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The recent incorporation of bacterial whole-genome sequencing (WGS) into Public Health laboratories has enhanced foodborne outbreak detection and source attribution. As a result, large volumes of publicly available datasets can be used to study the biology of foodborne pathogen populations at an unprecedented scale. To demonstrate the application of a heuristic and agnostic hierarchical population structure guided pan-genome enrichment analysis (PANGEA), we used populations of S. enterica lineage I to achieve two main objectives: (i) show how hierarchical population inquiry at different scales of resolution can enhance ecological and epidemiological inquiries; and (ii) identify population-specific inferable traits that could provide selective advantages in food production environments. Publicly available WGS data were obtained from NCBI database for three serovars of Salmonella enterica subsp. enterica lineage I (S. Typhimurium, S. Newport, and S. Infantis). Using the hierarchical genotypic classifications (Serovar, BAPS1, ST, cgMLST), datasets from each of the three serovars showed varying degrees of clonal structuring. When the accessory genome (PANGEA) was mapped onto these hierarchical structures, accessory loci could be linked with specific genotypes. A large heavy-metal resistance mobile element was found in the Monophasic ST34 lineage of S. Typhimurium, and laboratory testing showed that Monophasic isolates have on average a higher degree of copper resistance than the Biphasic ones. In S. Newport, an extra sugE gene copy was found among most isolates of the ST45 lineage, and laboratory testing of multiple isolates confirmed that isolates of S. Newport ST45 were on average less sensitive to the disinfectant cetylpyridimium chloride than non-ST45 isolates. Lastly, data-mining of the accessory genomic content of S. Infantis revealed two cryptic Ecotypes with distinct accessory genomic content and distinct ecological patterns. Poultry appears to be the major reservoir for Ecotype 1, and temporal analysis further suggested a recent ecological succession, with Ecotype 2 apparently being displaced by Ecotype 1. Altogether, the use of a heuristic hierarchical-based population structure analysis that includes bacterial pan-genomes (core and accessory genomes) can (1) improve genomic resolution for mapping populations and accessing epidemiological patterns; and (2) define lineage-specific informative loci that may be associated with survival in the food chain.

The common marmoset (Callithrix jacchus) is an omnivorous New World primate whose diet in the wild includes large amounts of fruit, seeds, flowers, and a variety of lizards and invertebrates. Marmosets also feed heavily on tree gums and exudates, and they have evolved unique morphological and anatomical characteristics to facilitate gum feeding (gummivory). In this study, we characterized the fecal microbiomes of adult and infant animals from a captive population of common marmosets at the Callitrichid Research Center at the University of Nebraska at Omaha under their normal dietary and environmental conditions. The microbiomes of adult animals were dominated by species of Bifidobacterium, Bacteroides, Prevotella, Phascolarctobacterium, Megamonas, and Megasphaera. Culturing and genomic analysis of the Bifidobacterium populations from adult animals identified four known marmoset-associated species (B. reuteri, B. aesculapii, B. myosotis, and B. hapali) and three unclassified taxa of Bifidobacterium that are phylogenetically distinct. Species-specific quantitative PCR (qPCR) confirmed that these same species of Bifidobacterium are abundant members of the microbiome throughout the lives of the animals. Genomic loci in each Bifidobacterium species encode enzymes to support growth and major marmoset milk oligosaccharides during breastfeeding; however, metabolic islands that can support growth on complex polysaccharide substrates in the diets of captive adults (pectin, xyloglucan, and xylan), including loci in B. aesculapii that can support its unique ability to grow on arabinogalactan-rich tree gums, were species-specific. IMPORTANCE Bifidobacterium species are recognized as important, beneficial microbes in the human gut microbiome, and their ability colonize individuals at different stages of life is influenced by host, dietary, environmental, and ecological factors, which is poorly understood. The common marmoset is an emerging nonhuman primate model with a short maturation period, making this model amenable to study the microbiome throughout a life history. Features of the microbiome in captive marmosets are also shared with human gut microbiomes, including abundant populations of Bifidobacterium species. Our studies show that several species of Bifidobacterium are dominant members of the captive marmoset microbiome throughout their life history. Metabolic capacities in genomes of the marmoset Bifidobacterium species suggest species-specific adaptations to different components of the captive marmoset diet, including the unique capacity in B. aesculapii for degradation of gum arabic, suggesting that regular dietary exposure in captivity may be important for preserving gum-degrading species in the microbiome.

This study addressed a knowledge gap about longitudinal changes in the gut microbiome metabolites during animal pairing. This research in the laboratory common marmoset can control for the confounding factors such as diet and other environmental conditions. Phascolarctobacterium showed the highest contribution to the sex-biased transmission of the female to the male after pairing. Here, we observed the sex difference in the increase in short-chain fatty acid concentration in the feces of newly paired marmosets, which may be caused by the sex-biased gut microbiome transmission after pairing., The role by which the gut microbiome influences host health (e.g., energy equilibrium and immune system) may be partly mediated by short-chain fatty acids, which are bacterial fermentation products from the dietary fibers. However, little is known about longitudinal changes in gut microbiome metabolites during cohabitation alongside social contact. In common marmosets (Callithrix jacchus), the gut microbiome community is influenced by social contact, as newly paired males and females develop convergent microbial profiles. Here, we monitored the dynamics of short-chain fatty acid concentrations in common marmoset feces from the prepairing (PRE) to postpairing (POST) stages. In males, we observed that the concentrations of acetate, propionate, isobutyrate, and isovalerate significantly increased in the POST stage compared to the PRE stage. However, no significant changes were found in females. We further found that the propionate concentration was significantly positively correlated with the abundance of Phascolarctobacterium in the male feces. Thus, the sex difference in the changes in the concentrations of short-chain fatty acids might be related to sex-biased gut microbiome transmission after pairing. We suggest that the significant changes in the gut microbiomes and some short-chain fatty acids of the common marmoset during cohabitation may contribute to physiological homeostasis during pairing. IMPORTANCE This study addressed a knowledge gap about longitudinal changes in the gut microbiome metabolites during animal pairing. This research in the laboratory common marmoset can control for the confounding factors such as diet and other environmental conditions. Phascolarctobacterium showed the highest contribution to the sex-biased transmission of the female to the male after pairing. Here, we observed the sex difference in the increase in short-chain fatty acid concentration in the feces of newly paired marmosets, which may be caused by the sex-biased gut microbiome transmission after pairing.

In this controlled study, we collected longitudinal fecal samples from 16 male and female marmoset monkeys for 2 weeks prior to and for 8 weeks after pairing in male-female dyads. We report for the first time that marmoset monkeys undergo significant changes to the gut microbiome following pairing and that these changes are sex-biased; i.e., females transmit more microbes to their social partners than males do. Marmosets exhibit pair bonding behavior such as spatial proximity, physical contact, and grooming, and sex biases in these behavioral patterns may contribute to the observed sex bias in social transmission of gut microbiomes., Social behavior can alter the microbiome composition via transmission among social partners, but there have been few controlled experimental studies of gut microbiome transmission among social partners in primates. We collected longitudinal fecal samples from eight unrelated male-female pairs of marmoset monkeys prior to pairing and for 8 weeks following pairing. We then sequenced 16S rRNA to characterize the changes in the gut microbiome that resulted from the pairing. Marmoset pairs had a higher similarity in gut microbiome communities after pairing than before pairing. We discovered sex differences in the degrees of change in gut microbiome communities following pairing. Specifically, the gut microbiome communities in males exhibited greater dissimilarity from the prepairing stage (baseline) than the gut microbiome communities in females. Conversely, females showed a gradual stabilization in the rate of the gut microbiome community turnover. Importantly, we found that the male fecal samples harbored more female-source gut microbes after pairing, especially early in pairing (paired test, P

Scope Previous studies have suggested that diets rich in omega-3 and low in omega-6 long-chain polyunsaturated fatty acids (PUFAs) can limit the development of metabolic syndrome (MetS). Transgenic soybeans yielding oils enriched for omega-3 PUFAs represent a new and readily-available option for incorporating omega-3 PUFAs into diets to provide health benefits. Methods and results Transgenic soybean oils, enriched for either stearidonic acid (SDA) or eicosapentaenoic acid (EPA), were incorporated in diets to test their effects on limiting the development of MetS in a mouse model of diet-induced obesity. Supplementation with SDA- but not EPA-enriched oils improved features of MetS compared to feeding a control wild-type oil. Because previous studies have linked the gut microorganism Akkermansia muciniphila to the metabolic effects of feeding omega-3 PUFAs, we investigated the causal contribution of A. muciniphila to mediating the metabolic benefits provided by SDA-enriched diets. Although A. muciniphila was not required for SDA-induced metabolic improvements, this microorganism did modulate levels of saturated and mono-unsaturated fatty acids in host adipose tissues. Conclusion Together, these findings support the utilization of SDA-enriched diets to modulate weight gain, glucose metabolism, and fatty acid profiles of liver and adipose tissue. This article is protected by copyright. All rights reserved.

AIMS: The objective of this study was to determine if the faecal microbiome has an association with Escherichia coli O157:H7 prevalence and enumeration. METHODS AND RESULTS: Pyrosequencing analysis of faecal microbiome was performed from feedlot cattle fed one of three diets: (i) 94 heifers fed low concentrate (LC) diet, (ii) 142 steers fed moderate concentrate (MC) diet, and (iii) 132 steers fed high concentrate (HC) diet. A total of 322 585 OTUs were calculated from 2,411,122 high‐quality sequences obtained from 368 faecal samples. In the LC diet group, OTUs assigned to the orders Clostridiales and RF39 (placed within the class Mollicutes) were positively correlated with both E. coli O157:H7 prevalence and enumeration. In the MC diet group, OTUs assigned to Prevotella copri were positively correlated with both E. coli O157:H7 prevalence and enumeration, whereas OTUs assigned to Prevotella stercorea were negatively correlated with both E. coli O157:H7 prevalence and enumeration. In both the MC diet group and the HC diet group, OTUs assigned to taxa placed within Clostridiales were both positively and negatively correlated with both E. coli O157:H7 prevalence and enumeration. However, all correlations were weak. In both the MC diet group and the HC diet group, stepwise linear regression through backward elimination analyses indicated that these OTUs were significantly correlated (P

Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.

The factors that govern assembly of the gut microbiota are insufficiently understood. Here, we test the hypothesis that inter-individual microbiota variation can arise solely from differences in the order and timing by which the gut is colonized early in life. Experiments in which mice were inoculated in sequence either with two complex seed communities or a cocktail of four bacterial strains and a seed community revealed that colonization order influenced both the outcome of community assembly and the ecological success of individual colonizers. Historical contingency and priority effects also occurred in Rag1-/- mice, suggesting that the adaptive immune system is not a major contributor to these processes. In conclusion, this study established a measurable effect of colonization history on gut microbiota assembly in a model in which host and environmental factors were strictly controlled, illuminating a potential cause for the high levels of unexplained individuality in host-associated microbial communities.

Inflammatory bowel diseases (IBD) are likely driven by aberrant immune responses directed against the resident microbiota. Although IBD is commonly associated with a dysbiotic microbiota enriched in putative pathobionts, the etiological agents of IBD remain unknown. Using a pathobiont-induced intestinal inflammation model and a defined bacterial community, we provide new insights into the immune-microbiota interactions during disease. In this model system, the pathobiont Helicobacter bilis instigates disease following sub-pathological dextran sulfate sodium treatment. We show that H. bilis causes mild inflammation in mono-associated mice, but severe disease in the presence of a microbiota, demonstrating synergy between the pathobiont and microbiota in exacerbating pathology. Remarkably, inflammation depends on the presence of H. bilis, but is marked by a predominant Th17 response against specific members of the microbiota and not the pathobiont, even upon the removal of the most immune-dominant taxa. Neither increases in pathobiont burden nor unique changes in immune-targeted microbiota member abundances are observed during disease. Collectively, our findings demonstrate that a pathobiont instigates inflammation without being the primary target of a Th17 response or by altering the microbiota community structure. Moreover, our findings point toward monitoring pathobiont-induced changes in microbiota immune targeting as a new concept in IBD diagnotics.

Soil microbial diversity is vast, and we lack even basic understanding of how this diversity is distributed ecologically. Using pyrosequencing and microarray methods, we quantified the structure of bacterial communities in two contrasting soils underlying Bornean rain forest (clay and sandy loam) that differ markedly in soil properties, aboveground tree flora, and leaf litter decomposition rates. We found significant soil-related taxonomic and phylogenetic differences between communities that, due to their proximity, are independent of climate. Bacterial communities showed distinct compositional and taxon-abundance distributions that were significantly correlated with the structure of the overlying tree community. Richness of bacteria was greater in the more resource-rich clay soil. Phylogenetic community analyses suggested that environmental filtering may be an important mechanism of community assembly in clay, compared to niche-competition in sandy loam. The Acidobacteria were the most abundant group in clay, but the Proteobacteria dominated in sandy loam. Of the ten most abundant classes, the Actinobacteria, Betaproteobacteria, Clostridia, Bacilli, and Gammaproteobacteria were more abundant in sandy loam than clay. Our study, which is the first to quantify edaphic variation in bacterial communities using high-throughput methods in soils underlying one of the most tree species rich forests on Earth, indicates an important role of plant–soil feedbacks linking the community structure of the trees and the underlying soil microbiome. We suggest the biochemical composition of carbon and nutrient resources in plant litter and soil pH and oxygen availability as important determinants of the distribution of bacterial diversity.

To study evolved resistance/tolerance in an insect model, we carried out an experimental evolution study using D. melanogaster and the opportunistic pathogen B. cereus as the agent of selection. The selected lines evolved a 3.0- to 3.3-log increase in the concentration of spores required for 50% mortality after 18–24 generations of selection. In the absence of any treatment, selected lines evolved an increase in egg production and delayed development time. The latter response could be interpreted as a cost of evolution. Alternatively, delayed development might have been a target of selection resulting in increased adult fat body function including production of antimicrobial peptides, and, incidentally, yolk production for oocytes and eggs. When treated with autoclaved spores, the egg production difference between selected and control lines was abolished, and this response was consistent with the hypothesis of a cost of an induced immune response. Treatment with autoclaved spores also reduced life span in some cases and elicited early-age mortality in the selected and wound-control lines both of which were consistent with the hypothesis of a cost associated with induction of immune responses. In general, assays on egg production yielded key outcomes including the negative effect of autoclaved spores on egg production.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn's disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1–V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

In this study, the association between genotypic and selected phenotypic characteristics was examined in a collection of Canadian Escherichia coli O157:H7 strains isolated from humans and cattle in the provinces of Alberta, Ontario, Saskatchewan, and Quebec. In a subset of 69 strains selected on the basis of specific phage types (PTs), a strong correlation between the lineage-specific polymorphism assay (LSPA6) genotype and PT was observed with all strains of PTs 4, 14, 21, 31, 33, and 87 belonging to the LSPA6 lineage I (LSPA6-LI) genotype, while those of PTs 23, 45, 67, and 74 belonged to LSPA6 lineage II (LSPA6-LII) genotypes. This correlation was maintained when additional strains of each PT were tested. E. coli O157:H7 strains with the LSPA6-LI genotype were much more common in the collection than were the LSPA6-LII or lineage I/II (LSPA6-LI/II)-related genotypes (82.6, 11.2, and 5.8%, respectively). Of the strains tested, proportionately more LSPA6-LI than LSPA6-LII genotype strains were isolated from humans (52.7% versus 19.7%) than from cattle (47.8% versus 80.2%). In addition, 96.7% of the LSPA6-LII strains carried the stx 2c variant gene, while only 50.0% of LSPA6-LI/II and 2.7% of LSPA6-LI strains carried this gene. LSPA6-LII strains were also significantly more likely to possess the colicin D gene, cda (50.8% versus 23.2%), and have combined resistance to streptomycin, sulfisoxazole, and tetracycline (72.1% versus 0.9%) than were LSPA6-LI strains. The LSPA6 genotype- and PT-related characteristics identified may be important markers of specific ecotypes of E. coli O157:H7 that have unique epidemiological and virulence characteristics.

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

Comparative genome hybridization of the Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica populations have shown that genome content is highly conserved, with relatively few genes in the F. tularensis subsp. tularensis genome being absent in other F. tularensis subspecies. To determine if organization of the genome differs between global populations of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica , we have used paired-end sequence mapping (PESM) to identify regions of the genome where synteny is broken. The PESM approach compares the physical distances between paired-end sequencing reads of a library of a wild-type reference F. tularensis subsp. holarctica strain to the predicted lengths between the reads based on map coordinates of two different F. tularensis genome sequences. A total of 17 different continuous regions were identified in the F. tularensis subsp. holarctica genome (CR holar c tica ) which are noncontiguous in the F. tularensis subsp. tularensis genome. Six of the 17 different CR holarctica are positioned as adjacent pairs in the F. tularensis subsp. tularensis genome sequence but are translocated in F. tularensis subsp. holarctica , implying that their arrangements are ancestral in F. tularensis subsp. tularensis and derived in F. tularensis subsp. holarctica . PCR analysis of the CR holarctica in 88 additional F. tularensis subsp. tularensis and F. tularensis subsp. holarctica isolates showed that the arrangements of the CR holarctica are highly conserved, particularly in F. tularensis subsp. holarctica , consistent with the hypothesis that global populations of F. tularensis subsp. holarctica have recently experienced a periodic selection event or they have emerged from a recent clonal expansion. Two unique F. tularensis subsp. tularensis -like strains were also observed which likely are derived from evolutionary intermediates and may represent a new taxonomic unit.

The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry theblaCMY-2gene. In the present study, the distribution of these four knownblaCMY-2-carrying plasmids among 35 ceftriaxone-resistantSalmonellaisolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates wereSalmonella entericaserotype Newport, 10 wereSalmonella entericaserotype Typhimurium, 1 wasSalmonella entericaserotype Agona, and 1 wasSalmonella entericaserotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surroundingblaCMY-2was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surroundingblaCMY-5from theKlebsiella oxytocaplasmid pTKH11. These findings are consistent with a model in whichblaCMY-2has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multipleSalmonellachromosomal backbones.

Fresh pork sausage is produced without a microbial kill step and therefore chilled or frozen to control microbial growth. In this report, the microbiota in a chilled fresh pork sausage model produced with or without an antimicrobial combination of sodium lactate and sodium diacetate was studied using a combination of traditional microbiological methods and deep pyrosequencing of 16S rRNA gene amplicons. In the untreated system, microbial populations rose from 10 2 to 10 6 CFU/g within 15 days of storage at 4°C, peaking at nearly 10 8 CFU/g by day 30. Pyrosequencing revealed a complex community at day 0, with taxa belonging to the Bacilli , Gammaproteobacteria , Betaproteobacteria , Actinobacteria , Bacteroidetes , and Clostridia . During storage at 4°C, the untreated system displayed a complex succession, with species of Weissella and Leuconostoc that dominate the product at day 0 being displaced by species of Pseudomonas ( P. lini and P. psychrophila ) within 15 days. By day 30, a second wave of taxa ( Lactobacillus graminis , Carnobacterium divergens , Buttiauxella brennerae , Yersinia mollaretti , and a taxon of Serratia ) dominated the population, and this succession coincided with significant chemical changes in the matrix. Treatment with lactate-diacetate altered the dynamics dramatically, yielding a monophasic growth curve of a single species of Lactobacillus ( L. graminis ), followed by a uniform selective die-off of the majority of species in the population. Of the six species of Lactobacillus that were routinely detected, L. graminis became the dominant member in all samples, and its origins were traced to the spice blend used in the formulation.

Cellulose is an important nutritional resource for a number of insect herbivores. Digestion of cellulose and other polysaccharides in plant-based diets requires several types of enzymes including a number of glycoside hydrolase (GH) families. In a previous study, we showed that a single GH45 gene is present in the midgut tissue of the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). However, the presence of multiple enzymes was also suggested by the lack of a significant biological response when the expression of the gene was silenced by RNA interference. In order to clarify the repertoire of cellulose-degrading enzymes and related GH family proteins in D. v. virgifera, we performed next-generation sequencing and assembled transcriptomes from the tissue of three different developmental stages (eggs, neonates, and third instar larvae). Results of this study revealed the presence of seventy-eight genes that potentially encode GH enzymes belonging to eight families (GH45, GH48, GH28, GH16, GH31, GH27, GH5, and GH1). The numbers of GH45 and GH28 genes identified in D. v. virgifera are among the largest in insects where these genes have been identified. Three GH family genes (GH45, GH48, and GH28) are found almost exclusively in two coleopteran superfamilies (Chrysomeloidea and Curculionoidea) among insects, indicating the possibility of their acquisitions by horizontal gene transfer rather than simple vertical transmission from ancestral lineages of insects. Acquisition of GH genes by horizontal gene transfers and subsequent lineage-specific GH gene expansion appear to have played important roles for phytophagous beetles in specializing on particular groups of host plants and in the case of D. v. virgifera, its close association with maize.

Cell differentiation in the Caulobacter crescentus cell cycle requires differential gene expression that is regulated primarily at the transcriptional level. Until now, however, a defined in vitro transcription system for the biochemical study of developmentally regulated transcription factors had not been available in this bacterium. We report here the purification of C. crescentus RNA polymerase holoenzymes and resolution of the core RNA polymerase from holoenzymes by chromatography on single-stranded DNA cellulose. The three RNA polymerase holoenzymes Esigma54, Esigma32, and Esigma73 were reconstituted exclusively from purified C. crescentus core and sigma factors. Reconstituted Esigma54 initiated transcription from the sigma54-dependent fljK promoter of C. crescentus in the presence of the transcription activator FlbD, and active Esigma32 specifically initiated transcription from the sigma32-dependent promoter of the C. crescentus heat-shock gene dnaK. For reconstitution of the Esigma73 holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coli and purified the full-length sigma73 protein. The reconstituted Esigma73 recognized the sigma70-dependent promoters of the E. coli lacUV5 and neo genes, as well as the sigma73-dependent housekeeping promoters of the C. crescentus pleC and rsaA genes. The ability of the C. crescentus Esigma73 RNA polymerase to recognize E. coli sigma70-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.

Although vertebrates harbor bacterial communities in their gastrointestinal tract whose composition is host-specific, little is known about the mechanisms by which bacterial lineages become selected. The goal of this study was to characterize the ecological processes that mediate host-specificity of the vertebrate gut symbiont Lactobacillus reuteri, and to systematically identify the bacterial factors that are involved. Experiments with monoassociated mice revealed that the ability of L. reuteri to form epithelial biofilms in the mouse forestomach is strictly dependent on the strain's host origin. To unravel the molecular basis for this host-specific biofilm formation, we applied a combination of transcriptome analysis and comparative genomics and identified eleven genes of L. reuteri 100-23 that were predicted to play a role. We then determined expression and importance of these genes during in vivo biofilm formation in monoassociated mice. This analysis revealed that six of the genes were upregulated in vivo, and that genes encoding for proteins involved in epithelial adherence, specialized protein transport, cell aggregation, environmental sensing, and cell lysis contributed to biofilm formation. Inactivation of a serine-rich surface adhesin with a devoted transport system (the SecA2-SecY2 pathway) completely abrogated biofilm formation, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity. In summary, this study established that the epithelial selection of bacterial symbionts in the vertebrate gut can be both specific and highly efficient, resulting in biofilms that are exclusively formed by the coevolved strains, and it allowed insight into the bacterial effectors of this process., Author Summary The bacterial communities found in the vertebrate gastrointestinal tract are remarkably stable and host-specific. However, the ecological and molecular processes that facilitate the selection of microbial symbionts and the exclusion of detrimental bacteria are not well understood. Here, we explore the mechanisms that underlie colonization and biofilm formation in specific strains of the gut symbiont Lactobacillus reuteri. When previously germ-free mice are colonized by individual strains of L. reuteri, only strains originating from rodents formed biofilms on the forestomach epithelium. Genomic, proteomic, and molecular analysis provide a detailed look into the host-specific molecular processes, such as adhesion, that contribute to colonization and biofilm formation. Our findings demonstrate high fidelity in the epithelial selection of a bacterial gut inhabitant, which can differentiate even between strains of the same species, strengthening the notion that some relationships between vertebrates and their microbial symbionts are highly coevolved and exclusive.

Background Individuality in the species composition of the vertebrate gut microbiota is driven by a combination of host and environmental factors that have largely been studied independently. We studied the convergence of these factors in a G10 mouse population generated from a cross between two strains to search for quantitative trait loci (QTLs) that affect gut microbiota composition or ileal Immunoglobulin A (IgA) expression in mice fed normal or high-fat diets. Results We found 42 microbiota-specific QTLs in 27 different genomic regions that affect the relative abundances of 39 taxa, including four QTL that were shared between this G10 population and the population previously studied at G4. Several of the G10 QTLs show apparent pleiotropy. Eight of these QTLs, including four at the same site on chromosome 9, show significant interaction with diet, implying that diet can modify the effects of some host loci on gut microbiome composition. Utilization patterns of IghV variable regions among IgA-specific mRNAs from ileal tissue are affected by 54 significant QTLs, most of which map to a segment of chromosome 12 spanning the Igh locus. Despite the effect of genetic variation on IghV utilization, we are unable to detect overlapping microbiota and IgA QTLs and there is no significant correlation between IgA variable pattern utilization and the abundance of any of the taxa from the fecal microbiota. Conclusions We conclude that host genetics and diet can converge to shape the gut microbiota, but host genetic effects are not manifested through differences in IgA production. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0552-6) contains supplementary material, which is available to authorized users.

Biosynthesis of the Caulobacter crescentus polar flagellum requires the expression of a large number of flagellar (fla) genes that are organized in a regulatory hierarchy of four classes (I to IV). The timing of fla gene expression in the cell cycle is determined by specialized forms of RNA polymerase and the appearance and/or activation of regulatory proteins. Here we report an investigation of the role of the C. crescentus transcriptional regulatory protein FlbD in the activation of sigma 54-dependent class III and class IV fla genes of the hierarchy by reconstituting transcription from these promoters in vitro. Our results demonstrate that transcription from promoters of the class III genes flbG, flgF, and flgI and the class IV gene fliK by Escherichia coli E sigma 54 is activated by FlbD or the mutant protein FlbDS140F (where S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation. In vitro studies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transcription regulation) sequence elements. We have now identified multiple ftr sequences that are conserved in both sequence and spatial architecture in all known class III and class IV promoters. These newly identified ftr elements are positioned ca. 100 bp from the transcription start sites of each sigma 54-dependent fla gene promoter, and our studies indicate that they play an important role in controlling the levels of transcription from different class III and class IV promoters. We have also used mutational analysis to show that the ftr sequences are required for full activation by the FlbD protein both in vitro and in vivo. Thus, our results suggest that FlbD, which is encoded by the class II flbD gene, is a global regulator that activates the cell cycle-regulated transcription from all identified sigma 54-dependent promoters in the C. crescentus fla gene hierarchy.

hile wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies., PLoS ONE, 7 (5), ISSN:1932-6203

The gastrointestinal tract harbors a complex and diverse microbiota that has an important role in host metabolism. Microbial diversity is influenced by a combination of environmental and host genetic factors and is associated with several polygenic diseases. In this study we combined next-generation sequencing, genetic mapping, and a set of physiological traits of the BXD mouse population to explore genetic factors that explain differences in gut microbiota and its impact on metabolic traits. Molecular profiling of the gut microbiota revealed important quantitative differences in microbial composition among BXD strains. These differences in gut microbial composition are influenced by host-genetics, which is complex and involves many loci. Linkage analysis defined Quantitative Trait Loci (QTLs) restricted to a particular taxon, branch or that influenced the variation of taxa across phyla. Gene expression within the gastrointestinal tract and sequence analysis of the parental genomes in the QTL regions uncovered candidate genes with potential to alter gut immunological profiles and impact the balance between gut microbial communities. A QTL region on Chr 4 that overlaps several interferon genes modulates the population of Bacteroides, and potentially Bacteroidetes and Firmicutes–the predominant BXD gut phyla. Irak4, a signaling molecule in the Toll-like receptor pathways is a candidate for the QTL on Chr15 that modulates Rikenellaceae, whereas Tgfb3, a cytokine modulating the barrier function of the intestine and tolerance to commensal bacteria, overlaps a QTL on Chr 12 that influence Prevotellaceae. Relationships between gut microflora, morphological and metabolic traits were uncovered, some potentially a result of common genetic sources of variation.

The flagellar (fla) genes in Caulobacter crescentus are organized into a regulatory hierarchy of four levels (I-IV) in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them. The periodicity of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. We have been particularly interested in understanding the function and regulation of one such transcription factor, FlbD. FlbD belongs to the NtrC family of bacterial response regulators that catalyse the initiation of transcription by sigma 54 RNA polymerase (E sigma 54) and its function is required for transcription of the class III and IV fla genes. Here we show that purified FlbD binds to ftr elements that are required for transcription from the sigma 54-dependent class III flbG promoter (ftr1) and repression of transcription from the class II fliF promoter (ftr4). Dimethylsulphate footprinting assays demonstrated that FlbD makes base-specific contacts at highly conserved guanine nucleotides in each half site of the ftr sequences. In a reconstituted in vitro transcription system using E. coli E sigma 54, we found that FlbD was clearly capable of driving transcriptional initiation from the flbG promoter and that this activity relied on the ftr1 binding site. Several observations suggest that phosphorylation plays a role in the regulation of FlbD activity. First, we found that a mutant form of FlbD (FlbDS140F) corresponding to the substitution found in a constitutively active NtrC protein (NtrCS160F), displayed a greater potential for activating E sigma 54-dependent transcription that the wildtype protein. We also observed that high energy-phosphate-containing molecules stimulate transcription activation by the wild type FlbD. Together, these results suggest that FlbD is responsible for mediating fla gene transcription initiation by E sigma 54 and that covalent modification is likely to play a role in governing FlbD activity during the cell cycle.

Colonization of the gastrointestinal tract and composition of the microbiota may be influenced by components of the diet, including trace elements. To understand how selenium regulates the intestinal microflora, we used high-throughput sequencing to examine the composition of gut microbiota of mice maintained on selenium-deficient, selenium-sufficient, and selenium-enriched diets. The microbiota diversity increased as a result of selenium in the diet. Specific phylotypes showed differential effects of selenium, even within a genus, implying that selenium had unique effects across microbial taxa. Conventionalized germ-free mice subjected to selenium diets gave similar results and showed an increased diversity of the bacterial population in animals fed with higher levels of selenium. Germ-free mice fed selenium diets modified their selenoproteome expression similar to control mice but showed higher levels and activity of glutathione peroxidase 1 and methionine-R-sulfoxide reductase 1 in the liver, suggesting partial sequestration of selenium by the gut microorganisms, limiting its availability for the host. These changes in the selenium status were independent of the levels of other trace elements. The data show that dietary selenium affects both composition of the intestinal microflora and colonization of the gastrointestinal tract, which, in turn, influence the host selenium status and selenoproteome expression.—Kasaikina, M. V., Kravtsova, M. A., Lee, B. C., Seravalli, J., Peterson, D. A., Walter, J., Legge, R., Benson, A. K., Hatfield, D. L., Gladyshev, V. N. Dietary selenium affects host selenoproteome expression by influencing the gut microbiota.

Recent research has provided mechanistic insight into the important contributions of the gut microbiota to vertebrate biology, but questions remain about the evolutionary processes that have shaped this symbiosis. In the present study, we showed in experiments with gnotobiotic mice that the evolution of Lactobacillus reuteri with rodents resulted in the emergence of host specialization. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and we identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and it identified genomic events that have shaped this process., Author Summary The gastrointestinal microbiota of vertebrates is important for nutrient utilization, resistance against pathogens, and immune maturation of its host, but little is known about the evolutionary relationships between vertebrates and individual bacterial members of these communities. Here we provide robust evidence that the evolution of the gut symbiont Lactobacillus reuteri with vertebrates resulted in the emergence of host specialization. Genomic approaches using a combination of genome sequence comparisons and microarray analysis were used to identify the host-specific genome content in rodent and human strains and the evolutionary events that resulted in host adaptation. The study revealed divergent patterns of genome evolution in rodent and human lineages and a distinct genome inventory in host-restricted sub-populations of L. reuteri that reflected the niche characteristics in the gut of their particular vertebrate hosts. The ecological significance of representative rodent-specific genes was demonstrated in gnotobiotic mice. In conclusion, this work provided evidence that the vertebrate gut symbiont Lactobacillus reuteri, despite the likelihood of horizontal transmission, has remained stably associated with related groups of vertebrate hosts over evolutionary time and has evolved a lifestyle specialized to these host animals.

sigma B is a secondary sigma factor of Bacillus subtilis. RNA polymerase containing sigma B transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the sigma B structural gene (sigB), regulate sigma B-dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the sigma B regulatory pathway. Evidence is now presented that RsbW inhibits sigma B-dependent transcription by binding to sigma B and blocking the formation of a sigma B-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or sigma B will coprecipitate both proteins from crude cell extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-filtration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of sigma B protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified sigma B and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a sigma B-dependent promoter (ctc) in vitro. RsbW efficiently blocked sigma B-dependent transcription but only if it was incubated with sigma B prior to the addition of the core enzyme.

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large ( n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genome-wide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

The mammalian gastrointestinal microbiota exerts a strong influence on host lipid and cholesterol metabolism. In this study, we have characterized the interplay among diet, gut microbial ecology, and cholesterol metabolism in a hamster model of hypercholesterolemia. Previous work in this model had shown that grain sorghum lipid extract (GSL) included in the diet significantly improved the high-density lipoprotein (HDL)/non-HDL cholesterol equilibrium (T. P. Carr, C. L. Weller, V. L. Schlegel, S. L. Cuppett, D. M. Guderian, Jr., and K. R. Johnson, J. Nutr. 135: 2236-2240, 2005). Molecular analysis of the hamsters' fecal bacterial populations by pyrosequencing of 16S rRNA tags, PCR-denaturing gradient gel electrophoresis, and Bifidobacterium -specific quantitative real-time PCR revealed that the improvements in cholesterol homeostasis induced through feeding the hamsters GSL were strongly associated with alterations of the gut microbiota. Bifidobacteria, which significantly increased in abundance in hamsters fed GSL, showed a strong positive association with HDL plasma cholesterol levels ( r = 0.75; P = 0.001). The proportion of members of the family Coriobacteriaceae decreased when the hamsters were fed GSL and showed a high positive association with non-HDL plasma cholesterol levels ( r = 0.84; P = 0.0002). These correlations were more significant than those between daily GSL intake and animal metabolic markers, implying that the dietary effects on host cholesterol metabolism are conferred, at least in part, through an effect on the gut microbiota. This study provides evidence that modulation of the gut microbiota-host metabolic interrelationship by dietary intervention has the potential to improve mammalian cholesterol homeostasis, which has relevance for cardiovascular health.

Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.

Twenty-five strains of Escherichia coli O157:H7 isolated from humans, cattle, and pigs were maintained in HCl (pH 2.5) and in a volatile fatty acid (VFA) mixture (pH 4.0) for up to 6 h at 37 degrees C to assess their ability to survive in acidic conditions that simulate those of the stomach and ileum, respectively. In HCl, the average group survival of bovine strains was significantly higher than that of porcine and human strains, whereas in VFAs, porcine strains were significantly more resistant than bovine and human strains. Bovine strains exhibited significantly higher average survival in HCl than in VFAs. The average survival of strains classified as octamer-based genome scanning (OBGS) lineage II was significantly superior to that of strains classified as OBGS lineage I in HCl. The group of lineage I strains was more resistant in VFAs compared with lineage II, but only after 6 h of challenge. The possible involvement of urease in acid resistance of E. coli O157:H7 was also examined. Although the strains possessed the ureC gene, as shown by PCR, this gene did not appear to contribute to acid resistance under the conditions tested. The data indicate that there is a relationship between acid resistance and source or lineage of O157:H7 strains.

While cattle in general have been identified as a reservoir of Escherichia coli O157:H7, there are limited data regarding the prevalence and clonality of this pathogen in downer dairy cattle and the potential impact to human health that may occur following consumption of meat derived from downer dairy cattle. In the present study, conducted at two slaughter facilities in Wisconsin between May and October of 2001, we established a higher prevalence of E. coli O157:H7 in fecal and/or tissue samples obtained aseptically from intact colons of downer dairy cattle (10 of 203, 4.9%) than in those from healthy dairy cattle (3 of 201, 1.5%). Analyses of 57 isolates, representing these 13 positive samples (one to five isolates per sample), by pulsed-field gel electrophoresis, revealed 13 distinct Xba I restriction endonuclease digestion profiles (REDP). Typically, isolates from different animals displayed distinct REDP and isolates from the same fecal or colon sample displayed indistinguishable REDP. However, in one sample, two different, but highly related, REDP were displayed by the isolates recovered. Antimicrobial susceptibility testing indicated that 10 of the 57 isolates, recovered from 2 (1 downer and 1 healthy animal) of the 13 positive samples, were resistant to at least 1 of 18 antimicrobials tested. However, there was no appreciable difference in the frequency of resistance of isolates recovered from downer and healthy dairy cattle, and not all isolates with the same REDP displayed the same antimicrobial susceptibility profile. Lastly, it was not possible to distinguish between isolates recovered from downer and healthy cattle based on their Xba I REDP or antimicrobial susceptibility. These results indicate that downer cattle had a 3.3-fold-higher prevalence of E. coli O157:H7 than healthy cattle within the time frame and geographic scope of this study.

Listeria monocytogenes is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight into the mechanisms used by L. monocytogenes to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated. In the studies presented here, we have cloned the sigB gene and several rsb genes from L. monocytogenes , encoding homologs of the alternative sigma factor ς B and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium Bacillus subtilis . The L. monocytogenes and B. subtilis sigB and rsb genes are similar in sequence and physical organization; however, we observed that the activity of ς B in L. monocytogenes was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium. The magnitude of the response was greatest after an osmotic upshift, suggesting a role for ς B in coordinating osmotic responses in L. monocytogenes . A null mutation in the sigB gene led to substantial defects in the ability of L. monocytogenes to use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of ς B reduced the ability of the cells to accumulate betaine. Thus, ς B coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of L. monocytogenes under conditions of high osmotic strength.

The flagellar genes (fla genes) in Caulobacter crescentus are organized into a regulatory hierarchy of four levels, I-IV, in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them. Timing of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. Here we report that purified FlbD activates transcription in vitro from the sigma 54-dependent class III flbG promoter and repress transcription from the class II fliF promoter by binding to ftr (flagellar transcription regulator) sequence elements required for their transcriptional regulation in vivo. The FlbD protein makes symmetrical base-specific contacts at three highly conserved guanine nucleotides in each half site of ftr1 and ftr1* at flbG and the single ftr4 site at fliF. The dual function of FlbD in activation of class III genes and repression of the class II fliF promoter is consistent with a central role of FlbD as a switch protein mediating the transition from level II to level III fla gene expression.

The sigB operon of Bacillus subtilis encodes sigma B plus three additional proteins (RsbV, RsbW, and RsbX) that regulate sigma B activity. Using an anti-sigma B monoclonal antibody to monitor the levels of sigma B protein, PSPAC to control the expression of the sigB operon, and a ctc-lacZ reporter system to monitor sigma B activity, we observed that the rsbV and rsbW products control sigma B activity at the ctc promoter independently of their effects on sigma B levels. In contrast, RsbX was found to have no effect on expression of ctc when the sigB operon was controlled by PSPAC. The data are consistent with RsbV and RsbW being regulators of sigma B activity and RsbX acting primarily as a negative regulator of sigB operon expression. Evidence that stationary-phase induction of the sigma B-dependent ctc promoter is accomplished by a reduction in RsbW-dependent inhibition of sigma B activity is also presented. In addition, Western blot (immunoblot) analyses of sigB operon expression demonstrated that sigma B accumulation is coupled to the synthesis of its primary inhibitor (RsbW). This finding is consistent with RsbW and sigma B being present within the cell in equivalent amounts, a circumstance that would permit RsbW to directly influence sigma B activity by a direct protein-protein interaction.

sigma B, a secondary sigma factor of Bacillus subtilis, was found to increase 5- to 10-fold when cultures were shifted from 37 to 48 degrees C. Western blot (immunoblot) analyses, in which monoclonal antibodies specific for the sigB operon products RsbV, RsbW, and sigma B were used to probe extracts from wild-type and mutant B. subtilis strains, revealed that all three proteins increased coordinately after heat shock and that this increase was dependent on sigma B but not RsbV, a positive regulator normally essential for sigma B-dependent sigB expression. Nuclease protection experiments of RNA synthesized after heat shock supported the notion that the shift to 48 degrees C enhanced transcription from the sigB operon's sigma B-dependent promoter. The level of mRNA initiating at the sigma B-dependent ctc promoter was also seen to increase approximately 5- to 10-fold after heat shock. Pulse-labeling of the proteins synthesized after a shift to 48 degrees C demonstrated that sigB wild-type and mutant strains produced the major heat-inducible proteins in similar amounts; however, at least seven additional proteins were present after the temperature shift in the wild-type strain but absent in the sigB null mutant. Thus, although sigma B is not required for the expression of essential heat shock genes, it is activated by heat shock to elevate its own synthesis and possibly the synthesis of several other heat-inducible proteins.

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.