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While 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.

The LEE pathogenicity island has been acquired on multiple occasions within the different lineages of enteropathogenic and enterohemorrhagic Escherichia coli. In each lineage, LEE expression is regulated by complex networks of pathways, including core pathways shared by all lineages and lineage-specific pathways. Within the O157:H7 lineage of enterohemorrhagic E. coli, strain-to-strain variation in LEE expression has been observed, implying that expression patterns can diversify even within highly related subpopulations. Using comparative genomics of E. coli O157:H7 subpopulations, we have identified one source of strain-level variation affecting LEE expression. The variation occurs in prophage-dense regions of the genome that lie immediately adjacent to the late regions of the pch prophage carrying pchA, pchB, pchC, and a newly identified pch gene, pchX. Genomic segments extending from the holin S region to the pchA, pchB, pchC, and pchX genes of their respective prophage are highly conserved but are nonetheless embedded within adjacent genomic segments that are extraordinarily variable, termed pch adjacent genomic regions (pch AGR). Despite the remarkable degree of variation, the pattern of variation in pch AGR is highly correlated with the distribution of phylogenetic markers on the backbone of the genome. Quantitative analysis of transcription from the LEE1 promoter further revealed that variation in the pch AGR has substantial effects on absolute levels and patterns of LEE1 transcription. Variation in the pch AGR therefore serves as a mechanism to diversify LEE expression patterns, and the lineage-specific pattern of pch AGR variation could ultimately influence ecological or virulence characteristics of subpopulations within each lineage.

Comparative genuine hybridization of the Francisella tularensis subsp, tularensis and F. tularensis subsp. holarctica populations have shown that genuine content is highly conserved, with relatively few genes in the F. tularensis subsp. tularensis genuine being absent in other F. tularensis subspecies. To determine if organization of the genuine 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 genuine 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 genuine sequences. A total of 17 different continuous regions were identified in the F. tularensis subsp, holarctica genuine ([CR.sub.holarctica]) which are noncontiguous in the F. tularensis subsp, tularensis genuine. Six of the 17 different [CR.sub.holarctica] are positioned as adjacent pairs in the F. tularensis subsp, tularensis genuine 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.sub.holarctica] in 88 additional F. tularensis subsp, tularensis and F. tularensis subsp, holarctica isolates showed that the arrangements of the [CR.sub.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 (Imo0421). 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 [P.sub.SPAC] 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.

Incorporation of six markers into a multilocus genotyping assay, termed lineage-specific polymorphism assay-6 (LSPA-6) is described. Analysis of over 1,400 0157:H7 strains with the LSPA-6 demonstrated that five genotypes comprise over 91% of the strains, suggesting that these subpopulations may be widespread.

Listeria monocytogenes is 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 in L. monocytogenes which serve to scavenge the preferred compatible solutes. The general stress response regulator [[sigma].sup.B] has been shown to play an important role in osmotic adaptation in L. 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 the opuC, gbuA, and betL genes. All three genes are osmotically inducible to some degree, betL is transcribed from a [[sigma].sup.B]-independent promoter, while gbuA is transcribed from dual promoters, one of which is [[sigma].sup.B] dependent, opuC is transcribed exclusively from a [[sigma].sup.B]-dependent promoter. The betL promoter is similar in sequence to the [[sigma].sup.B]-independent gbuAP1 promoter. Kinetic analysis of transcript accumulation after osmotic upshift, demonstrated that [[sigma].sup.B]-dependent transcripts from gbuAP2 and sigB accumulate for an extended period after upshift, suggesting that [[sigma].sup.B] activity may provide a mechanism for sustained high-level expression during osmotic challenge. In contrast to osmotic upshift, expression from the [[sigma].sup.B]-dependent opuC and gbuAP2 promoters after temperature upshift and ethanol stress was minimal, suggesting that additional mechanisms may also participate in regulating transcription from these [[sigma].sup.B]-dependent promoters.

Thirteen different serotypes of Listeria monocytogenes can 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.

The lineage of Escherichia coli O157 reveals divergence and phylogeographic variation due to the incidence of subclones and polymorphisms among subpopulations as evidenced by octamer-based genome scanning, marker sorting, and DNA sequence analysis. Furthermore, genome diversity is a result of random drift and bacteriophage-mediated events.

Research has been conducted on the sigma (super)B in Listeria monocytogenes, the activity of which is stimulated by high osmolarity. The effect of the cold shock on sigma (super)B causing its adaptation in a growth-phase manner has been investigated.

Phosphotransferase system (PTS) complementation assays were performed to establish the effects of serine 46 phosphorylation on PTS activity in Listeria monocytogenes. Results indicated that conditions which favor phosphorylation of serine 46 decrease the PTS activity of L monocytogenes HPr and provide a mechanism for regulating the PTS. This was borne out by the substantial decrease in L monocytogenes HPr activity under conditions favoring serine 46 phosphorylation. HPr phosphorylated at serine 46 also does not seem to possess self-phosphatase activity.

Genes encoding the HPr (ptsH) and enzyme I (ptsI) components of the Listeria monocytogenes phosphotransferase system (PTS) were isolated to examine the mechanisms of sugar transport in L. monocytogenes. The overexpressed and purified ptsH product was observed to complement PTS activity in Staphylococcus aureus extracts taken from ptsH mutants. Results indicate that the genes form an operon which is regulated by glucose and pH or by the by-products of glucose metabolism.

The Listeria monocytogenes rsbVW-sigB-rsbX transcription unit in L. monocytogenes is structurally similar to its counterpart in Bacillus subtilis. Its activity in L. monocytogenes is likewise responsive to many physical and environmental signals. These were concluded in an examination of the role of the sigma(super B) regulon in adaptive responses of L. monocytogenes, wherein the genes encoding homologs of sigma(super B) and several of the Rsb proteins were cloned. It was found that the sigma(super B) regulon is involved in responses to physiological and chemical signals and may function as a primary osmosensor in the bacterium.

A study has been conducted to examine the role of the transcriptional regulatory protein FlbD in the activation of sigma54-dependent class III and class IV fla genes during transcription in Caulobacter crescentus. Transcription assays show that transcription from promoters of the class III and class IV genes is activated by FlbD. In vitro experiments confirmed that ftr sequences located ca. 100 bp from the transcription start sites of each sigma54-dependent fla gene promoter have an important role in controlling transcription in class III and class IV promoters.

The roles of RsbV, RsbW and RsbX in controlling the levels and activity of sigma(super B) in Bacillus subtilis were examined. It was observed that RsbV and RsbW regulate the expression of ctc independently of their influence on sigma(super B) levels. In contrast, RsbX had no effect on expression of ctc when the sigB operon was controlled by an inducible promoter. The results suggest that RsbV and RsbW directly regulate sigma(super B) activity, while RsbX acts primarily as a negative regulator of sigB operon expression.

The Bacillus subtilis RNA polymerase holoenzyme containing sigma-B is activated only upon entry into stationary phase or after heat shock. RsbW is a sporulation protein which is involved in the regulation of sigma-B activation. The nature of the interaction between sigma-B and RsbW was investigated. The results showed that RsbW is capable of binding directly to sigma-B, which blocks the formation of an active RNA polymerase.

The regulation of sigma(super B) synthesis by products encoded by orfV, orfW and orfX of Bacillus subtilis which all reside in a single operon (sigB) was analyzed. Mutation studies involving combinations of null mutations within the three orfs showed that the regulation of sigma(super B) is accomplished through a single pathway of negative control. OrfW is the primary inhibitor of sigB transcription, while OrV acts as an inhibitor of OrfW. OrfX might function as an inhibitor of OrfV. Overall, OrfW is the terminal regulatory molecule.

Abstract 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.

Abstract 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.