Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33

<p>Abstract</p> <p>Background</p> <p>Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency.</p> <p>Results</p> <p>In this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus <it>Aspergillus fumigatus </it>Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, <it>bgl3</it>, was cloned based on the peptide sequences obtained from the LC-MS/MS results. <it>bgl</it>3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of <it>bgl</it>3 in <it>Pichia pastoris </it>X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg^-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan.</p> <p>Conclusions</p> <p>An native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of <it>A. fumigatus </it>Z5. The gene <it>bgl</it>3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene <it>bgl3 </it>was expressed in <it>Pichia pastoris </it>X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.</p

Comparative transcriptomics in Yersinia pestis: a global view of environmental modulation of gene expression

<p>Abstract</p> <p>Background</p> <p>Environmental modulation of gene expression in <it>Yersinia pestis </it>is critical for its life style and pathogenesis. Using cDNA microarray technology, we have analyzed the global gene expression of this deadly pathogen when grown under different stress conditions <it>in vitro</it>.</p> <p>Results</p> <p>To provide us with a comprehensive view of environmental modulation of global gene expression in <it>Y. pestis</it>, we have analyzed the gene expression profiles of 25 different stress conditions. Almost all known virulence genes of <it>Y. pestis </it>were differentially regulated under multiple environmental perturbations. Clustering enabled us to functionally classify co-expressed genes, including some uncharacterized genes. Collections of operons were predicted from the microarray data, and some of these were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Several regulatory DNA motifs, probably recognized by the regulatory protein Fur, PurR, or Fnr, were predicted from the clustered genes, and a Fur binding site in the corresponding promoter regions was verified by electrophoretic mobility shift assay (EMSA).</p> <p>Conclusion</p> <p>The comparative transcriptomics analysis we present here not only benefits our understanding of the molecular determinants of pathogenesis and cellular regulatory circuits in <it>Y. pestis</it>, it also serves as a basis for integrating increasing volumes of microarray data using existing methods.</p

Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The zinc uptake regulator Zur is a Zn²⁺-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in <it>Y. pestis</it>.</p> <p>Results</p> <p>We constructed a <it>zur </it>null mutant of <it>Y. pestis </it>biovar <it>microtus </it>strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of <it>Y. pestis </it>upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons <it>znuA, znuCB </it>and <it>ykgM</it>-<it>RpmJ2</it>. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes.</p> <p>Conclusion</p> <p>Zur as a repressor directly controls the transcription of <it>znuA, znuCB </it>and <it>ykgM</it>-<it>RpmJ2 </it>in <it>Y. pestis </it>by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in <it>Y. pestis </it>likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.</p

Identification and characterization of PhoP regulon members in Yersinia pestis biovar Microtus

<p>Abstract</p> <p>Background</p> <p>The transcription regulator PhoP has been shown to be important for <it>Y. pestis </it>survival in macrophages and under various <it>in vitro </it>stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in <it>Y. pestis</it>. A series of biochemical experiments were done herein to study members of the PhoP regulon of <it>Y. pestis </it>biovar <it>Microtus</it>.</p> <p>Results</p> <p>By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in <it>Y. pestis</it>. Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation.</p> <p>Conclusion</p> <p>The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which <it>Y. pestis </it>survives the antibacterial strategies employed by host macrophages.</p

Bacterial Microbiota in Unfed Ticks ( Dermacentor nuttalli ) From Xinjiang Detected Through 16S rDNA Amplicon Sequencing and Culturomics

Ticks are a major arthropod vector of zoonotic diseases affecting both humans and domestic animals worldwide. Thus, studying tick microbiota would aid in understanding of the potential threats posed by ticks. Approximately 8,000 unfed ticks, identified as Dermacentor nuttalli , were collected from the sylvosteppe in the western Tianshan mountains. To investigate their potential pathogens, we divided the ticks into 36 groups of 200–300 individuals each for examination with culturomics and 16S rDNA amplicon sequencing. A total of 237 bacterial genera were identified with the two methods. Culturomics identified 46 bacterial species from 23 genera, predominantly Pseudomonas , Pantoea , and Bacillus , whereas 16S rDNA sequencing identified 461 OTUs from 233 genera, predominantly Pseudomonas (53.8%), Coxiella (17.2%), and Pantoea (6.4%). Coxiella , Rickettsia , and ten other genera were discovered only by sequencing, because optimal cultivating conditions were not used for their isolation, whereas Arthrobacter and three other genera were discovered only through culturomics. Several of the identified bacteria, such as line-related sepsis-causing Delftia acidovorans and the pneumonia agent Acinetobacter pittii , can cause human diseases. Thus, both sequencing and culturomics methods are crucial for comprehensive understanding of the microbiota of D. nuttalli

Genotyping and Phylogenetic Analysis of Yersinia pestis by MLVA: Insights into the Worldwide Expansion of Central Asia Plague Foci

BACKGROUND: The species Yersinia pestis is commonly divided into three classical biovars, Antiqua, Medievalis, and Orientalis, belonging to subspecies pestis pathogenic for human and the (atypical) non-human pathogenic biovar Microtus (alias Pestoides) including several non-pestis subspecies. Recent progress in molecular typing methods enables large-scale investigations in the population structure of this species. It is now possible to test hypotheses about its evolution which were proposed decades ago. For instance the three classical biovars of different geographical distributions were suggested to originate from Central Asia. Most investigations so far have focused on the typical pestis subspecies representatives found outside of China, whereas the understanding of the emergence of this human pathogen requires the investigation of strains belonging to subspecies pestis from China and to the Microtus biovar. METHODOLOGY/PRINCIPAL FINDINGS: Multi-locus VNTR analysis (MLVA) with 25 loci was performed on a collection of Y. pestis isolates originating from the majority of the known foci worldwide and including typical rhamnose-negative subspecies pestis as well as rhamnose-positive subspecies pestis and biovar Microtus. More than 500 isolates from China, the Former Soviet Union (FSU), Mongolia and a number of other foci around the world were characterized and resolved into 350 different genotypes. The data revealed very close relationships existing between some isolates from widely separated foci as well as very high diversity which can conversely be observed between nearby foci. CONCLUSIONS/SIGNIFICANCE: The results obtained are in full agreement with the view that the Y. pestis subsp. pestis pathogenic for humans emerged in the Central Asia region between China, Kazakhstan, Russia and Mongolia, only three clones of which spread out of Central Asia. The relationships among the strains in China, Central Asia and the rest of the world based on the MLVA25 assay provide an unprecedented view on the expansion and microevolution of Y. pestis

Epidemic clones, oceanic gene pools and eco-LD in the free living marine pathogen Vibrio parahaemolyticus

We investigated global patterns of variation in 157 whole genome sequences of Vibrio parahaemolyticus, a free-living and seafood associated marine bacterium. Pandemic clones, responsible for recent outbreaks of gastroenteritis in humans have spread globally. However, there are oceanic gene pools, one located in the oceans surrounding Asia and another in the Mexican Gulf. Frequent recombination means that most isolates have acquired the genetic profile of their current location. We investigated the genetic structure in the Asian gene pool by calculating the effective population size in two different ways. Under standard neutral models, the two estimates should give similar answers but we found a thirty fold difference. We propose that this discrepancy is caused by the subdivision of the species into a hundred or more ecotypes which are maintained stably in the population. To investigate the genetic factors involved, we used 51 unrelated isolates to conduct a genome-wide scan for epistatically interacting loci. We found a single example of strong epistasis between distant genome regions. A majority of strains had a type VI secretion system associated with bacterial killing. The remaining strains had genes associated with biofilm formation and regulated by c-di-GMP signaling. All strains had one or other of the two systems and none of isolate had complete complements of both systems, although several strains had remnants. Further top-down analysis of patterns of linkage disequilibrium within frequently recombining species will allow a detailed understanding of how selection acts to structure the pattern of variation within natural bacterial populations

Open-source genomic analysis of Shiga-toxin–producing E. coli O104:H4

An outbreak caused by Shiga-toxin–producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance

Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism

Recent mixing of<i> Vibrio parahaemolyticus</i> populations

Humans have profoundly affected the ocean environment but little is known about anthropogenic effects on the distribution of microbes. Vibrio parahaemolyticus is found in warm coastal waters and causes gastroenteritis in humans and economically significant disease in shrimps. Based on data from 1103 genomes of environmental and clinical isolates, we show that V. parahaemolyticus is divided into four diverse populations, VppUS1, VppUS2, VppX and VppAsia. The first two are largely restricted to the US and Northern Europe, while the others are found worldwide, with VppAsia making up the great majority of isolates in the seas around Asia. Patterns of diversity within and between the populations are consistent with them having arisen by progressive divergence via genetic drift during geographical isolation. However, we find that there is substantial overlap in their current distribution. These observations can be reconciled without requiring genetic barriers to exchange between populations if long-range dispersal has increased dramatically in the recent past. We found that VppAsia isolates from the US have an average of 1.01% more shared ancestry with VppUS1 and VppUS2 isolates than VppAsia isolates from Asia itself. Based on time calibrated trees of divergence within epidemic lineages, we estimate that recombination affects about 0.017% of the genome per year, implying that the genetic mixture has taken place within the last few decades. These results suggest that human activity, such as shipping, aquatic products trade and increased human migration between continents, are responsible for the change of distribution pattern of this species