Search

Members of the Streptococcus bovis group are important causes of endocarditis. However, factors associated with their pathogenicity, such as adhesins, remain uncharacterized. We recently demonstrated that endocarditis-derived Streptococcus gallolyticus subsp, gallolyticus isolates frequently adhere to extracellular matrix (ECM) proteins. Here, we generated a draft genome sequence of an ECM protein-adherent S. gallolyticus subsp. gallolyticus strain and found, by genome-wide analyses, 11 predicted LPXTG-type cell wall-anchored proteins with characteristics of MSCRAMMs, including a modular architecture of domains predicted to adopt immunoglobulin (Ig)-like folding. A recombinant segment of one of these, Acb, showed high-affinity binding to immobilized collagen, and cell surface expression of Acb correlated with the presence of acb and collagen adherence of isolates. Three of the 11 proteins have similarities to major pilus subunits and are organized in separate clusters, each including a second Ig-fold-containing MSCRAMM and a class C sortase, suggesting that the sequenced strain encodes three distinct types of pili. Reverse transcription-PCR demonstrated that all three genes of one cluster, acb-sbs7-srtC1, are cotranscribed, consistent with pilus operons of other grampositive bacteria. Further analysis detected expression of all 11 genes in cells grown to mid to late exponential growth phases. Wide distribution of 9 of the 11 genes was observed among S. gallolyticus subsp, gallolyticus isolates with fewer genes present in other S. bovis group species/subspecies. The high prevalence of genes encoding putative MSCRAMMs and pili, including a collagen-binding MSCRAMM, among S. gallolyticus subsp, gallolyticus isolates may play an important role in the predominance of this subspecies in S. bovis endocarditis. doi: 10.1128/JB.00909-09

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformabUity of DH10B.

The draft genome sequence of Mannheimia haemolytica A1, the causative agent of bovine respiratory disease complex (BRDC), is presented. Strain ATCC BAA-410, isolated from the lung of a calf with BRDC, was the DNA source. The annotated genome includes 2,839 coding sequences, 1,966 of which were assigned a function and 436 of which are unique to M. haemolytica. Through genome annotation many features of interest were identified, including bacteriophages and genes related to virulence, natural competence, and transcriptional regulation. In addition to previously described virulence factors, M. haemolytica encodes adhesins, including the filamentous hemagglutinin FhaB and two trimeric autotransporter adhesins. Two dual-function immunoglobulin-protease/adhesins are also present, as is a third immunoglobulin protease. Genes related to iron acquisition and drug resistance were identified and are likely important for survival in the host and virulence. Analysis of the genome indicates that M. haemolytica is naturally competent, as genes for natural competence and DNA uptake signal sequences (USS) are present. Comparison of competence loci and USS in other species in the family Pasteurellaceae indicates that M. haemolytica, Actinobacillus pleuropneumoniae, and Haemophilus ducreyi form a lineage distinct from other Pasteurellaceae. This observation was supported by a phylogenetic analysis using sequences of predicted housekeeping genes.

The [gamma]-proteobacterium Francisella tularensis is one of the most infectious human pathogens, and the highly virulent organism F. tularensis subsp, tularensis (type A) and less virulent organism F. tularensis subsp. holarctica (type B) are most commonly associated with significant disease in humans and animals. Here we report the complete genome sequence and annotation for a low-passage type B strain (OSU18) isolated from a dead beaver found near Red Rock, Okla., in 1978. A comparison of the F. tularensis subsp, holarctica sequence with that of F. tularensis subsp, tularensis strain Schu4 (P. Larsson et al., Nat. Genet. 37:153-159, 2005) highlighted genetic differences that may underlie different pathogenicity phenotypes and the evolutionary relationship between type A and type B strains. Despite extensive DNA sequence identity, the most significant difference between type A and type B isolates is the striking amount of genomic rearrangement that exists between the strains. All but two rearrangements can be attributed to homologous recombination occurring between two prominent insertion elements, ISFtu1 and ISFtu2. Numerous pseudogenes have been found in the genomes and are likely contributors to the difference in virulence between the strains. In contrast, no rearrangements have been observed between the OSU18 genome and the genome of the type B live vaccine strain (LVS), and only 448 polymorphisms have been found within non-transposase-coding sequences whose homologs are intact in OSU18. Nonconservative differences between the two strains likely include the LVS attenuating mutation(s).

Enterococcus faecalis, a common cause of endocarditis and known for its capacity to transfer antibiotic resistance to other pathogens, has recently emerged as an important, multidrug-resistant nosocomial pathogen. However, knowledge of its lineages and the potential of particular clones of this species to disseminate and cause disease is limited. Using a nine-gene multilocus sequence typing (MLST) scheme, we identified an evolving and widespread clonal complex of E. faecalis that has caused outbreaks and life-threatening infections. Moreover, this unusual clonal complex was found to contain isolates of unexpected relatedness, including the first known U.S. vancomycin-resistant enterococcus (E. faecalis strain V583), the first known penicillinase-producing ([Bla.sup.+]) E. faecalis isolate, and the previously described widespread clone of penicillinase producers, a trait found in

RNA transcript levels in the syphilis spirochete Treponema pallidum subsp. pallidum (Nichols) isolated from experimentally infected rabbits were determined by the use of DNA microarray technology. This characterization of the T. pallidum transcriptome during experimental infection provides further insight into the importance of gene expression levels for the survival and pathogenesis of this bacterium.

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.

The cjrB and cjrC genes from Js-sensitive enteroinvasive Escherichia coli strains and from Shigella flexneri are similar with one or two base pair per gene difference between them. The genes encode identical proteins. The temperature and iron regulation of colicin Js uptake is mediated by VirB and Fur proteins respectively.

Research describes the details of the colicin Js system in Shigella sonnei 9colicin type 7). Results illustrate the structure of the colicin js coding region on plasmid ColJs, colicin activity gene identification, and Js polypeptide molecular characterization.

Results demonstrate that fsrB gene is essential for fsr regulator's functions and the expression of fsrA, fsrB, fsrC, gelE, and sprE promoters is directed by three different promoters. The expression of fsrB and fsrC is dependent on cell density. Two repeats of 7-basepair length regulate fsrB and gelE expression.

Treponema pallidum is different from other spirochetes because it contains cytoplasmic filaments underneath the cytoplasmic membrane. Cloning and expression of the cytoplasmic filament protein gene cfpA of Treponema pallidum subsp. pallidum Nichols identified three components of the gene: a 2,034-nucleotide coding region; a putative promoter region; and a putative ribosome-binding site. Sequence analysis indicated a protein of 678 residues with a molecular mass of 78.5 kilodaltons and an isoelectric potential of 6.15. It was suggested that T. pallidum cytoplasmic filaments may be a unique form of prokaryotic intracytoplasmic inclusions.

Increase in the expression of cea-lacZ fusions and production of colicin E1 in bacteria during the stationary phase of the cells are caused by the lack of nutrients from the medium. Expression of the fusion increased when cells were starved in 10 mM MgSO4 and when they were grown in conditioned medium in which cells have been grown previously. The increase is not due to SOS response, anaerobiosis, catabolite repression, integration host factor and the stationary-phase regulators encoded by rpoS and lrp.

A novel method is used to compare the pairwise structures of RecA protein sequences from 62 eubacterial sources. Results show that the eubacterial sources can be classified into two major groups, those of mostly vertebrate hosts and those of mostly soil habitat. The data also suggests that the bacteriophage T4 UvX protein aligns best with RecA sequences on two segments disjoint from the ATP-binding domain.

The feasibility of using allelic replacement to generate enterococcal pyr and pur auxotrophs of Enterococcus faecalis is evaluated by a combination of DNA techniques, mutagenesis, restriction mapping and subcloning. Results indicate that the preposed allelic replacement method is very useful for generating enterococcal pur and pyr auxotrophs which can be used infections caused by Enterococcus faecalis.

Rhodobacter sphaeroides strain 2.4.1deltaS mutagenized with a derivative of Tn5 produced eight UV-sensitive strains. Contour-clamped homogeneous electric field pulsed-field gel electrophoresis of the strains revealed at least five insertion sites in chromosome I, which were assumed to lack a DNA repair mechanism. DNA sequence analysis of these mutants confirmed a deficiency in three loci, which showed insertions to be within genes for subunits of the UV damage excision repair.

Restriction fragments produced by NotI, SfiI and SrfI formulated a physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols) of the length of approximately 1,030 to 1,080 kbp is size, which is the causative agent of syphilis. This restriction cleaved the T. pallidum genome in to 16,8 and 15 fragments. The process of construction of this physical map is discussed.

Auxotrophic mutants aid the analysis of the structure and function of chromosomes I and II of Rhodobacter sphaeroides 2.4.1T. The presence of histidine, thymine and p-aminobenzoic acid (pABA)-dihydroxybenzoic acid (dhBA) at low frequencies causes a return to prototrophy. Cosmids possessing the region of transposon location substitute the serine, uracil and pABA-dHBA mutants. The conclusions obtained from these experiments indicate a similarity among chromosomes in bacteria.

Amino acid requirements of Enterococcus faecalis OG1 are determined by a defined synthetic medium, which is used to reveal the unnecessary exogeneous purines or the pyrimidines that are absent. Genomic libraries prepared from strain OG1 are transduced into Escherichia coli auxotrophic mutants, and cloned DNAs with known functions are localized on the restriction map of OG1. The OG1 is generated using the SfiI, AscI and NotI fragments. The size of the OG1 chromosome is revised from the previous value of 2750kb to 2825kb.

The ColE1 plasmid cea gene codes for colicin E1, and is the first gene in an operon whose regulation is complex. The effects of anaerobiosis on the expression of cea was studied using cea-lacZ gene fusions. The results showed that the expression of cea increased by as much as 45-fold under anaerobic conditions. Both transcript and protein levels increased, indicating that the stimulation occured at a transcriptional level. The anaerobic expression of cea also required the SOS response, catabolite expression and the Fnr regulatory function.

A genetic map based on NotI restriction sites in Escherichia coli strain MG1655 was constructed. The NotI sites were placed on the genetic and physical map using several approaches which involved the use of transposons to introduce auxotrophic markers and NotI sites, Southern hybridization using NotI fragments as probes on an ordered lambda library, hybridization using known clones as probes on NotI digests and analysis of sequenced sites for NotI recognition sites. Comparisons of the derived map with maps of other E. coli strains showed some differences, but indicated an overall consistency.

Presents novel details about colicin J, plasmid-encoded toxic exoproteins that are produced by calicinogenic strains of Escherichia coli and some related species of Enterobactriaceae. Structure of the colicin Js coding region on plasmid ColJs; Identification of genes for colicin activity, immunity and release; Molecular characterization of the Js peptide.

Tests the possibility of generating enterococcal pyr and pur auxotrophs by allelic replacement. Bacterial strains, plasmids and phage; Media; Routine DNA techniques; Complementation; Transposon mutagenesis; Restriction mapping and subcloning; Allelic replacement; Growth curves.

Reports that the two genes in fsr locus, fsrB and fsrC, in Esterococcus faecalis are highly expressed in the post-exponential growth phase and that their expression is dependent on cell density. Control of gelatinase (gelE) and serine protease expression (ptrE); Bacterial strains and plasmids; DNA sequencing and sequence analysis; Deletion mutagenesis.

The DNA sequences of chromosomes I and II of Rhodobactersphaeroides strain 2.4.1 have been revised, and the annotation of the entire genomic sequence, including both chromosomes and the five plasmids, has been updated. Errors in the originally published sequence have been corrected, and ~ 11% of the coding regions in the original sequence have been affected by the revised annotation. [ABSTRACT FROM AUTHOR]