of the feasibility of using PBP to recover P.
    Keywords: Environmental Microbiology
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    Cosmetics-triggered percutaneous remote control of transgene expression in mice (2015)
    Oxford University Press
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    Publication Date: 2015-08-18
    Description: Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator O PmeR , that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Tight regulation of plant immune responses by combining promoter and suicide exon elements (2015)
    Oxford University Press
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    Publication Date: 2015-08-18
    Description: Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR151-dependent HR in Nicotiana benthamiana and Nicotiana tabacum , respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.
    Keywords: Synthetic Biology and Assembly Cloning
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    Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae (2015)
    Oxford University Press
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    Publication Date: 2015-07-25
    Description: We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae . Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by ‘VEGAS adapter’ (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae , each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.
    Keywords: Synthetic Biology and Assembly Cloning
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    YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae (2015)
    Oxford University Press
    Details
    Publication Date: 2015-07-25
    Description: It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae . We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts.
    Keywords: Synthetic Biology and Assembly Cloning
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    Microfluidic droplet enrichment for targeted sequencing (2015)
    Oxford University Press
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    Publication Date: 2015-07-25
    Description: Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications.
    Keywords: Synthetic Biology and Assembly Cloning
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