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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.

Listeria monocytogenes strains belonging to phylogenetic lineage II (serotypes 1/2a, 1/2c, and 3a) carry a lineage-specific genome segment encoding a putative sigma subunit of RNA polymerase (lmo0423, herein referred to as sigC), a gene of unknown function (lmo0422) similar to the padR family of regulators, and a gene that is similar to the rodA-ftsW family of cell wall morphology genes (lmo0421). To understand the function of this set of genes, their expression patterns and the effects of null mutations in the lineage II L. monocytogenes strain 10403S were examined. The data are consistent with the three genes comprising an operon (the sigC operon) that is highly induced by temperature upshift. The operon is transcribed from three different promoters, the proximal of which (P1) depends upon sigC itself. Null mutations in sigC or lmo0422 increase the death rate at lethal temperatures and cause loss of thermal adaptive response, whereas the lmo0421 mutation causes only a loss of the adaptive response component. Only the sigC mutation affects transcription from the P1 promoter, whereas ectopic expression of lmo0422 from the PSPAC promoter complements the individual lmo0422 and sigC null mutations, showing that lmo0422 is the actual thermal resistance regulator or effector while sigC provides a mechanism for temperature-dependent transcription of lmo0422 from P1. Our genetic and phylogenetic analyses are consistent with lmo0422—renamed lstR (for lineage-specific thermal regulator)—and sigC comprising a system of thermal resistance that was ancestral to the genus Listeria and was subsequently lost during divergence of the lineage I L. monocytogenes population. Although only a relatively small number of cases of human listeriosis occur each year in the United States (estimated at 2,500 per year), the associated rates of morbidity and mortality are substantial, with nearly 30% mortality in some outbreaks (40, 58). Because the causative agent of the disease, Listeria monocytogenes, is ubiquitous in nature and possesses durable physiological characteristics, the organism is one of the most significant food safety problems in the food production industry.

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.

As the first series of genetic analyses of gut microbiome composition in humans is now emerging, the results should be met with enthusiasm, but also with caution. Findings from the initial offerings demonstrate how population-scale approaches can provide deeper insights into host-microbiome interactions while at the same time illustrating that our understanding of the architecture of highly complex microbiome 'traits' is still rudimentary.

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.

The activity of ς B in Listeria monocytogenes is stimulated by high osmolarity and is necessary for efficient uptake of osmoprotectants. Here we demonstrate that, during cold shock, ς B contributes to adaptation in a growth phase-dependent manner and is necessary for efficient accumulation of betaine and carnitine as cryoprotectants.

Multilocus-genotyping methods have shown that Escherichia coli O157:H7 is a geographically disseminated clone. However, high-resolution methods such as pulse-field gel electrophoresis demonstrate significant genomic diversity among different isolates. To assess the genetic relationship of human and bovine isolates of E. coli O157:H7 in detail, we have developed an octamer-based genome-scanning methodology, which compares the distance between over-represented, strand-biased octamers that occur in the genome. Comparison of octamer-based genome-scanning products derived from >1 megabase of the genome demonstrated the existence of two distinct lineages of E. coli O157:H7 that are disseminated within the United States. Human and bovine isolates are nonrandomly distributed among the lineages, suggesting that one of these lineages may be less virulent for humans or may not be efficiently transmitted to humans from bovine sources. Restriction fragment length polymorphism analysis with lambdoid phage genomes indicates that phage-mediated events are associated with divergence of the lineages, thereby providing one explanation for the degree of diversity that is observed among E. coli O157:H7 by other molecular-fingerprinting methods.

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) utilizes high-energy phosphate present in PEP to drive the uptake of several different carbohydrates in bacteria. In order to examine the role of the PTS in the physiology of Listeria monocytogenes , we identified the ptsH and ptsI genes encoding the HPr and enzyme I proteins, respectively, of the PTS. Nucleotide sequence analysis indicated that the predicted proteins are nearly 70% similar to HPr and enzyme I proteins from other organisms. Purified L. monocytogenes HPr overexpressed in Escherichia coli was also capable of complementing an HPr defect in heterologous extracts of Staphylococcus aureus ptsH mutants. Additional studies of the transcriptional organization and control indicated that the ptsH and ptsI genes are organized into a transcription unit that is under the control of a consensus-like promoter and that expression of these genes is mediated by glucose availability and pH or by by-products of glucose metabolism.

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.

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.

Background We propose a sequence clustering algorithm and compare the partition quality and execution time of the proposed algorithm with those of a popular existing algorithm. The proposed clustering algorithm uses a grammar-based distance metric to determine partitioning for a set of biological sequences. The algorithm performs clustering in which new sequences are compared with cluster-representative sequences to determine membership. If comparison fails to identify a suitable cluster, a new cluster is created. Results The performance of the proposed algorithm is validated via comparison to the popular DNA/RNA sequence clustering approach, CD-HIT-EST, and to the recently developed algorithm, UCLUST, using two different sets of 16S rDNA sequences from 2,255 genera. The proposed algorithm maintains a comparable CPU execution time with that of CD-HIT-EST which is much slower than UCLUST, and has successfully generated clusters with higher statistical accuracy than both CD-HIT-EST and UCLUST. The validation results are especially striking for large datasets. Conclusions We introduce a fast and accurate clustering algorithm that relies on a grammar-based sequence distance. Its statistical clustering quality is validated by clustering large datasets containing 16S rDNA sequences.

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.

Populations of the food- and waterborne pathogen Escherichia coli O157:H7 are comprised of two major lineages. Recent studies have shown that specific genotypes within these lineages differ substantially in the frequencies with which they are associated with human clinical disease. While the nucleotide sequences of the genomes of lineage I strains E. coli O157 Sakai and EDL9333 have been determined, much less is known about the genomes of lineage II strains. In this study, suppression subtractive hybridization (SSH) was used to identify genomic features that define lineage II populations. Three SSH experiments were performed, yielding 1,085 genomic fragments consisting of 811 contigs. Bacteriophage sequences were identified in 11.3% of the contigs, 9% showed insertions and 2.3% deletions with respect to E. coli O157:H7 Sakai, and 23.2% did not have significant identity to annotated sequences in GenBank. In order to test for the presence of these novel loci in lineage I and II strains, 27 PCR primer sets were designed based on sequences from these contigs. All but two of these PCR targets were found in the majority (51.9% to 100%) of 27 lineage II strains but in no more than one (E. coli O157:H7 strains isolated from human and animal sources.

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.

Non-sorbitol-fermenting, β-glucuronidase-negative Escherichia coli O157:H7 strains are regarded as a clone complex, and populations from different geographical locations are believed to share a recent common ancestor. Despite their relatedness, high-resolution genotyping methods can detect significant genome variation among different populations. Phylogenetic analysis of high-resolution genotyping data from these strains has shown that subpopulations from geographically unlinked continents can be divided into two primary phylogenetic lineages, termed lineage I and lineage II, and limited studies of the distribution of these lineages suggest there could be differences in their propensity to cause disease in humans or to be transmitted to humans. Because the genotyping methods necessary to discriminate the two lineages are tedious and subjective, these methods are not particularly suited for studying the large sets of strains that are required to systematically evaluate the ecology and transmission characteristics of these lineages. To overcome this limitation, we have developed a lineage-specific polymorphism assay (LSPA) that can readily distinguish between the lineage I and lineage II subpopulations. In the studies reported here, we describe the development of a six-marker test (LSPA-6) and its validation in a side-by-side comparison with octamer-based genome scanning. Analysis of over 1,400 O157:H7 strains with the LSPA-6 demonstrated that five genotypes comprise over 91% of the strains, suggesting that these subpopulations may be widespread.

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.

Previous studies have suggested that carbohydrates may affect expression of virulence genes in Listeria monocytogenes. Which carbohydrates influence virulence gene expression and how carbohydrates mediate expression, however, is not clear. The goal of this work was to examine how carbohydrates affect virulence gene expression in L. monocytogenes 10403S. Growth studies were conducted in medium containing glucose and various sugars. Metabolism of arbutin, arabitol, cellobiose, mannose, maltose, trehalose, and salicin were repressed in the presence of glucose. Only when glucose was consumed were these sugars fermented, indicating that catabolite repression by glucose had occurred. To determine whether virulence gene expression was also influenced by catabolite repression, we performed primer extension experiments, using primers for hly and prfA, which encode for a hemolysin and the regulator protein PrfA, respectively. In the presence of cellobiose and arbutin, transcription of hemolysin was reduced. However, none of the sugars affected transcription of prfA. The results demonstrate that catabolite repression occurs in L. monocytogenes and suggests that, at least in strain 10403S, cellobiose and arbutin repress expression of hemolysin.

Listeria monocytogenesis well known for its durable physiological characteristics, which allow the organism to grow at low temperature and pH and high osmolarity. Growth under high osmolarity depends on the accumulation of compatible solutes, among which glycine betaine and carnitine are the preferred solutes for this organism. Three different transport systems, Gbu, BetL, and OpuC, have been identified inL. monocytogeneswhich serve to scavenge the preferred compatible solutes. The general stress response regulator σBhas been shown to play an important role in osmotic adaptation inL. monocytogenes, presumably by directing transcription from one or more of the solute transport genes. In the studies presented here, we have used primer extension analyses to identify the promoter elements responsible for transcription of theopuC,gbuA, andbetLgenes. All three genes are osmotically inducible to some degree.betLis transcribed from a σB-independent promoter, whilegbuAis transcribed from dual promoters, one of which is σBdependent.opuCis transcribed exclusively from a σB-dependent promoter. ThebetLpromoter is similar in sequence to the σB-independentgbuAP1 promoter. Kinetic analysis of transcript accumulation after osmotic upshift demonstrated that σB-dependent transcripts fromgbuAP2 andsigBaccumulate for an extended period after upshift, suggesting that σBactivity may provide a mechanism for sustained high-level expression during osmotic challenge. In contrast to osmotic upshift, expression from the σB-dependentopuCandgbuAP2 promoters after temperature upshift and ethanol stress was minimal, suggesting that additional mechanisms may also participate in regulating transcription from these σB-dependent promoters.

Thirteen different serotypes ofListeria monocytogenescan be distinguished on the basis of variation in somatic and flagellar antigens. Although the known virulence genes are present in all serotypes, greater than 90% of human cases of listeriosis are caused by serotypes 1/2a, 1/2b, and 4b and nearly all outbreaks of food-borne listeriosis have been caused by serotype 4b strains. Phylogenetic analysis of these three common clinical serotypes places them into two different lineages, with serotypes 1/2b and 4b belonging to lineage I and 1/2a belonging to lineage II. To begin examining evolution of the genome in these serotypes, DNA microarray analysis was used to identify lineage-specific and serotype-specific differences in genome content. A set of 44 strains representing serotypes 1/2a, 1/2b, and 4b was probed with a shotgun DNA microarray constructed from the serotype 1/2a strain 10403s. Clones spanning 47 different genes in 16 different contiguous segments relative to the lineage II 1/2a genome were found to be absent in all lineage I strains tested (serotype 4b and 1/2b) and an additional nine were altered exclusively in 4b strains. Southern hybridization confirmed that conserved alterations were, in all but two loci, due to absence of the segments from the genome. Genes within these contiguous segments comprise five functional categories, including genes involved in synthesis of cell surface molecules and regulation of virulence gene expression. Phylogenetic reconstruction and examination of compositional bias in the regions of difference are consistent with a model in which the ancestor of the two lineages had the 1/2 somatic serotype and the regions absent in the lineage I genome arose by loss of ancestral sequences.

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.

The O157:H7 lineage of enterohemorrhagic Escherichia coli is a geographically disseminated complex of highly related genotypes that share common ancestry. The common clone that is found worldwide carries several markers of events in its evolution, including markers for acquisition of virulence genes and loss of physiological characteristics, such as sorbitol fermentation ability and β-glucuronidase production. Populations of variants that are distinct with respect to motility and the sorbitol and β-glucuronidase markers appear to have diverged at several points along the inferred evolutionary pathway. In addition to these variants, distinct subpopulations of the contemporary non-sorbitol-fermenting, β-glucuronidase-negative O157:H7 clone were recently detected among bovine and human clinical isolates in the United Stares by using high-resolution genome comparison. In order to determine if these recently described subpopulations were derived from a regional or ancestral divergence event, we used octamer-based genome scanning, marker sorting, and DNA sequence analysis to examine their phylogenetic relationship to populations of non-sorbitol-fermenting, β-glucuronidase negative O157:H7 and O157:H− strains from Australia. The inferred phylogeny is consistent with the hypothesis that subpopulations on each continent resulted from geographic spread of an ancestral divergence event and subsequent expansion of distinct subpopulations. Marker sorting and DNA sequence analyses identified sets of monophyletic markers consistent with the pattern of divergence and demonstrated that phylogeographic variation occurred through emergence of regional subclones and concentration of regional polymorphisms among distinct subpopulations. DNA sequence analysis of representative polyphyletic markers showed that genome diversity accrued through random drift and bacteriophage-mediated events.

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes . By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.

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.

G A A b st ra ct s 3 minority groups, with pancreatic cancer nationally has remained unchanged since 2002. Among all 5261 visits of minority patients to our medical center over the past 8 years, 1.3% (66) were in the Emergency Department, 11.1% (585) were in the inpatient setting, and 1.6% (84) were for surgery. These differences are statistically significant for the 3 sites of care (p

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.

The sigB operon of Bacillus subtilis encodes sigma B and three additional open reading frames (orfV, orfW, and orfX). Having previously mapped several mutations that alter the induction pattern of a sigma B-dependent promoter (ctc) to regions of cloned B. subtilis DNA which contain these three open reading frames, we directly tested the regulatory potential of orfV, orfW, and orfX by creating null alleles of each of these genes and examining the effects of the mutations, either singly or in pairs, on transcription of ctc and the sigB operon. Using lacZ reporter gene fusions and Northern (RNA) blot analyses, we have determined that all three genes modulate the activation of the sigma B-dependent promoters at both the sigB operon and ctc. Our data are consistent with the three gene products participating in a single pathway of negative control. orfW and orfX single-mutant strains have high levels of sigB and ctc transcription. sigB and ctc transcription in an orfV strain is similar to that found in mutant strains which lack sigma B itself. The orfV mutation is dominant to orfX but recessive to orfW. These results suggest that OrfW is the primary inhibitor of sigma B-dependent transcription and that OrfV is capable of counteracting the negative control of OrfW but is prevented from doing this by the orfX gene product.

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.

As an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes in Staphylococcus aureus and other Gram-positive bacteria. The use of a restriction-deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) in S. aureus NCTC 8325-4. Plasmid pPQ126 (13.6 kb) is a novel, temperature-sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30 degrees C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39 degrees C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination-deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30 degrees C), and excision results in derivatives of pPQ126 that harbour DNA of chromosomal origin.

Background Functional assignments for short-read metagenomic data pose a significant computational challenge due to perceived unpredictability of alignment behavior and the inability to infer useful functional information from translated protein-fragments/peptides. To address this problem, we have examined the predictability of short peptide alignments by systematically studying alignment behavior of large sets of short peptides generated from well-characterized proteins as well as hypothetical proteins in the KEGG database. Results Using test sets of peptides modeling the length and phylogenetic distributions of short-read metagenomic data, we observed that peptides from well-characterized proteins had indistinguishable alignments to proteins from the same orthologous family and proteins from different families. Nonetheless, the patterns contained remarkable phylogenetic and structural signals, with alignments of even very short peptides naturally restricted to their orthologous family and/or proteins having similar structural folds. In stark contrast, peptides from “hypothetical proteins” had only sparse hit patterns with low frequencies and much lower identities. By weighting the structure-driven alignments and filtering peptides with behaviors similar to those derived from “hypothetical proteins”, we demonstrate that the accuracy of abundance predictions of protein families is dramatically improved. Conclusions Evolutionary processes have dispersed protein folds across multiple protein families, precluding accurate functional assignment to short peptides, whose alignment behavior is non-random and driven by structure. Algorithms that filter sparse peptides and weight hit patterns of peptides from “known space” dramatically improve quantification of functions from diverse mixtures of peptides and should substantially improve applications of metagenomic analyses requiring accurate quantitative measures of functional families. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2272-z) contains supplementary material, which is available to authorized users.

Background Genetic analysis of Escherichia coli O157:H7 strains has shown divergence into two distinct lineages, lineages I and II, that appear to have distinct ecological characteristics, with lineage I strains more commonly associated with human disease. In this study, microarray-based comparative genomic hybridization (CGH) was used to identify genomic differences among 31 E. coli O157:H7 strains that belong to various phage types (PTs) and different lineage-specific polymorphism assay (LSPA) types. Results A total of 4,084 out of 6,057 ORFs were detected in all E. coli O157:H7 strains and 1,751 were variably present or absent. Based on this data, E. coli O157:H7 strains were divided into three distinct clusters, which consisted of 15 lineage I (LSPA type 111111), four lineage I/II (designated in this study) (LSPA type 211111) and 12 lineage II strains (LSPA 222222, 222211, 222212, and 222221), respectively. Eleven different genomic regions that were dominant in lineage I strains (present in ≥80% of lineage I and absent from ≥ 92% of lineage II strains) spanned segments containing as few as two and up to 25 ORFs each. These regions were identified within E. coli Sakai S-loops # 14, 16, 69, 72, 78, 83, 85, 153 and 286, Sakai phage 10 (S-loops # 91, 92 and 93) and a genomic backbone region. All four lineage I/II strains were of PT 2 and possessed eight of these 11 lineage I-dominant loci. Several differences in virulence-associated loci were noted between lineage I and lineage II strains, including divergence within S-loop 69, which encodes Shiga toxin 2, and absence of the non-LEE encoded effector genes nleF and nleH1-2 and the perC homologue gene pchD in lineage II strains. Conclusion CGH data suggest the existence of two dominant lineages as well as LSPA type and PT-related subgroups within E. coli O157:H7. The genomic composition of these subgroups supports the phylogeny that has been inferred from other methods and further suggests that genomic divergence from an ancestral form and lateral gene transfer have contributed to their evolution. The genomic features identified in this study may contribute to apparent differences in the epidemiology and ecology of strains of different E. coli O157:H7 lineages.