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Maintenance of membrane function is essential and regulated at the genomic, transcriptional, and translational levels. Bacterial pathogens have a variety of mechanisms to adapt their membrane in response to transmission between environment, vector, and human host. Using a well-characterized model of lipid A diversification (Francisella), we demonstrate temperature-regulated membrane remodeling directed by multiple alleles of the lipid A-modifying N-acyltransferase enzyme, LpxD. Structural analysis of the lipid A at environmental and host temperatures revealed that the LpxD1 enzyme added a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme added a 3-OH C16 acyl group at 18 °C (environment). Mutational analysis of either of the individual Francisella lpxD genes altered outer membrane (OM) permeability, antimicrobial peptide, and antibiotic susceptibility, whereas only the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. Additionally, growth-temperature analysis revealed transcriptional control of the lpxD genes and posttranslational control of the LpxD1 and LpxD2 enzymatic activities. These results suggest a direct mechanism for LPS/lipid A-level modifications resulting in alterations of membrane fluidity, as well as integrity and may represent a general paradigm for bacterial membrane adaptation and virulence-state adaptation.

We sequenced four strains of Bacillus subtilis and the type strains for two closely related species, Bacillus vallismortis and Bacillus mojavensis. We report the high-quality Sanger genome sequences of B. subtilis subspecies subtilis RO-NN-1 and AUSI98, B. subtilis subspecies spizizenii TU-B-10(T) and DV1-B-1, Bacillus mojavensis RO-H-1(T), and Bacillus vallismortis DV1-F-3(T).

Enterotoxigenic Escherichia coli (ETEC) is an important pathogenic variant (pathovar) of E. coli in developing countries from a human health perspective, causing significant morbidity and mortality. Previous studies have examined specific regulatory networks in ETEC, although little is known about the global effects of inter- and intrakingdom signaling on the expression of virulence and colonization factors in ETEC. In this study, an E. coli/Shigella pan-genome microarray, combined with quantitative reverse transcriptase PCR (qRT-PCR) and RNA sequencing (RNA-seq), was used to quantify the expression of ETEC virulence and colonization factors. Biologically relevant chemical signals were combined with ETEC isolate E24377A during growth in either Luria broth (LB) or Dulbecco's modified Eagle medium (DMEM), and transcription was examined during different phases of the growth cycle; chemical signals examined included glucose, bile salts, and preconditioned media from E. coli/Shigella isolates. The results demonstrate that the presence of bile salts, which are found in the intestine and thought to be bactericidal, upregulates the expression of many ETEC virulence factors, including heat-stable (estA) and heat-labile (eltA) enterotoxin genes. In contrast, the ETEC colonization factors CS1 and CS3 were downregulated in the presence of bile, consistent with findings in studies of other enteric pathogens. RNA-seq analysis demonstrated that one of the most differentially expressed genes in the presence of bile is a unique plasmid-encoded AraC-like transcriptional regulator (peaR); other previously unknown genetic elements were found as well. These results provide transcriptional targets and putative mechanisms that should help improve understanding of the global regulatory networks and virulence expression in this important human pathogen.

Here, we describe the draft genome sequence of Vibrio fischeri SR5, a squid symbiotic isolate from Sepiola robusta in the Mediterranean Sea. This 4.3-Mbp genome sequence represents the first V. fischeri genome from an S. robusta symbiont and the first from outside the Pacific Ocean.

Acinetobacter baumannii is a Gram-negative bacterium that causes nosocomial infections worldwide, with recent prevalence and higher frequency in wounded military personnel. Four A. baumannii strains from the Walter Reed Army Medical Center (WRAMC) isolated between 2008 and 2009 were sequenced, representing diverse, multidrug-resistant isolates from osteomyelitis or septic patients.

Background: Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood., Methods: We characterized 121 EAEC strains isolated in 2008 as part of a case-control study of moderate to severe acute diarrhea among children 0-59 months of age in Bamako, Mali. We applied multiplex polymerase chain reaction and comparative genome hybridization to identify potential virulence factors among the EAEC strains, coupled with classification and regression tree modeling to reveal combinations of factors most strongly associated with illness., Results: The gene encoding the autotransporter protease SepA, originally described in Shigella species, was most strongly associated with diarrhea among the EAEC strains tested (odds ratio, 5.6 [95% confidence interval, 1.92-16.17]; P = .0006). In addition, we identified 3 gene combinations correlated with diarrhea: (1) a clonal group positive for sepA and a putative hemolysin; (2) a group harboring the EAST-1 enterotoxin and the flagellar type H33 but no other previously identified EAEC virulence factor; and (3) a group carrying several of the typical EAEC virulence genes., Conclusion: Our data suggest that only a subset of EAEC strains are pathogenic in Mali and suggest that sepA may serve as a valuable marker for the most virulent isolates.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC encodes the sRNA chaperone Hfq, which is important in posttranscriptional regulation. In EHEC strain EDL933, Hfq acts as a negative regulator of the locus of enterocyte effacement (LEE), which encodes most of the proteins involved in type III secretion and attaching and effacing (AE) lesions. Here, we deleted hfq in the EHEC strain 86-24 and compared global transcription profiles of the hfq mutant and wild-type (WT) strains in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler, the transcriptional activator of all the LEE genes, as well as genes encoded in the LEE2 to -5 operons. Decreased expression of the LEE genes in the hfq mutant occurred at middle, late, and stationary growth phases. We also confirmed decreased regulation of the LEE genes by examining the proteins secreted and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC, which is involved in interkingdom signaling and virulence gene regulation in EHEC, as well as an increase in expression of stx(2AB), which encodes the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a regulatory role in EHEC 86-24 that is different from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE.

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax-a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a "species" DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Acinetobacter baumannii has recently emerged as a significant global pathogen, with a surprisingly rapid acquisition of antibiotic resistance and spread within hospitals and health care institutions. This study examines the genomic content of three A. baumannii strains isolated from distinct body sites. Isolates from blood, peri-anal, and wound sources were examined in an attempt to identify genetic features that could be correlated to each isolation source., Results: Pulsed-field gel electrophoresis, multi-locus sequence typing and antibiotic resistance profiles demonstrated genotypic and phenotypic variation. Each isolate was sequenced to high-quality draft status, which allowed for comparative genomic analyses with existing A. baumannii genomes. A high resolution, whole genome alignment method detailed the phylogenetic relationships of sequenced A. baumannii and found no correlation between phylogeny and body site of isolation. This method identified genomic regions unique to both those isolates found on the surface of the skin or in wounds, termed colonization isolates, and those identified from body fluids, termed invasive isolates; these regions may play a role in the pathogenesis and spread of this important pathogen. A PCR-based screen of 74 A. baumanii isolates demonstrated that these unique genes are not exclusive to either phenotype or isolation source; however, a conserved genomic region exclusive to all sequenced A. baumannii was identified and verified., Conclusions: The results of the comparative genome analysis and PCR assay show that A. baumannii is a diverse and genomically variable pathogen that appears to have the potential to cause a range of human disease regardless of the isolation source.

The replacement of the bladder with a neobladder made from ileal tissue is the prescribed treatment in some cases of bladder cancer or trauma. Studies have demonstrated that individuals with an ileal neobladder have recurrent colonization by Escherichia coli and other species that are commonly associated with urinary tract infections; however, pyelonephritis and complicated symptomatic infections with ileal neobladders are relatively rare. This study examines the genomic content of two E. coli isolates from individuals with neobladders using comparative genomic hybridization (CGH) with a pan-E. coli/Shigella microarray. Comparisons of the neobladder genome hybridization patterns with reference genomes demonstrate that the neobladder isolates are more similar to the commensal, laboratory-adapted E. coli and a subset of enteroaggregative E. coli than they are to uropathogenic E. coli isolates. Genes identified by CGH as exclusively present in the neobladder isolates among the 30 examined isolates were primarily from large enteric isolate plasmids. Isolations identified a large plasmid in each isolate, and sequencing confirmed similarity to previously identified plasmids of enteric species. Screening, via PCR, of more than 100 isolates of E. coli from environmental, diarrhoeagenic and urinary tract sources did not identify neobladder-specific genes that were widely distributed in these populations. These results taken together demonstrate that the neobladder isolates, while distinct, are genomically more similar to gastrointestinal or commensal E. coli, suggesting why they can colonize the transplanted intestinal tissue but rarely progress to acute pyelonephritis or more severe disease.

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in children less than 5 years of age in low- and middle-income nations, whereas it is an emerging enteric pathogen in industrialized nations. Despite being an important cause of diarrhea, little is known about the genomic composition of ETEC. To address this, we sequenced the genomes of five ETEC isolates obtained from children in Guinea-Bissau with diarrhea. These five isolates represent distinct and globally dominant ETEC clonal groups. Comparative genomic analyses utilizing a gene-independent whole-genome alignment method demonstrated that sequenced ETEC strains share approximately 2.7 million bases of genomic sequence. Phylogenetic analysis of this "core genome" confirmed the diverse history of the ETEC pathovar and provides a finer resolution of the E. coli relationships than multilocus sequence typing. No identified genomic regions were conserved exclusively in all ETEC genomes; however, we identified more genomic content conserved among ETEC genomes than among non-ETEC E. coli genomes, suggesting that ETEC isolates share a genomic core. Comparisons of known virulence and of surface-exposed and colonization factor genes across all sequenced ETEC genomes not only identified variability but also indicated that some antigens are restricted to the ETEC pathovar. Overall, the generation of these five genome sequences, in addition to the two previously generated ETEC genomes, highlights the genomic diversity of ETEC. These studies increase our understanding of ETEC evolution, as well as provide insight into virulence factors and conserved proteins, which may be targets for vaccine development.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG, the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD, encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.

Murine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans.

In this study, we identified critically ill patients with Acinetobacter baumannii bacteremia and examined perirectal surveillance cultures for the presence of genetically related A baumannii strains using pulsed-field gel electrophoresis to determine whether gut colonization preceded clinical infection. Seven patients with imipenem-resistant A baumannii bacteremia were identified from January to June of 2008. Six of 7 (86%) patients were colonized in the gastrointestinal tract with genetically similar strains preceding their bacteremia., (Copyright © 2010 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.)

Swarming motility by the urinary tract pathogen Proteus mirabilis has been a long-studied but little understood phenomenon. On agar, a P. mirabilis colony grows outward in a bull's-eye pattern formed by consecutive waves of rapid swarming followed by consolidation into shorter cells. To examine differential gene expression in these growth phases, a microarray was constructed based on the completed genome sequence and annotation. RNA was extracted from broth-cultured, swarming, and consolidation-phase cells to assess transcription during each of these growth states. A total of 587 genes were differentially expressed in broth-cultured cells versus swarming cells, and 527 genes were differentially expressed in broth-cultured cells versus consolidation-phase cells (consolidate). Flagellar genes were highly upregulated in both swarming cells and consolidation-phase cells. Fimbriae were downregulated in swarming cells, while genes involved in cell division and anaerobic growth were upregulated in broth-cultured cells. Direct comparison of swarming cells to consolidation-phase cells found that 541 genes were upregulated in consolidate, but only nine genes were upregulated in swarm cells. Genes involved in flagellar biosynthesis, oligopeptide transport, amino acid import and metabolism, cell division, and phage were upregulated in consolidate. Mutation of dppA, oppB, and cysJ, upregulated during consolidation compared to during swarming, revealed that although these genes play a minor role in swarming, dppA and cysJ are required during ascending urinary tract infection. Swarming on agar to which chloramphenicol had been added suggested that protein synthesis is not required for swarming. These data suggest that the consolidation phase is a state in which P. mirabilis prepares for the next wave of swarming.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes the large intestine, causing attaching and effacing (AE) lesions. Most of the genes involved in AE lesion formation are encoded within a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). The LysR-type transcriptional factor QseA regulates the LEE by binding to the regulatory region of ler. We performed transcriptome analyses comparing wild-type (WT) EHEC and the qseA mutant to elucidate QseA's role in gene regulation. During both growth phases, several genes carried in O-islands were activated by QseA, whereas genes involved in cell metabolism were repressed. During late-logarithmic growth, QseA activated expression of the LEE genes as well as non-LEE-encoded effector proteins. We also performed electrophoretic mobility shift assays, competition experiments and DNase I footprints. The results demonstrated that QseA directly binds both the ler proximal and distal promoters, its own promoter, as well as promoters of genes encoded in EHEC-specific O-islands. Additionally, we mapped the transcriptional start site of qseA, leading to the identification of two promoter sequences. Taken together, these results indicate that QseA acts as a global regulator in EHEC, co-ordinating expression of virulence genes.

The mammalian gastrointestinal (GI) tract is colonized by a complex consortium of bacterial species. Bacteria engage in chemical signaling to coordinate population-wide behavior. However, it is unclear if chemical sensing plays a role in establishing mammalian host-bacterial commensal relationships. Enterohemorrhagic Escherichia coli (EHEC) is a deadly human pathogen but is a member of the GI flora in cattle, its main reservoir. EHEC harbors SdiA, a regulator that senses acyl-homoserine lactones (AHLs) produced by other bacteria. Here, we show that SdiA is necessary for EHEC colonization of cattle and that AHLs are prominent within the bovine rumen but absent in other areas of the GI tract. We also assessed the rumen metagenome of heifers, and we show that it is dominated by Clostridia and/or Bacilli but also harbors Bacteroidetes. Of note, some members of the Bacteroidetes phyla have been previously reported to produce AHLs. SdiA-AHL chemical signaling aids EHEC in gauging these GI environments, and promotes adaptation to a commensal lifestyle. We show that chemical sensing in the mammalian GI tract determines the niche specificity for colonization by a commensal bacterium of its natural animal reservoir. Chemical sensing may be a general mechanism used by commensal bacteria to sense and adapt to their mammalian hosts. Additionally, because EHEC is largely prevalent in cattle herds, interference with SdiA-mediated cattle colonization is an exciting alternative to diminish contamination of meat products and cross-contamination of produce crops because of cattle shedding of this human pathogen.

Antibiotic resistance is one of the greatest challenges of the twenty-first century. However, the increasing understanding of bacterial pathogenesis and intercellular communication has revealed many potential strategies to develop novel drugs to treat bacteria-mediated disease. Interference with bacterial virulence and/or cell-to-cell signalling pathways is an especially compelling approach, as it is thought to apply less selective pressure for the development of bacterial resistance than traditional strategies, which are aimed at killing bacteria or preventing their growth. Here, we discuss the mechanisms of bacterial virulence and present promising anti-virulence strategies and compounds for the future treatment of bacterial infections.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in developing countries, and perennially the most common cause of traveller's diarrhea. ETEC constitute a diverse pathotype that elaborate heat-labile and/or heat-stable enterotoxins. Recent molecular pathogenesis studies reveal sophisticated pathogen-host interactions that might be exploited in efforts to prevent these important infections. While vaccine development for these important pathogens remains a formidable challenge, extensive efforts that attempt to exploit new genomic and proteomic technology platforms in discovery of novel targets are presently ongoing., (Published by Elsevier SAS.)

Background: Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation., Methods: In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12., Conclusion: This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies.

An outbreak of severe diarrheal illness was recently reported in northeastern Oklahoma, and Shiga toxin-producing Escherichia coli serotype O111 was identified as the etiological agent. Our results indicated that this isolate is unable to decarboxylate lysine, a characteristic that is shared with other outbreak-linked O111 isolates. Therefore, further investigation is recommended to determine whether the lysine decarboxylase test could be used to identify a subset of pathogenic E. coli, particularly Shiga toxin-producing E. coli O111 isolates, that have the potential of causing human infections and outbreaks.

In all likelihood, it is only a matter of time before our public health system will face a major biological threat, whether intentionally dispersed or originating from a known or newly emerging infectious disease. It is necessary not only to increase our reactive "biodefense," but also to be proactive and increase our preparedness. To achieve this goal, it is essential that the scientific and public health communities fully embrace the genomic revolution, and that novel bioinformatic and computing tools necessary to make great strides in our understanding of these novel and emerging threats be developed. Genomics has graduated from a specialized field of science to a research tool that soon will be routine in research laboratories and clinical settings. Because the technology is becoming more affordable, genomics can and should be used proactively to build our preparedness and responsiveness to biological threats. All pieces, including major continued funding, advances in next-generation sequencing technologies, bioinformatics infrastructures, and open access to data and metadata, are being set in place for genomics to play a central role in our public health system., Competing Interests: The authors have declared that no competing interests exist.

Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [bla(CMY-2) and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.

The ability to respond to stress is at the core of an organism's survival. The hormones epinephrine and norepinephrine play a central role in stress responses in mammals, which require the synchronized interaction of the whole neuroendocrine system. Mammalian adrenergic receptors are G-coupled protein receptors (GPCRs); bacteria, however, sense these hormones through histidine sensor kinases (HKs). HKs autophosphorylate in response to signals and transfer this phosphate to response regulators (RRs). Two bacterial adrenergic receptors have been identified in EHEC, QseC and QseE, with QseE being downstream of QseC in this signaling cascade. Here we mapped the QseC signaling cascade in the deadly pathogen enterohemorrhagic E. coli (EHEC), which exploits this signaling system to promote disease. Through QseC, EHEC activates expression of metabolic, virulence and stress response genes, synchronizing the cell response to these stress hormones. Coordination of these responses is achieved by QseC phosphorylating three of the thirty-two EHEC RRs. The QseB RR, which is QseC's cognate RR, activates the flagella regulon which controls bacteria motility and chemotaxis. The QseF RR, which is also phosphorylated by the QseE adrenergic sensor, coordinates expression of virulence genes involved in formation of lesions in the intestinal epithelia by EHEC, and the bacterial SOS stress response. The third RR, KdpE, controls potassium uptake, osmolarity, and also the formation of lesions in the intestine. Adrenergic regulation of bacterial gene expression shares several parallels with mammalian adrenergic signaling having profound effects in the whole organism. Understanding adrenergic regulation of a bacterial cell is a powerful approach for studying the underlying mechanisms of stress and cellular survival.

Background: Capsule expression may be important during ascending Escherichia coli urinary tract infections (UTIs)., Methods: An isogenic ksl(k2)ABCDE mutant of extraintestinal pathogenic E. coli (ExPEC) strain CFT073 that could not synthesize the K2 capsule was compared with wild-type CFT073, to determine virulence in a murine model of ascending UTI and in vitro killing assays., Results: No significant differences were observed regarding the abilities of the mutant and the wild-type CFT073 strains to colonize the murine urinary tract in single-challenge infection experiments. However, in competitive-colonization experiments, the mutant was significantly outcompeted by the wild-type strain in urine and the kidneys. The mutant strain was also more susceptible to human serum. Complementation of the mutant with a plasmid containing the ksl(k2)ABCDE genes restored capsule expression, enhanced survival in the murine urinary tract, and restored serum resistance., Conclusion: These results indicate that expression of the K2 capsule is important for the pathogenesis of UTI and provides protection against complement-mediated killing. To our knowledge, this is the first study in which the E. coli capsule has been proven to play a role in infection by use of isogenic mutants and genetic complementation.

Bacterial pathogens sense host cues to activate expression of virulence genes. Most of these signals are sensed through histidine kinases (HKs), which comprise the main sensory mechanism in bacteria. The host stress hormones epinephrine (Epi) and norepinephrine are sensed through the QseC HK, which initiates a complex signaling cascade to regulate virulence gene expression in enterohemorrhagic Escherichia coli (EHEC). Epi signaling through QseC activates expression of the genes encoding the QseEF 2-component system. QseE is an HK, and QseF is a response regulator. Here, we show that QseE is a second bacterial adrenergic receptor that gauges the stress signals Epi, sulfate, and phosphate. The qseEF genes are organized within an unusual operonic structure, in that a gene is encoded between qseE and qseF. This gene was renamed qseG, and it was shown to encode an outer membrane (OM) protein. EHEC uses a type III secretion system (TTSS) to translocate effector proteins to the epithelial cells that rearrange the host cytoskeleton to form pedestal-like structures that cup the bacterium. QseE, QseG, and QseF are necessary for pedestal formation. Although QseE and QseF are involved in the transcriptional control of genes necessary for pedestal formation, QseG is necessary for translocation of effectors into epithelial cells. QseG is an OM protein necessary for translocation of TTSS effectors that also works in conjunction with a 2-component signaling system that senses host stress signals.

Bacillus cereus UW85 produces the linear aminopolyol antibiotic zwittermicin A (ZmA). This antibiotic has diverse biological activities, such as suppression of disease in plants caused by protists, inhibition of fungal and bacterial growth, and amplification of the insecticidal activity of the toxin protein from Bacillus thuringiensis. ZmA has an unusual chemical structure that includes a d amino acid and ethanolamine and glycolyl moieties, as well as having an unusual terminal amide that is generated from the modification of the nonproteinogenic amino acid beta-ureidoalanine. The diverse biological activities and unusual structure of ZmA have stimulated our efforts to understand how this antibiotic is biosynthesized. Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. A nearly identical gene cluster is identified on a plasmid from B. cereus AH1134, and we show that this strain is also capable of producing ZmA. Bioinformatics and biochemical analyses of the ZmA biosynthesis enzymes strongly suggest that ZmA is initially biosynthesized as part of a larger metabolite that is processed twice, resulting in the formation of ZmA and two additional metabolites. Additionally, we propose that the biosynthesis gene cluster for the production of the amino sugar kanosamine is contained within the ZmA biosynthesis gene cluster in B. cereus UW85.

The identification of cell-to-cell signaling inhibitors among bacteria is a novel mechanism used to control the virulence of pathogens. Signaling inhibitors have a significant potential to be used as therapeutics in that they will exert less biological pressure to develop resistance than classic antibiotics, which attempt to eradicate or significantly inhibit bacterial growth. Decreasing the virulence of pathogens by inhibiting signaling provides a mechanism by which the bacteria will be "misled" into not activating virulence factors. If virulence factors are not activated, these pathogens will either pass through the host without incidence or be controlled by the host's immune system. In this unit, we discuss the general principles for the design and implementation of a high-throughput screen to identify inhibitors of bacterial cell-to-cell communication. We also provide a detailed protocol using a specific example of signaling inhibitor identification using enterohemorrhagic Escherichia coli as a model system.

The pathogenic bacterium Bacillus anthracis has become the subject of intense study as a result of its use in a bioterrorism attack in the United States in September and October 2001. Previous studies suggested that B. anthracis Ames Ancestor, the original Ames fully virulent plasmid-containing isolate, was the ideal reference. This study describes the complete genome sequence of that original isolate, derived from a sample kept in cold storage since 1981.

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

Whole-genome sequencing has been skewed toward bacterial pathogens as a consequence of the prioritization of medical and veterinary diseases. However, it is becoming clear that in order to accurately measure genetic variation within and between pathogenic groups, multiple isolates, as well as commensal species, must be sequenced. This study examined the pangenomic content of Escherichia coli. Six distinct E. coli pathovars can be distinguished using molecular or phenotypic markers, but only two of the six pathovars have been subjected to any genome sequencing previously. Thus, this report provides a seminal description of the genomic contents and unique features of three unsequenced pathovars, enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. We also determined the first genome sequence of a human commensal E. coli isolate, E. coli HS, which will undoubtedly provide a new baseline from which workers can examine the evolution of pathogenic E. coli. Comparison of 17 E. coli genomes, 8 of which are new, resulted in identification of approximately 2,200 genes conserved in all isolates. We were also able to identify genes that were isolate and pathovar specific. Fewer pathovar-specific genes were identified than anticipated, suggesting that each isolate may have independently developed virulence capabilities. Pangenome calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors. This comparative study of the species E. coli, while descriptive, should provide the basis for future functional work on this important group of pathogens.

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.

Francisella tularensis is a gram-negative intracellular bacterium and the causative agent of the zoonotic disease tularemia. F. tularensis is a category A select agent and thus a potential agent of bioterrorism. Whereas an F. tularensis live, attenuated vaccine strain (LVS) is the basis of an investigational vaccine, this vaccine is not licensed for human use because of efficacy and safety concerns. In the present study, we immunized mice with isolated native outer membrane proteins (OMPs), ethanol-inactivated LVS (iLVS), or purified LVS lipopolysaccharide (LPS) and assessed the ability of each vaccine preparation to protect mice against pulmonary challenge with the virulent type A F. tularensis strain SchuS4. Antibody isotyping indicated that both Th1 and Th2 antibody responses were generated in mice after immunization with OMPs or iLVS, whereas LPS immunization resulted in only immunoglobulin A production. In survival studies, OMP immunization provided the greatest level of protection (50% survival at 20 days after infection with SchuS4), and there were associated 3-log reductions in the spleen and liver bacterial burdens (compared to nonvaccinated mice). Cytokine quantitation for the sera of SchuS4-challenged mice indicated that OMP and iLVS immunizations induced high levels of tumor necrosis factor alpha and interleukin-2 (IL-2) production, whereas only OMP immunization induced high levels of IL-10 production. By comparison, high levels of proinflammatory cytokines, including RANTES, granulocyte colony-stimulating factor, IL-6, IL-1alpha, IL-12p40, and KC, in nonvaccinated mice indicated that these cytokines may facilitate disease progression. Taken together, the results of this study demonstrate the potential utility of an OMP subunit (acellular) vaccine for protecting mammals against type A F. tularensis.

The nucleotide sequence from the genome of Moraxella catarrhalis ATCC 43617 was annotated and used both to assess the metabolic capabilities and limitations of this bacterium and to design probes for a DNA microarray. An absence of gene products for utilization of exogenous carbohydrates was noteworthy and could be correlated with published phenotypic data. Gene products necessary for aerobic energy generation were present, as were a few gene products generally ascribed to anaerobic systems. Enzymes for synthesis of all amino acids except proline and arginine were present. M. catarrhalis DNA microarrays containing 70-mer oligonucleotide probes were designed from the genome-derived nucleotide sequence data. Analysis of total RNA extracted from M. catarrhalis ATCC 43617 cells grown under iron-replete and iron-restricted conditions was used to establish the utility of these DNA microarrays. These DNA microarrays were then used to analyze total RNA from M. catarrhalis cells grown in a continuous-flow biofilm system and in the planktonic state. The genes whose expression was most dramatically increased by growth in the biofilm state included those encoding a nitrate reductase, a nitrite reductase, and a nitric oxide reductase. Real-time reverse transcriptase PCR analysis was used to validate these DNA microarray results. These results indicate that growth of M. catarrhalis in a biofilm results in increased expression of gene products which can function not only in energy generation but also in resisting certain elements of the innate immune response.

Intrakingdom cell-to-cell communication and interkingdom cell-to-cell communication play essential roles in the virulence of enterohemorrhagic Escherichia coli (EHEC). Four signals, autoinducer 2 (AI-2), AI-3, and the human hormones epinephrine and norepinephrine, are important in this communication. The effect of these signaling compounds on the transcriptome of EHEC was examined in this study. We demonstrated that the luxS mutation affects primarily central metabolic genes in both pathogenic and nonpathogenic strains of E. coli and that addition of exogenous AI-2 does not fully restore the expression profile in a luxS-deficient strain lacking the ability to synthesize AI-2. Addition of AI-3 or epinephrine increased expression of the locus of enterocyte effacement regulon, which is known to play a pivotal role in EHEC virulence. Moreover, when epinephrine was added to the culture medium, the greatest number of gene alterations was observed. These alterations included a greater proportion of alterations in EHEC genes than in MG1655 genes, suggesting that epinephrine may be a global virulence signal. Detailed examination with real-time reverse transcriptase PCR (RT-PCR) confirmed the increases in virulence gene expression with addition of AI-3 and epinephrine. Additional studies with real-time RT-PCR examining the EHEC secreted effectors and putative fimbrial gene expression showed a variable expression profile, indicating that there is differential regulation of the secreted molecules. This study began to examine the global signaling networks in EHEC and revealed expression profiles that are signal and pathogen specific.

Francisella tularensis is a category A bioterror pathogen which in some cases can cause a severe and fatal human infection. Very few virulence factors are known in this species due to the difficulty in working with it as well as the lack of tools for genetic manipulation. This work describes the construction of a shuttle vector that can replicate in Escherichia coli and F. tularensis as well as two distinct promoter trap constructs based on the shuttle vector backbone. Replication in F. tularensis is based on the promiscuous origin of replication from the Staphylococcus aureus plasmid pC194. We demonstrate the novel plasmids can coexist with established F. tularensis vectors based on the pFNL10 plasmid, the current workhorse of F. tularensis genetics. Our promoter trap can identify promoters that are activated during intracellular growth and survival. These new vectors provide additional tools for the genetic manipulation of F. tularensis.

The first reported Far East scarlet-like fever (FESLF) epidemic swept the Pacific coastal region of Russia in the late 1950s. Symptoms of the severe infection included erythematous skin rash and desquamation, exanthema, hyperhemic tongue, and a toxic shock syndrome. The term FESLF was coined for the infection because it shares clinical presentations with scarlet fever caused by group A streptococci. The causative agent was later identified as Yersinia pseudotuberculosis, although the range of morbidities was vastly different from classical pseudotuberculosis symptoms. To understand the origin and emergence of the peculiar clinical features of FESLF, we have sequenced the genome of the FESLF-causing strain Y. pseudotuberculosis IP31758 and compared it with that of another Y. pseudotuberculosis strain, IP32953, which causes classical gastrointestinal symptoms. The unique gene pool of Y pseudotuberculosis IP31758 accounts for more than 260 strain-specific genes and introduces individual physiological capabilities and virulence determinants, with a significant proportion horizontally acquired that likely originated from Enterobacteriaceae and other soil-dwelling bacteria that persist in the same ecological niche. The mobile genome pool includes two novel plasmids phylogenetically unrelated to all currently reported Yersinia plasmids. An icm/dot type IVB secretion system, shared only with the intracellular persisting pathogens of the order Legionellales, was found on the larger plasmid and could contribute to scarlatinoid fever symptoms in patients due to the introduction of immunomodulatory and immunosuppressive capabilities. We determined the common and unique traits resulting from genome evolution and speciation within the genus Yersinia and drew a more accurate species border between Y. pseudotuberculosis and Y. pestis. In contrast to the lack of genetic diversity observed in the evolutionary young descending Y. pestis lineage, the population genetics of Y. pseudotuberculosis is more heterogenous. Both Y. pseudotuberculosis strains IP31758 and the previously sequenced Y. pseudotuberculosis strain IP32953 have evolved by the acquisition of specific plasmids and by the horizontal acquisition and incorporation of different genetic information into the chromosome, which all together or independently seems to potentially impact the phenotypic adaptation of these two strains., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Transcription of the locus of enterocyte effacement (LEE) genes in enterohemorrhagic Escherichia coli (EHEC) is regulated by the LEE-encoded Ler and GrlR/GrlA proteins as well as the non-LEE-encoded regulator QseA. This work demonstrates that GrlR/GrlA activate LEE2 transcription in a Ler-independent fashion, whereas transcription of grlRA is activated by QseA in both Ler-dependent and -independent manners.

Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were utilized in comparative genomic hybridization (CGH) analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis and four cystitis) isolates and three fecal/commensal strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found by CGH to be present in all UPEC strains but in none of the fecal/commensal strains. When the sequences of three additional sequenced UPEC strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains were identified. Seven previously unrecognized genomic islands (>30 kb) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the importance of horizontal transfer for UPEC and the mosaic structure of the genome. UPEC strains contain a greater number of iron acquisition systems than do fecal/commensal strains, which is reflective of the adaptation to the iron-limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC specific, strongly suggests that many urovirulence factors remain uncharacterized.

Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern.

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique "genomic islands" identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

The genetic relatedness of the Bacillus anthracis typing phages Gamma and Cherry was determined by nucleotide sequencing and comparative analysis. The genomes of these two phages were identical except at three variable loci, which showed heterogeneity within individual lysates and among Cherry, Wbeta, Fah, and four Gamma bacteriophage sequences.

Prochloron spp. are obligate cyanobacterial symbionts of many didemnid family ascidians. It has been proposed that the cyclic peptides of the patellamide class found in didemnid extracts are synthesized by Prochloron spp., but studies in which host and symbiont cells are separated and chemically analyzed to identify the biosynthetic source have yielded inconclusive results. As part of the Prochloron didemni sequencing project, we identified patellamide biosynthetic genes and confirmed their function by heterologous expression of the whole pathway in Escherichia coli. The primary sequence of patellamides A and C is encoded on a single ORF that resembles a precursor peptide. We propose that this prepatellamide is heterocyclized to form thiazole and oxazoline rings, and the peptide is cleaved to yield the two cyclic patellamides, A and C. This work represents the full sequencing and functional expression of a marine natural-product pathway from an obligate symbiont. In addition, a related cluster was identified in Trichodesmium erythraeum IMS101, an important bloom-forming cyanobacterium.

Members of the Bacillus cereus group of organisms include Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis. Collectively, these organisms represent microbes of high economic, medical and biodefense importance. Given this significance, this group contains the highest number of closely related fully sequenced genomes, giving the unique opportunity for thorough comparative genomic analyses. Much of the disease and host specificity of members of this group can be attributed to their plasmids, which vary in size and number. Chromosomes exhibit a high level of synteny and protein similarity, with limited differences in gene content, questioning the speciation of the group members. Genomic data have spurred functional studies that combined microarrays and proteomics. Recent advances are reviewed in this article and highlight the advantages of genomic approaches to the study of this important group of bacteria.

Background: The first microbial genome sequence, Haemophilus influenzae, was published in 1995. Since then, more than 400 microbial genome sequences have been completed or commenced. This massive influx of data provides the opportunity to obtain biological insights through comparative genomics. However few tools are available for this scale of comparative analysis., Results: The BLAST Score Ratio (BSR) approach, implemented in a Perl script, classifies all putative peptides within three genomes using a measure of similarity based on the ratio of BLAST scores. The output of the BSR analysis enables global visualization of the degree of proteome similarity between all three genomes. Additional output enables the genomic synteny (conserved gene order) between each genome pair to be assessed. Furthermore, we extend this synteny analysis by overlaying BSR data as a color dimension, enabling visualization of the degree of similarity of the peptides being compared., Conclusions: Combining the degree of similarity, synteny and annotation will allow rapid identification of conserved genomic regions as well as a number of common genomic rearrangements such as insertions, deletions and inversions. The script and example visualizations are available at: http://www.microbialgenomics.org/BSR/.

Sequencing and comparative genome analysis of four strains of Campylobacter including C. lari RM2100, C. upsaliensis RM3195, and C. coli RM2228 has revealed major structural differences that are associated with the insertion of phage- and plasmid-like genomic islands, as well as major variations in the lipooligosaccharide complex. Poly G tracts are longer, are greater in number, and show greater variability in C. upsaliensis than in the other species. Many genes involved in host colonization, including racR/S, cadF, cdt, ciaB, and flagellin genes, are conserved across the species, but variations that appear to be species specific are evident for a lipooligosaccharide locus, a capsular (extracellular) polysaccharide locus, and a novel Campylobacter putative licABCD virulence locus. The strains also vary in their metabolic profiles, as well as their resistance profiles to a range of antibiotics. It is evident that the newly identified hypothetical and conserved hypothetical proteins, as well as uncharacterized two-component regulatory systems and membrane proteins, may hold additional significant information on the major differences in virulence among the species, as well as the specificity of the strains for particular hosts.

Since the recognition of prokaryotes as essential components of the oceanic food web, bacterioplankton have been acknowledged as catalysts of most major biogeochemical processes in the sea. Studying heterotrophic bacterioplankton has been challenging, however, as most major clades have never been cultured or have only been grown to low densities in sea water. Here we describe the genome sequence of Silicibacter pomeroyi, a member of the marine Roseobacter clade (Fig. 1), the relatives of which comprise approximately 10-20% of coastal and oceanic mixed-layer bacterioplankton. This first genome sequence from any major heterotrophic clade consists of a chromosome (4,109,442 base pairs) and megaplasmid (491,611 base pairs). Genome analysis indicates that this organism relies upon a lithoheterotrophic strategy that uses inorganic compounds (carbon monoxide and sulphide) to supplement heterotrophy. Silicibacter pomeroyi also has genes advantageous for associations with plankton and suspended particles, including genes for uptake of algal-derived compounds, use of metabolites from reducing microzones, rapid growth and cell-density-dependent regulation. This bacterium has a physiology distinct from that of marine oligotrophs, adding a new strategy to the recognized repertoire for coping with a nutrient-poor ocean.