Showing papers by "George M. Weinstock published in 1998"

Complete Genome Sequence of Treponema pallidum, the Syphilis Spirochete

Abstract: The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.

Generation and Testing of Mutants of Enterococcus faecalis in a Mouse Peritonitis Model

Abstract: A previously described mouse peritonitis model was used to study derivatives of Enterococcus faecalis strain OG1RF. The addition of sterile rat fecal extracts (SRFE) lowered the LD50 of OG1RF >10-fold. Hemolysin production caused a 35-fold lower LD50 and a much shorter survival, similar to previous results using a peritonitis model without SRFE. A purine (but not a pyrimidine) auxotroph was considerably less lethal than wild type; gelatinase mutants were also attenuated. A suicide vector was generated with an enterococcal selectable marker in order to disrupt a gene encoding an E. faecalis antigen; the resulting mutant was not attenuated despite a slower growth rate. In conclusion, this model allows attenuated mutants to be detected, corroborates prior reports that hemolysin is a virulence factor, and suggests a role for gelatinase in virulence of E. faecalis in mice; the attenuated purine auxotroph may provide a system for developing vectors for in vivo expression systems.

In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification

Abstract: Disruption of the previously described efaA (from Enterococcus faecalis antigen A) gene was generated in E. faecalis strain OG1RF and loss of an ∼37-kDa immunoreactive band from the mutant was demonstrated in Western blots. In a mouse peritonitis model, mice infected with the efaAfs (fs=from Enterococcus faecalis) mutant showed more prolonged survival than mice infected with the parent strain OG1RF. These results suggest that efaAfs encodes a function important for infection of mice by enterococci. An efaA-like gene was also identified in E. faecium DNA libraries and its deduced amino acid sequence showed 73% similarity to EfaA of E. faecalis and 42–63% similarities to a group of streptococcal virulence and adhesion associated proteins that are components of ATP-binding cassette transport systems. Intragenic probes representing efaAfs, recAfs, efaAfm (fm=from E. faecium) and gyrAfm were tested for their ability to identify E. faecalis and E. faecium using colony lysates of 133 enterococci and one Streptococcus sp. Probes of E. faecium and E. faecalis origin hybridized to all isolates of E. faecium and E. faecalis, respectively, regardless of their clinical source but not to any of 29 other enterococci. These results suggest that the use of gene probes may prove helpful in identification of isolates of E. faecium and E. faecalis.

Bacterial genomes : physical structure and analysis

Abstract: Part One: Genome Structure, Stability, and Evolution. Genome structure. Genome stability. Genome evolution. Part Two: Strategies for Genome Analysis. Physical mapping strategies. Genomic fingerprinting methods. Genomic sequencing projects. Part Three: Physical Maps of Bacteria and Their Methods for Construction. Index.

A Cluster of Genes Involved in Polysaccharide Biosynthesis from Enterococcus faecalis OG1RF

Abstract: Our previous work identified a cosmid clone containing a 43-kb insert from Enterococcus faecalis OG1RF that produced a nonprotein antigen in Escherichia coli. In the present work, we studied this clone in detail. Periodate treatment of lysates of the clone confirmed that the antigen was carbohydrate in nature. Analysis of DNA sequences and transposon insertion mutants suggested that the insert contained a multicistronic gene cluster. Database comparison showed that the cluster contained genes similar to genes involved in the biosynthesis of dTDP-rhamnose, glycosyltransferases, and ABC transporters involved in the export of sugar polymers from both gram-positive and gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of the clone. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide.

Repetitive Sequence-Based PCR versus Pulsed-Field Gel Electrophoresis for Typing of Enterococcus faecalis at the Subspecies Level

Abstract: Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate Enterococcus faecalis isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in Streptococcus pneumoniae, was applied to 41 isolates of E. faecalis collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were β-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.

The genome of Treponema pallidum: new light on the agent of syphilis

Abstract: Treponema pallidum subsp, pallidum, the causative agent of the sexually transmitted disease syphilis, is a fastidious, microaerophilic obligate parasite of humans. This bacterium is one of the few prominent infectious agents that has not been cultured continuously in vitro and consequently relatively little is known about its virulence mechanisms at the molecular level. T. pallidum therefore represented an attractive candidate for genomic sequencing. The complete genome sequence of T. pallidum has now been completed and comprises 1,138,006 base pairs containing 1041 predicted protein coding sequences. An important goal of this project is to identify possible virulence factors. Analysis of the genome indicates a number of potential virulence factors including a family of 12 proteins related to the Msp protein of Treponema denticola, a number of putative hemolysins, as well as several other classes of proteins of interest. The results of this analysis are reviewed in this article and indicate the value of whole genome sequences for rapidly advancing knowledge of infectious agents.

Effect of Disruption of a Gene Encoding an Autolysin of Enterococcus faecalis OG1RF

Abstract: A mutant (TX5127) of Enterococcus faecalis OG1RF was generated by disruption mutagenesis of a previously described autolysin gene. TX5127 formed longer chains (2 to 10 cells per chain) than wild-type OG1RF (mainly single cells) during growth in broth even though it had a growth rate similar to that of the parental strain as measured by turbidity and cell count. Autolysin activity, as defined by the ability to lyse heat-killed Micrococcus lysodeikticus cells, was absent in TX5127, while this activity was easily detectable in OG1RF. However, disruption of this autolysin gene did not block the ability of TX5127 to hydrolyze E. faecalis cell walls compared to that of OG1RF. The autolysis rate of cells of TX5127 in 10 mM sodium phosphate buffer (pH 6.8) was slower than that of wild-type OG1RF. TX5127 also showed a decreased rate of lysis in the presence of penicillin, as measured by changes in the turbidity of the culture during 24 h of incubation at 37°C and a slightly decreased effect of penicillin as measured by time-kill curves. The virulence of TX5127 was similar to that of OG1RF in the mouse peritonitis model, indicating that the autolysin of E. faecalis is not important for infection in this model.

Conditional adherence of Enterococcus faecalis to extracellular matrix proteins

Abstract: The adherence of 44 clinical isolates of Enterococcus faecalis, a common cause of endocarditis, and 13 Enterococcus faecium to substrates of six extracellular matrix (ECM) proteins was examined using 35S-labeled bacteria. One E. faecalis strain, isolated from a patient with endocarditis, adhered to collagen types I and IV and another E. faecalis strain adhered to laminin and to collagen types I and IV. However, most isolates showed little adherence (<5% of added cells adhered) when grown at 37°C regardless of their source (endocarditis, urine or fecal sample). When grown at 46°C (but not when grown in CO2 or nutrient limited media), most isolates of E. faecalis increased their adherence to immobilized laminin, collagen types I and IV but not to fibronectin, fibrinogen or bovine serum albumin, whereas none of the E. faecium increased adherence when grown at 46°C or 50°C. The adherence of E. faecalis was eliminated by digestion with trypsin, suggesting that a protein is somehow important, directly or indirectly, for adherence to occur. Pre-incubation of bacteria with soluble collagen types I and IV inhibited the adherence to these ECM proteins. These results demonstrate that in E. faecalis, adherence to ECM proteins is produced during routine in vitro growth conditions by occasional isolates and can be produced during certain stressful growth conditions by others. Whether this adherence relates to the propensity of E. faecalis to cause endocarditis remains to be determined.

Illuminating the agent of syphilis: the Treponema pallidum genome project.

Abstract: As the causative agent of the common sexually transmitted disease syphilis and a fastidious, microaerophilic obligate parasite of humans, Treponema pallidum subsp. pallidum is one of the few prominent infectious agents that has not been cultured continuously in vitro. T. pallidum therefore represents an attractive candidate for genomic sequencing. Preliminary sequence results from the 1.13 million base pair genome are consistent with the expected limited metabolic capabilities of this spirochete, but indicate that the bacterium may express toxins and surface proteins that have not been identified previously.

Targeted mutagenesis of enterococcal genes

Abstract: A series of methods was developed for targeted mutagenesis in enterococci. First, a transposon mutagenesis system, miniγδ-200 (mγδ), which was used previously to make insertion mutants in streptococci, was shown to be useful for generation of mutants in enterococci. After mutagenesis of cosmid clones carrying enterococcal DNA inserts in Escherichia coli with mγδ, we were able to isolate the mutants by phenotype or to screen for them by immunoblotting or comparison of restriction diges­tion patterns. Clones with mγδ insertions in targeted enterococcal genes were then introduced into enterococci by electroporation to generate targeted disruption mutations. Allelic replacement en masse, that is, electroporation of enterococci with DNA from a pool of mutagenized cosmid clones, was shown to be an efficient method to obtain mutations in genes with detectable phenotypes in enterococci. We next constructed a vector for mutagenesis using small intragenic fragments of enterococcal genes to disrupt targeted genes. This vector was modified from pBluescript SK (—) by cloning the kanamycin resis­tance determinant from mγδ into the ScaI site internal to the ampicillin resistance gene. It was then used to generate insertion mutants in Enterococcus faecalis with an intragenic fragment as small as about 500 bp. A third method, based on the conjugation system reported by Trieu-Cuot et al. [1], was devel­oped in order to circumvent difficulties in the elec­troporation of some enterococcal strains and to improve the efficiency with which targeted mutations can be generated in enterococci. This system was capable of mobilizing both small plasmids and large cosmids into enterococci by conjugation, and produced disruption mutations by homologous recombination.

Characterization of RecA1332 in vivo and in vitro. A role for alpha-helix E as a liaison between the subunit-subunit interface and the DNA and ATP binding domains of RecA protein.

Abstract: Background: The RecA protein of Escherichia coli is essential for homologous recombination and induction of the SOS response. RecA has three cysteines located at positions 90, 116 and 129. Chemical modification of these residues abolishes ATP hydrolysis and repressor cleavage, and causes a reduction in the DNA strand exchange and DNA strand annealing activities. Several mutants at each of these positions were isolated and partially characterized. One of these, recA1332, replaces cysteine 129 with methionine. Although this is a relatively conservative mutation based on hydrophobicity, recA1332 was completely defective for DNA repair but the purified protein was active for ATPase in vitro. Results: In vivo, strains containing this mutant allele were shown to be defective when assayed for all RecA-dependent activities. In vitro, RecA1332 protein possessed DNA-dependent ATP hydrolysis activity that showed an increased sensitivity to inhibition by monovalent cations, and whose kcat was reduced 3- to 12-fold. In addition, RecA1332 was unable to use oligodeoxyribonulceotides as ssDNA cofactors in the ATPase reaction. RecA1332 showed altered binding to single- and double-stranded DNA and, although it was able to perform DNA strand exchange, it was slowed in its ability to both form joint molecule intermediates and to convert these species to product. Conclusions: Our results are consistent with a defect in intermolecular interactions between RecA monomers. We propose that α-helix E (which includes C129M) is a liaison that connects the subunit–subunit interactions to DNA and ATP binding, thereby creating filament stability and cooperativity.

UTP is a cofactor for the DNA strand exchange reaction performed by the RecA protein of Escherichia coli.

Abstract: The RecA protein of Escherichia coli is required for homologous genetic recombination and induction of the SOS regulon. In order for RecA protein to function in these two roles, a nucleoside triphosphate cofactor, usually ATP or dATP, is required. We have examined the ability of UTP to substitute for (r,d)ATP as nucleoside triphosphate cofactor. We have found that although UTP is hydrolyzed by RecA protein in the presence of long DNA molecules, it is not hydrolyzed in reactions in which the cofactors are oligodeoxyribonucleotides less than ∼50 nt in length. We show that UTP can efficiently substitute for ATP as nucleoside triphosphate cofactor for the DNA strand exchange reaction in vitro. The RecA1332 protein (Cys129 → Met), which was originally shown to be defective for homologous recombination in vivo, is able to perform DNA strand exchange in vitro with ATP, but is unable to do so with UTP. These results suggest that UTP may be a cofactor for DNA strand exchange in vivo. The inability of RecA protein ...

Sequence Skimming of Chromosome II of Rhodobacter sphaeroides 2.4.1 T

Abstract: Rhodobacter sphaeroides is a photosynthetic member of the α-3 subgroup of Gram-negative bacteria (Woese, 1987). It is distinguished by a number of important characteristics which include at least six modes of growth. These accompany the ability of the organism to display a diverse range of metabolic activities, as well as other notable characteristics with respect to genome organization, evolution, and other processes (Table 43-1). This metabolic diversity may have evolved from a need to synthesize de novo a different range of compounds under each set of growth conditions.

Isolation of enterococcal antigen-encoding genes from genomic libraries

Abstract: We have devised a procedure using immune sera to identify antigen-encoding genes of strains of Enterococcus faecalis. First, genomic cosmid libraries containing large inserts were constructed and screened with sera from patients with enterococcal infectious endocarditis and with serum from a rabbit immunized with surface proteins of an enterococcal endocarditis isolate. Immunopositive cosmid clones were analyzed by restriction enzyme digestions and clones containing distinct inserts were chosen for subcloning. Sublibraries were screened with one of the five sera, and immunopositive subclones were subjected to DNA sequencing. BLASTX and BLASTN at NCBI were used to search for database similarities.