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PubMed2025

Genomic and transcriptomic insights into the virulence and adaptation of shock syndrome-causing Streptococcus anginosus.

Streptococcus anginosus is a common isolate of the oral cavity and an opportunistic pathogen for systemic infections. Although the pyogenic infections caused by S. anginosus are similar to those caused by Streptococcus pyogenes, S. anginosus lacks most of the well-characterized virulence factors of S. pyogenes. To investigate the pathogenicity of S. anginosus, we analysed the genome of a newly identified S. anginosus strain, KH1, which was associated with toxic shock-like syndrome in an immunocompetent adolescent. The genome of KH1 contains nine genomic islands, two Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated systems and many phage-related proteins, indicating that the genome is influenced by prophages and horizontal gene transfer. Comparative genome analysis of 355 S. anginosus strains revealed a significant difference between the sizes of the pan genome and core genome, reflecting notable strain variations. We further analysed the transcriptomes of KH1 under conditions mimicking either the oral cavity or the bloodstream. We found that in an artificial saliva medium, the expression of a putative quorum quenching system and pyruvate oxidase for H2O2 production was upregulated, which could optimize the competitiveness of S. anginosus in the oral ecosystem. Conversely, in a modified serum medium, purine and glucan biosynthesis, competence and bacteriocin production were significantly upregulated, likely facilitating the survival of KH1 in the bloodstream. These findings indicate that S. anginosus can utilize diverse mechanisms to adapt to different environmental niches and establish infection, despite its lack of toxin production.

Streptococcus anginosus↗
PubMed2006

Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome: isolation of unique sequences and demonstration of significant similarity to the chromosome of E. coli K-12.

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E. coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E. coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. blast searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E. coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain B7A, which is attenuated in virulence and lacked several H10407-specific sequences. Restriction-enzyme-based optical maps of strain H10407 were compared to in silico restriction maps of strain MG1655 and related E. coli pathogens. The 5.1 Mb ETEC chromosome was approximately 500 kb greater in length than the chromosome of E. coli K-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H10407 chromosome is expected to demonstrate approximately 96 % identity with that of E. coli K-12.

Bacteriophages↗
PubMed2003

Microarray analyses of Xylella fastidiosa provide evidence of coordinated transcription control of laterally transferred elements.

Genetically distinct strains of the plant bacterium Xylella fastidiosa (Xf) are responsible for a variety of plant diseases, accounting for severe economic damage throughout the world. Using as a reference the genome of Xf 9a5c strain, associated with citrus variegated chlorosis (CVC), we developed a microarray-based comparison involving 12 Xf isolates, providing a thorough assessment of the variation in genomic composition across the group. Our results demonstrate that Xf displays one of the largest flexible gene pools characterized to date, with several horizontally acquired elements, such as prophages, plasmids, and genomic islands (GIs), which contribute up to 18% of the final genome. Transcriptome analysis of bacteria grown under different conditions shows that most of these elements are transcriptionally active, and their expression can be influenced in a coordinated manner by environmental stimuli. Finally, evaluation of the genetic composition of these laterally transferred elements identified differences that may help to explain the adaptability of Xf strains to infect such a wide range of plant species.

Chromosome Mapping↗
PubMed2005

Integration and excision by the large serine recombinase phiRv1 integrase.

The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.

Attachment Sites, Microbiological↗
PubMed2005

Potential photosynthesis gene recombination between Prochlorococcus and Synechococcus via viral intermediates.

Genes (psbA and psbD) encoding for photosynthetically important proteins were recently found in a number of cultured cyanophage genomes. This phenomenon may be a beneficial trait to the viruses or their photosynthetic cyanobacterial hosts, or may represent an untapped pool of genes involved in the formation of the photosynthetic apparatus that are prone to lateral gene transfer. Here we show analyses of psbA genes from uncultured environmental viruses and prophage populations. We observe a statistically significant separation between viral genes and their potential Synechococcus hosts' genes, and statistical analyses under models of codon evolution indicate that the psbA genes of viruses are evolving under levels of purifying selection that are virtually indistinguishable from their hosts. Furthermore, our data also indicate the possible exchange and reshuffling of psbA genes between Synechococcus and Prochlorococcus via phage intermediates. Overall, these observations raise the possibility that marine viruses serve as a potential genetic pool in shaping the evolution of cyanobacterial photosynthesis.

Bacterial Proteins↗
PubMed2003

Holin locus characterisation from lysogenic Xenorhabdus nematophila and its involvement in Escherichia coli SheA haemolytic phenotype.

Lysogeny has previously been described in the entomopathogenic bacteria of the genus Xenorhabdus. Screening of a X. nematophila prophage DNA library on blood agar resulted in the identification of a 5.7-kb locus that caused a haemolytic phenotype when cloned in Escherichia coli, but not in the E. coli sheA null mutant, lacking the SheA cryptic haemolysin. This locus exhibited similarity to lysis genes from lambdoid phages. In particular, it encoded a functional holin able to complement a lambda Sam7 mutant. It is the second time that a locus encoding a functional holin is shown to reveal the SheA haemolytic phenotype in E. coli. The possible role of the holin in extracellular release of SheA is discussed.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of EspK, a type III secreted effector protein of enterohaemorrhagic Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea in humans and deploys a type III secretion system (T3SS) encoded by the locus of enterocyte effacement to elicit the formation of attaching and effacing (AE) lesions on intestinal epithelia. Here, we report the identification of a new secreted substrate of this system, z1829, which is encoded by cryptic prophage CP-933N. Elevated secretion of a beta-lactamase-z1829 fusion protein was detected upon mutation of sepD in EHEC O157:H7 and the fusion protein was translocated into infected epithelial cells in a T3SS-dependent manner; accordingly, we named the protein EspK. In common with the related Salmonella enterica type III secreted effector GogB, we observed that EspK localized to the cytoplasm when transiently expressed in COS-7 cells using EspK-specific antiserum. Inactivation of espK did not impair adherence or actin nucleation during infection of HeLa cells but affected persistence of EHEC O157:H7 in the intestines of orally inoculated calves. Inactivation of an orthologue of espK in the murine AE pathogen Citrobacter rodentium did not impair intestinal colonization in mice.

Animals↗
PubMed2006

Global molecular and morphological effects of 24-hour chromium(VI) exposure on Shewanella oneidensis MR-1.

The biological impact of 24-h ("chronic") chromium(VI) [Cr(VI) or chromate] exposure on Shewanella oneidensis MR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic characterization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (peptidoglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressed under conditions of 24-h chromate treatment.

Bacterial Proteins↗
PubMed2006

Characterization of the global transcriptional responses to different types of DNA damage and disruption of replication in Bacillus subtilis.

DNA damage and perturbations in DNA replication can induce global transcriptional responses that can help organisms repair the damage and survive. RecA is known to mediate transcriptional responses to DNA damage in several bacterial species by inactivating the repressor LexA and phage repressors. To gain insight into how Bacillus subtilis responds to various types of DNA damage, we measured the effects of DNA damage and perturbations in replication on mRNA levels by using DNA microarrays. We perturbed replication either directly with p-hydroxyphenylazo-uracil (HPUra), an inhibitor of DNA polymerase, or indirectly with the DNA-damaging reagents mitomycin C (MMC) and UV irradiation. Our results indicate that the transcriptional responses to HPUra, MMC, and UV are only partially overlapping. recA is the major transcriptional regulator under all of the tested conditions, and LexA appears to directly repress the expression of 63 genes in 26 operons, including the 18 operons previously identified as LexA targets. MMC and HPUra treatments caused induction of an integrative and conjugative element (ICEBs1) and resident prophages (PBSX and SPbeta), which affected the expression of many host genes. Consistent with previous results, the induction of these mobile elements required recA. Induction of the phage appeared to require inactivation of LexA. Unrepaired UV damage and treatment with MMC also affected the expression of some of the genes that are controlled by DnaA. Furthermore, MMC treatment caused an increase in origin-proximal gene dosage. Our results indicate that different types of DNA damage have different effects on replication and on the global transcriptional profile.

Bacillus Phages↗
PubMed2006

Genome rearrangements, deletions, and amplifications in the natural population of Bartonella henselae.

Cats are the natural host for Bartonella henselae, an opportunistic human pathogen and the agent of cat scratch disease. Here, we have analyzed the natural variation in gene content and genome structure of 38 Bartonella henselae strains isolated from cats and humans by comparative genome hybridizations to microarrays and probe hybridizations to pulsed-field gel electrophoresis (PFGE) blots. The variation in gene content was modest and confined to the prophage and the genomic islands, whereas the PFGE analyses indicated extensive rearrangements across the terminus of replication with breakpoints in areas of the genomic islands. We observed no difference in gene content or structure between feline and human strains. Rather, the results suggest multiple sources of human infection from feline B. henselae strains of diverse genotypes. Additionally, the microarray hybridizations revealed DNA amplification in some strains in the so-called chromosome II-like region. The amplified segments were centered at a position corresponding to a putative phage replication initiation site and increased in size with the duration of cultivation. We hypothesize that the variable gene pool in the B. henselae population plays an important role in the establishment of long-term persistent infection in the natural host by promoting antigenic variation and escape from the host immune response.

Animals↗
PubMed2006

DNA adenine methylation regulates virulence gene expression in Salmonella enterica serovar Typhimurium.

Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam(-) mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam(-) mutants in the mouse model may be caused by altered patterns of gene expression.

Bacterial Proteins↗
PubMed2003

Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18.

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.

Adenosine Triphosphatases↗
PubMed2004

Bacteriophage Tuc2009 encodes a tail-associated cell wall-degrading activity.

Tuc2009 is a P335-type member of the tailed-phage supergroup Siphoviridae and was originally identified as a resident prophage of the gram-positive bacterium Lactococcus lactis UC509. A Tuc2009 gene designated tal2009 which is located within the morphogenic module was shown to specify a lytic activity within the 3' portion of its coding region. Comparative sequence analysis indicated that the cell wall-degrading part of Tal2009 is a member of the M37 protein family and that Tal2009 lacks a cell-binding domain, a finding supported by binding studies. Tal2009 appears to undergo self-mediated posttranslational processing in both L. lactis and Escherichia coli. Antibodies directed against a purified C-terminal portion of Tal2009 were used for immunoelectron microscopy, which showed that Tal2009 is located at the tail tip of Tuc2009. Antibody neutralization studies demonstrated that Tal2009-directed antibodies inhibited the ability of phage to mediate host lysis by more than 100-fold. These data indicate that tal2009 encodes a tail-associated lysin involved in localized cell wall degradation, thus allowing the Tuc2009 DNA injection machinery access to the membrane of its bacterial host.

Catalysis↗
PubMed2004

Sequence tolerance of the phage lambda PRM promoter: implications for evolution of gene regulatory circuitry.

Much of the gene regulatory circuitry of phage lambda centers on a complex region called the O(R) region. This approximately 100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a lambda cloning vector that allowed us to clone fragments spanning the O(R) region. By using this vector, we carried out intensive mutagenesis of the P(RM) promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the -35 and -10 regions were randomized and cloned this pool into the vector, making a pool of P(RM) variant phage. About 10% of the P(RM) variants were able to lysogenize, suggesting that the lambda regulatory circuitry is compatible with a wide range of P(RM) sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.

Bacteriophage lambda↗
PubMed2005

The LEE1 promoters from both enteropathogenic and enterohemorrhagic Escherichia coli can be activated by PerC-like proteins from either organism.

The PerC protein of enteropathogenic Escherichia coli (EPEC), encoded by the pEAF plasmid, is an activator of the locus of enterocyte effacement (LEE) pathogenicity island via the LEE1 promoter. It has been assumed that the related LEE-containing pathogen enterohemorrhagic E. coli (EHEC) lacks PerC-dependent activation due to utilization of an alternative LEE1 promoter and lack of a perC gene. However, we show here that EPEC PerC can activate both the EPEC and EHEC LEE1 promoters and that the major transcriptional start site is similarly located in both organisms. Moreover, a PerC-like protein family identified from EHEC genome analyses, PerC1 (also termed PchABC), can also activate both promoters in a manner similar to that of EPEC PerC. The perC1 genes are carried by lambdoid prophages, which exist in multiple copies in different EHEC strains, and have a variable flanking region which may affect their expression. Although individual perC1 copies appear to be poorly expressed, the total perC1 expression level from a strain encoding multiple copies approaches that of perC in EPEC and may therefore contribute significantly to LEE1 activation. Alignment of the protein sequences of these PerC homologues allows core regions of the PerC protein to be identified, and we show by site-directed mutagenesis that these core regions are important for function. However, purified PerC protein shows no in vitro binding affinity for the LEE1 promoter, suggesting that other core E. coli proteins may be involved in its mechanism of activation. Our data indicate that the nucleoid-associated protein IHF is one such protein.

5' Flanking Region↗
PubMed2006

Novel mechanism of Escherichia coli porin regulation.

A novel mechanism of Escherichia coli porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated ipeX for inhibition of porin expression, and to DNA containing a 3'-truncated ompR gene. The major effect of ipeX on ompC expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced ompC transcription. ipeX was localized within an untranslated region of 247 base pairs between the stop codon of nmpC-a remnant porin gene from the cryptic phage qsr' (DLP12) genome-and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from lc identical to that of ipeX. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the lc transcript, whose 3' end contains the corresponding ipeX sequence, inhibits OmpC expression. Overexpression of ipeX RNA inhibited both OmpC and OmpF expression but not that of OmpA. ompC-phoA chimeric gene constructs revealed a 248-bp untranslated region of ompC required for ipeX-mediated inhibition. However, no sequence complementarity was found between ipeX and this region of ompC, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of ipeX on ompC, but not on ompF, was independent of the RNA chaperone Hfq.

Bacterial Proteins↗
PubMed1964

HOST RANGE OF CERTAIN VIRULENT AND TEMPERATE BACTERIOPHAGES ATTACKING GROUP D STREPTOCOCCI.

Brock, Thomas D. (Indiana University, Bloomington). Host range of certain virulent and temperate bacteriophages attacking group D streptococci. J. Bacteriol. 88:165-171. 1964.-Host range studies with five bacteriophages isolated from sewage showed that a high degree of strain specificity occurred among the species Streptococcus faecalis, S. faecalis var. zymogenes, and S. faecalis var. liquefaciens. Certain phages were able to attack various members of these subgroups but not other members. A high degree of strain specificity was also found with respect to eight phages attacking strains of S. faecium. Many strains of this latter group were resistant to all the phages available. The only firm conclusion was that no phage attacked both S. faecium and S. faecalis strains. In several selected strains of the S. faecalis-liquefaciens-zymogenes group, resistance to several virulent phages seemed to be related to the presence of certain prophages in these strains. A search for transduction with the temperate phages was unsuccessful. Mutants of S. faecium 8043 resistant to certain growth-factor analogues showed unpredictable changes in resistance and sensitivity to two virulent phages.

Bacteriophage Typing↗
PubMed2006

Predominance of clones carrying Panton-Valentine leukocidin genes among methicillin-resistant Staphylococcus aureus strains isolated in Japanese hospitals from 1979 to 1985.

We examined 97 strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated between 1979 and 1985, the period of time when the appearance of MRSA strains increased, and we determined that these strains are distinct from the MRSA clones predominating in today's Japanese hospitals. Type IV staphylococcal cassette chromosome mec (SCCmec) strains were the most frequent, comprising 53.6% of all strains, followed by type I (22.7%) and type II (21.6%) SCCmec strains. Among the type IV SCCmec strains, the frequencies of two new subtypes, type IV.3 (IVc) and type IV.4 (IVd), were very high, comprising 38.1 and 10.3% of all strains, respectively. Forty-four of the 97 strains (45.3%) were Panton-Valentine leukocidin (PVL) positive. Among the PVL-positive strains, sequence type 30 (ST30)-SCCmec type IV strains producing type 4 coagulase were the most frequent. This is in striking contrast to the MRSA strains isolated in the 1990s, most of which were ST5-SCCmec type II strains producing type 2 coagulase and positive for the toxic shock syndrome toxin 1 gene. We also identified a new PVL-carrying prophage lysogenized in a type IV.3 SCCmec strain, 81/108. phi108PVL was distinct from the three extant PVL-carrying phages and was presumed to be carried by ST30-type IV.3 SCCmec strains isolated in Japan. These results provide genetic bases for the transition of MRSA clones in Japan that is commonly considered the transition from coagulase type 4 MRSA strains to coagulase type 2 MRSA strains. The results also suggested that MRSA strains that predominated between 1979 and 1985 were generated from PVL-positive methicillin-susceptible S. aureus strains through the integration of SCCmec elements.

Bacterial Toxins↗