Subscribe: pubmed: 0099-2240
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pubmed: 0099-2240



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Functional characterization and unique diversity of genes and microorganisms involved in arsenite oxidation from the tailings of a realgar mine.

Functional characterization and unique diversity of genes and microorganisms involved in arsenite oxidation from the tailings of a realgar mine.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Zeng XC, E G, Wang J, Wang N, Chen X, Mu Y, Li H, Yang Y, Liu Y, Wang Y

Abstract
The tailings of Shimen realgar mine have unique geochemical features. Arsenite oxidation is one of the major biogeochemical processes occurred in the tailings. However, little is known about the functional and molecular aspects of the microbial community involved in arsenite oxidation. Here, we fully explored the functional and molecular features of the microbial communities from the tailings of Shimen realgar mine. We collected six samples from the sites A, B, C, D, E and F of the tailings, respectively. Microcosm assays indicated that all the six sites contain both chemoautotrophic and heterotrophic arsenite-oxidizing microorganisms; their activities differed considerably from each other. The microbial arsenite-oxidizing activities show a positive correlation with the soluble arsenic concentrations. The microbial communities of the six sites contain 40 phyla of bacteria and 2 phyla of archaea that show extremely high diversity. Soluble arsenic, sulfate, pH and TOC are the key environmental factors in shaping the microbial communities. We further identified 114 unique arsenite oxidase genes from the samples; all of them code for new or new-type arsenite oxidases. We also isolated 10 novel arsenite-oxidizers from the samples, of which 4 are chemoautotrophic and 6 are heterotrophic. These data highlight the unique diversities of the arsenite-oxidizing microorganisms and their oxidase genes from the tailings of Shimen realgar mine. To the best of our knowledge, this is the first report describing the functional and molecular features of the microbial communities from the tailings of a realgar mine.
IMPORTANCE: This study was focused on the functional and molecular characterization of the microbial communities from the tailings of Shimen realgar mine. We fully explored for the first time the arsenite-oxidizing activities and the functional gene diversities of the microorganisms from the tailings, as well as the correlation of the microbial activities/diversities with environmental factors. The findings of this study help us better understanding the diversities of the arsenite-oxidizing bacteria and the geochemical cycle of arsenic in the tailings of Shimen realgar mine, and gain insights into the microbial mechanisms by which the secondary minerals of the tailings were formed. This work also offers a set of unique arsenite-oxidizing bacteria for the basic research of the molecular regulation of arsenite oxidation in bacterial cells, and for the environment-friendly bioremediation of arsenic-contaminated groundwater.

PMID: 27663031 [PubMed - as supplied by publisher]




A Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus B-6 Exhibiting Endo-Xylanase, β-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

A Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus B-6 Exhibiting Endo-Xylanase, β-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Teeravivattanakit T, Baramee S, Phitsuwan P, Waeonukul R, Pason P, Tachaapaikoon C, Sakka K, Ratanakhanokchai K

Abstract
The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli Recombinant PcAxy43A consisting of a family 43 glycoside hydrolase and a family 6 carbohydrate-binding module exhibited endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited β-xylosidase activity toward a chromogenic substrate, p-nitrophenyl-β-d-xylopyranoside, and xylobiose while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, α-L-Araf-(1→2)-D-Xylp or α-L-Araf-(1→3)-D-Xylp and α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-β-D-Xylp Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for saccharification of arabinoxylan into simple sugars for many applications.
IMPORTANCE: In this study, the GH43 intracellular multifunctional endo-xylanase, β-xylosidase and AXH from P. curdlanolyticus B-6 was characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)2 and C(O)3 positions of di-arabinosyl-substituted xylose residues, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxh43A and endo-xylanase Xyn10C from strain B-6 providing novel metabolic potential for processing arabinoxylans to xylose and arabinose.

PMID: 27663030 [PubMed - as supplied by publisher]




Production and Characterization of Neutralizing Antibodies against Bungarus multicinctus Snake Venom.

Production and Characterization of Neutralizing Antibodies against Bungarus multicinctus Snake Venom.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Lee CH, Lee YC, Leu SJ, Lin LT, Chiang JR, Hsu WJ, Yang YY

Abstract
Banded krait (Bungarus multicinctus; BM), one of the major envenomation species in Taiwan, contains neurotoxic venom proteins (BM proteins), that poses a serious medical problem in tropical and sub-tropical countries. Even though horse-derived serum is an efficient therapy against snake venom, it is associated with high cost and side effects. Therefore, developing a more cost-effective alternative treatment option is highly envisaged. In this study, chickens were immunized with BM proteins, and polyclonal immunoglobulin Y (IgY) antibodies were purified from eggs. IgY showed a similar binding activity to BM proteins as horse antivenin, and its titer in chickens lasted for at least 6 months. We constructed two antibody libraries by phage display antibody technology containing 1.0 ×10(7) and 2.9 × 10(8) transformants, respectively. After bio-panning, phage-based ELISA indicated specific clones were enriched. Thirty randomly selected clones expressing monoclonal single-chain variable-fragment (scFv) antibodies were classified into four groups with a short linker and two with a long linker. These selected scFv antibodies showed specific binding activities to BM proteins but not to venomous proteins of other snakes. Most importantly, polyclonal IgY demonstrated a similar neutralization efficiency as did horse-derived antivenin in mice injected with a minimum lethal dosage (MLD) of venom proteins. A mixture of several monoclonal anti-BM scFv antibodies was also able to partially inhibit the lethal effect on mice. We profoundly believe that the IgY and scFv antibodies can be applied in developing diagnostic agents for wound exudates and as an alternative treatment for snakebite envenomations in the future.
IMPORTANCE: Snake envenomation is one of global medical issues in concern. Horse-derived antivenin is an effective way to treat snakebites but it is costly and occasionally causes severe side effects. In this study, we first generated and characterized IgY antibodies with neutralization activity in chickens. Subsequently, we generated a panel of monoclonal scFv antibodies using phage display antibody technology. Mixture of scFv antibodies was able to partially inhibit the lethal effect in mice injected with lethal dosage of venom proteins and prolong their survival time. We believe the chicken-derived IgY and scFv antibodies have great potential to develop diagnostic agents for wound exudates and therapeutic agents against snake envenomation in the future.

PMID: 27663029 [PubMed - as supplied by publisher]




Two outer membrane proteins contribute to cellular fitness in Caulobacter crescentus by preventing intracellular S-layer protein accumulation.

Two outer membrane proteins contribute to cellular fitness in Caulobacter crescentus by preventing intracellular S-layer protein accumulation.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Overton KW, Park DM, Yung MC, Dohnalkova AC, Smit J, Jiao Y

Abstract
Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport S-layer protein from the cytoplasm to the cell exterior. Caulobacter crescentus is unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFa and RsaFb, that, together with other components, form a type I protein translocation pathway for S-layer export. These proteins have homology to E. coli TolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFa and RsaFb led to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and RNA-seq, we show that loss of both RsaFa and RsaFb led to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein mis-folding and degradation pathways. These findings provide new insight into the requirement for RsaFa and RsaFb in cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in C. crescentus IMPORTANCE: Decreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell largely due to lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural systems. As one example of a system evolved to prevent intracellular protein accumulation, our study demonstrates that Caulobacter crescentus has two homologous outer membrane transporter proteins that are involved in S-layer export. This is an interesting case study that demonstrates how bacteria can evolve redundancy to ensure adequate protein export functionality and maintain high cellular fitness. Moreover, we provide evidence that these two outer membrane proteins, although being the closest C. crescentus homologs to TolC in E. coli, do not process TolC functionality in C. crescentus.

PMID: 27663028 [PubMed - as supplied by publisher]




Adaptation of Akkermansia muciniphila to the oxic-anoxic interface of the mucus layer.

Adaptation of Akkermansia muciniphila to the oxic-anoxic interface of the mucus layer.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Ouwerkerk JP, van der Ark KC, Davids M, Claassens NJ, Robert Finestra T, de Vos WM, Belzer C

Abstract
Akkermansia muciniphila colonizes the mucus layer of the gastrointestinal tract where the organism can be exposed to the oxygen that diffuses from epithelial cells. To understand how A. muciniphila is able to survive and grow at this oxic-anoxic interface, its oxygen tolerance, response and reduction capacities were studied. A. muciniphila was found to be oxygen-tolerant. On top of this, under aerated conditions, A. muciniphila showed significant oxygen reduction capacities and its growth rate and yield were increased as compared to strict anaerobic conditions. Transcriptome analysis revealed an initial oxygen stress response upon exposure to oxygen. Hereafter, genes related to respiration were expressed, including those coding for the cytochrome bd complex, which can function as terminal oxidase. The functionality of A. muciniphila cytochrome bd genes was proven by successfully complementing the cytochrome-deficient Escherichia coli strain ECOM4. We conclude that A. muciniphila can use oxygen when present at nanomolar concentrations.
IMPORTANCE: The manuscript explains how Akkermansia muciniphila previously described, as a strictly anaerobic bacterium is able to tolerate and even benefit from low levels of oxygen. Interestingly, we measured growth enhancement of A. muciniphila and changes in metabolism as a result of the oxygen exposure. In this manuscript we discuss the similarities and differences of this oxygen responsive mechanism compared to other intestinal anaerobic isolates. Taken together we think these are valuable data that indicate how anaerobic intestinal colonizing bacterium can exploit low levels of oxygen present in the mucus layer. Prompting also a direct relevance for applicability of our results, as addition of low oxygen concentrations could benefit the in vitro growth of certain anaerobic organisms.

PMID: 27663027 [PubMed - as supplied by publisher]




Response of germ-free mice to colonization with O. formigenes and altered Schaedler flora.

Response of germ-free mice to colonization with O. formigenes and altered Schaedler flora.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Li X, Ellis ML, Dowell AE, Kumar R, Morrow CD, Schoeb TR, Knight J

Abstract
Colonization with Oxalobacter formigenes may reduce the risk of calcium oxalate kidney stone disease. To improve our limited understanding of host/O.formigenes and microbe/O.formigenes interactions, germ-free or altered Schaedler flora (ASF) mice were colonized with O.formigenes Germ-free mice were stably colonized with O.formigenes suggesting O.formigenes does not require other organisms to sustain its survival. Examination of intestinal material indicated no viable O.formigenes in the small intestine, ∼4 × 10(6) O.formigenes per 100mg contents in the cecum and proximal colon, and ∼0.02% of total cecal O. formigenes cells were tightly associated to the mucosa. O.formigenes did not alter the overall microbial composition of ASF, and ASF did not impact O.formigenes capacity to degrade dietary oxalate in the cecum. 24-hour urine and fecal collections within metabolic cages in semi-rigid isolators demonstrated that introduction of ASF into germ-free mice significantly reduced urinary oxalate excretion. These experiments also showed that mono-colonized O.formigenes mice excrete significantly more urinary calcium compared to germ-free mice, which may be due to degradation of calcium oxalate crystals by O.formigenes and the subsequent intestinal absorption of free calcium. In conclusion, the successful establishment of defined-flora O.formigenes mouse models should improve our understanding of O.formigenes host and microbe interactions. These data support the use of O.formigenes as a probiotic that has limited impact on the composition of the resident microbiota but providing efficient oxalate degrading function.
IMPORTANCE: Despite evidence suggesting a lack of O. formigenes colonization is a risk factor for calcium oxalate stone formation, little is known about O. formigenes biology. This study is the first to utilize germ-free mice to examine the response to mono-colonization with O. formigenes and the impact of a defined bacterial cocktail, altered Schaedler flora, on O. formigenes colonization. This study demonstrates that germ-free mice on their regular diet remain mono-colonized with O. formigenes, and suggests that further studies with O. formigenes gnotobiotic mouse models could improve our understanding of O. formigenes biology and host/O. formigenes and microbe/O. formigenes interactions.

PMID: 27663026 [PubMed - as supplied by publisher]




Crz1p regulates pH homeostasis in Candida glabrata by altering membrane lipid composition.

Crz1p regulates pH homeostasis in Candida glabrata by altering membrane lipid composition.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Yan D, Lin X, Qi Y, Liu H, Chen X, Liu L, Chen J

Abstract
The asexual facultative aerobic haploid yeast Candida glabrata is widely used in the industrial production of various organic acids. To elucidate the physiological function of the transcription factor CgCrz1p and its role in tolerance to acid stress we deleted or overexpressed the corresponding gene CgCRZ1 Deletion of CgCRZ1 resulted in a 60% decrease in dry cell weight (DCW) and a 50% drop in cell viability compared to the wild type at pH 2.0. Expression of lipid metabolism-associated genes was also significantly down-regulated. Consequently, the proportion of C18:1 fatty acids, ratio of unsaturated to saturated fatty acids, and ergosterol content decreased by 30%, 46%, and 30%, respectively. Additionally, membrane integrity, fluidity, and H(+)-ATPase activity were reduced by 45%, 9%, and 50%, respectively. In contrast, overexpression of CgCrz1p increased C18:1 and ergosterol content by 16% and 40%, respectively. Overexpression also enhanced membrane integrity, fluidity, and H(+)-ATPase activity by 31%, 6%, and 20%, respectively. Moreover, in the absence of pH buffering, DCW and pyruvate titer increased by 48% and 60%, respectively, compared to the wild type. Together, these results suggest that CgCrz1p regulates tolerance to acidic conditions by altering membrane lipid composition in C. glabrata IMPORTANCE: The present study provides an insight into the metabolism of Candida glabrata under acidic conditions, such as those encountered during industrial production of organic acids. We found that overexpression of the transcription factor CgCrz1p improved viability, biomass, and pyruvate yields at low pH. Analysis of plasma membrane lipid composition indicated that CgCrz1p might play an important role in its integrity and fluidity, and enhanced the pumping of protons in acidic environments. We propose that altering the structure of the cell membrane may provide a successful strategy for increasing C glabrata productivity at low pH.

PMID: 27663025 [PubMed - as supplied by publisher]




A clonal lineage of Fusarium oxysporum circulates in tap water of different French hospitals.

A clonal lineage of Fusarium oxysporum circulates in tap water of different French hospitals.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Edel-Hermann V, Sautour M, Gautheron N, Laurent J, Aho S, Bonnin A, Sixt N, Hartemann P, Dalle F, Steinberg C

Abstract
Fusarium oxysporum is typically a soilborne fungus but can also be found in aquatic environments. In hospitals, the water distribution system may be a reservoir for fungi responsible for nosocomial infections. F. oxysporum was previously detected in the water distribution system of five French hospitals. Sixty-eight isolates from water representative of all hospital units previously sampled and characterized by translation elongation factor 1α sequence typing were subjected to microsatellite analysis and full-length ribosomal intergenic spacer (IGS) sequence typing. All but three isolates shared common microsatellite loci and a common two-locus sequence type (ST). This ST has an international geographical distribution in both water networks of hospitals and among clinical isolates. The ST dominant in water was not detected among 300 isolates of F. oxysporum originating from surrounding soils. Further characterization of fifteen isolates by vegetative compatibility testing allowed us to conclude that a clonal lineage of F. oxysporum circulates in tap water of the different hospitals.
IMPORTANCE: We demonstrated that a clonal lineage of Fusarium oxysporum inhabits the water distribution systems of several French hospitals. This clonal lineage, which appears to be particularly adapted to water networks, represents a potential risk for human infection and raises questions about its worldwide distribution.

PMID: 27663024 [PubMed - as supplied by publisher]




Innovative Use of Palladium Compounds to Selectively Detect Live Enterobacteriaceae Cells in Milk by Polymerase Chain Reaction.

Innovative Use of Palladium Compounds to Selectively Detect Live Enterobacteriaceae Cells in Milk by Polymerase Chain Reaction.

Appl Environ Microbiol. 2016 Sep 23;

Authors: Soejima T, Iwatsuki KJ

Abstract
Ethidium- and propidium-monoazide (EMA and PMA) have been used in combination with PCR for more than a decade to facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving lower limits of detection and quantification of targeted live cells, fewer laborious procedures, lower costs, and potentially higher throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry 3 times higher costs because of the requirement for much larger amounts for LD discrimination than palladium compounds. Palladium compounds can penetrate dead (compromised) but not live bacteria and can be chelated primarily by chromosomal DNA and cell wall transmembrane proteins, with small amounts of DNA-binding proteins in vivo The new mechanism for palladium compounds is obviously different from that of platinum compounds which primarily target DNA. Combining palladium compounds with PCR in water resulted in much clearer discrimination between live and dead Enterobacteriaceae bacteria compared with the PMA method. Palladium-PCR correlated with reference plating or with the currently used PMA-PCR method for pasteurized milk, based on EN ISO 16140:2003 validation. Palladium-PCR enabled us to specifically detect and assay viable Enterobacteriaceae cells at concentrations of 5 to 10 CFU/ml in milk following USA/EU regulations after a 4.5-h process in a typical laboratory exposed to natural/electric light, as specified by USA/EU regulations.
IMPORTANCE: Ethidium- and propidium-monoazide (EMA and PMA) facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving fewer laborious procedures, lower costs, and potentially higher throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry 3 times higher costs because of the requirement for much larger amounts for LD discrimination than palladium compounds which have also a novel reaction mechanism different from platinum compounds. In view of testing cost, palladium compound here is also very useful compared with platinum compounds. Ultimately, the innovative Pd-PCR may be also substituted for the currently used reference plating methods.

PMID: 27663023 [PubMed - as supplied by publisher]