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G M Weinstock

Publications and source records attributed to G M Weinstock.

At least 19 recordsLinked to original sources
PubMed2006

Comparative analysis of the bovine MHC class IIb sequence identifies inversion breakpoints and three unexpected genes.

The bovine major histocompatibility complex (MHC) or BoLA is organized differently from typical mammalian MHCs in that a large portion of the class II region, called class IIb, has been transposed to a position near the centromere on bovine chromosome 23. Gene mapping indicated that the rearrangement resulted from a single inversion, but the boundaries and gene content of the inverted segment have not been fully determined. Here, we report the genomic sequence of BoLA IIb. Comparative sequence analysis with the human MHC revealed that the proximal inversion breakpoint occurred approximately 2.5 kb from the 3' end of the glutamate-cysteine ligase, catalytic subunit (GCLC) locus and that the distal breakpoint occurred about 2 kb from the 5' end from a divergent class IIDRbeta-like sequence designated DSB. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared with the corresponding region of the human class II MHC (HLA class II). Differences with HLA include the presence of a single histone H2B gene located between the proteasome subunit, beta type, 9 (PSMB9) and DMB loci and a duplicated TAP2 with a variant splice site. BoLA IIb spans approximately 450 kb DNA, with 20 apparently intact genes and no obvious pseudogenes. The region contains 227 simple sequence repeats (SSRs) and approximately 167 kb of retroviral-related repetitive DNA. Nineteen of the 20 genes identified in silico are supported by bovine EST data indicating that the functional gene content of BoLA IIb has not been diminished because it has been transposed from the remainder of BoLA genes.

Animals↗
PubMed2006

Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis.

Genome sequences offer a broad view of host-pathogen interactions at the systems biology level. With the completion of the sequence of the honey bee, interest in the relevant pathogens is heightened. Here we report the genome sequences of two of the major pathogens of honey bees, the bacterium Paenibacillus larvae (causative agent for American foulbrood disease) and the fungus Ascosphaera apis. (causative agent for chalkbrood disease). Ongoing efforts to characterize the genomes of these species can be used to understand and mitigate the effects of two important pathogens, and will provide a contrast with pathogenic, benign and freeliving relatives.

Animals↗
PubMed2003

The Escherichia fergusonii iucABCD iutA genes are located within a larger chromosomal region similar to pathogenicity Islands.

Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.

Bacterial Outer Membrane Proteins↗
PubMed2001

Comparing vertebrate whole-genome shotgun reads to the human genome.

Multi-species sequence comparisons are a very efficient way to reveal conserved genes. Because sequence finishing is expensive and time consuming, many genome sequences are likely to stay incomplete. A challenge is to use these fragmented data for understanding the human genome. Methods for using cross-species whole-genome shotgun sequence (WGS) for genome annotation are described in this paper. About one-half million high-quality rat WGS reads (covering 7.5% of the rat genome) generated at the Baylor College of Medicine Human Genome Sequencing Center were compared with the human genome. Using computer-generated random reads as a negative control, a set of parameters was determined for reliable interpretation of BLAST search results. About 10% of the rat reads contain regions that are conserved in the human genomic sequence and about one-third of these include known gene-coding regions. Mapping the conserved regions to human chromosomes showed a 23-fold enrichment for coding regions compared with noncoding regions. This approach can also be applied to other mammalian genomes for gene finding. These data predicted approximately 42,500 genes in the human, slightly more than reported previously.

Animals↗
PubMed2001

Characterization of emeA, a NorA homolog and multidrug resistance efflux pump, in Enterococcus faecalis.

We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).

Amino Acid Sequence↗
PubMed2001

Characterization of fsr, a regulator controlling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF.

We have previously identified a locus, fsr, a homologue of staphylococcal agr loci, which positively regulates the expression of gelatinase and serine protease (encoded by gelE and sprE, respectively) in Enterococcus faecalis OG1RF. The expression of the three genes in the fsr locus, fsrA, fsrB, and fsrC, appears to be autoregulated, and we have shown that mutants with insertion disruptions in each of these three genes were significantly attenuated in a mouse peritonitis model compared to the parent strain. In the present study, we showed that fsrB and fsrC are highly expressed in the postexponential growth phase and that their expression is cell density dependent. Reverse transcriptase PCR using primers covering the intergenic regions in the fsr/gelE loci confirmed that fsrB and fsrC, as well as gelE and sprE, are cotranscribed. We also showed, using a nonpolar fsrB deletion mutant, that fsrB, the homologue of agrB of staphylococci with unknown function, is required for the regulatory function of fsr. Primer extension and analysis of transcriptional fusions indicated the presence of promoters immediately upstream of fsrA, of fsrB, and of gelE and that the fsrB and gelE promoters are fsr dependent, while the fsrA promoter is an fsr-independent weak constitutive promoter. Two conserved 7-bp direct repeats were found immediately upstream of the fsrB and gelE promoters, similar to the repeats found upstream of P2 and P3 promoters of the agr locus; deletions and mutations in the repeated sequences completely abolished the fsrB and gelE promoter activities, suggesting that the repeats are important for the regulatory function in the fsrB and gelE promoter regions.

Amino Acid Sequence↗
PubMed2001

Genetic organization of plasmid ColJs, encoding colicin Js activity, immunity, and release genes.

The 5.2-kb ColJs plasmid of a colicinogenic strain of Shigella sonnei (colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the rep, mob, and cer regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (cja), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (cji) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (cjl) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the cjl gene was found upstream from cja. Moreover, the promoter upstream from cjl was similar to promoters described upstream from several colicin activity genes. The cji gene was found to be located downstream from cja with a transcription polarity opposite to that of the cjl and cja genes. The cja, cji, and cjl genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus.

Bacterial Proteins↗
PubMed2001

The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake.

A cosmid library of DNA from colicin Js-sensitive enteroinvasive Escherichia coli (EIEC) strain O164 was made in colicin Js-resistant strain E. coli VCS257, and colicin Js-sensitive clones were identified. Sensitivity to colicin Js was associated with the carriage of a three-gene operon upstream of and partially overlapping senB. The open reading frames were designated cjrABC (for colicin Js receptor), coding for proteins of 291, 258, and 753 amino acids, respectively. Tn7 insertions in any of them led to complete resistance to colicin Js. A near-consensus Fur box was found upstream of cjrA, suggesting regulation of the cjr operon by iron levels. CjrA protein was homologous to iron-regulated Pseudomonas aeruginosa protein PhuW, whose function is unknown; CjrB was homologous to the TonB protein from Pseudomonas putida; and CjrC was homologous to a putative outer membrane siderophore receptor from Campylobacter jejuni. Cloning experiments showed that the cjrB and cjrC genes are sufficient for colicin Js sensitivity. Uptake of colicin Js into sensitive bacteria was dependent on the ExbB protein but not on the E. coli K-12 TonB and TolA, -B, and -Q proteins. Sensitivity to colicin Js is positively regulated by temperature via the VirB protein and negatively controlled by the iron source through the Fur protein. Among EIEC strains, two types of colicin Js-sensitive phenotypes were identified that differed in sensitivity to colicin Js by 1 order of magnitude. The difference in sensitivity to colicin Js is not due to differences between the sequences of the CjrB and CjrC proteins.

Bacterial Proteins↗
PubMed2001

Biology of Treponema pallidum: correlation of functional activities with genome sequence data.

Aspects of the biology of T. pallidum subsp. pallidum, the agent of syphilis, are examined in the context of a century of experimental studies and the recently determined genome sequence. T. pallidum and a group of closely related pathogenic spirochetes have evolved to become highly invasive, persistent pathogens with little toxigenic activity and an inability to survive outside the mammalian host. Analysis of the genome sequence confirms morphologic studies indicating the lack of lipopolysaccharide and lipid biosynthesis mechanisms, as well as a paucity of outer membrane protein candidates. The metabolic capabilities and adaptability of T. pallidum are minimal, and this relative deficiency is reflected by the absence of many pathways, including the tricarboxylic acid cycle, components of oxidative phosphorylation, and most biosynthetic pathways. Although multiplication of T. pallidum has been obtained in a tissue culture system, continuous in vitro culture has not been achieved. The balance of oxygen utilization and toxicity is key to the survival and growth of T. pallidum, and the genome sequence reveals a similarity to lactic acid bacteria that may be useful in understanding this relationship. The identification of relatively few genes potentially involved in pathogenesis reflects our lack of understanding of invasive pathogens relative to toxigenic organisms. The genome sequence will provide useful raw data for additional functional studies on the structure, metabolism, and pathogenesis of this enigmatic organism.

Animals↗
PubMed2001

Enterococcus faecalis multi-drug resistance transporters: application for antibiotic discovery.

Using bioinformatics approaches, 34 potential multidrug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both with single disruption mutants and with 2-gene mutants built upon a delta norA deleted strain background.

ATP-Binding Cassette Transporters↗
PubMed2000

From microbial genome sequence to applications.

Whole genome sequences of microbial pathogens present new opportunities for clinical applications. Chief among these are development of antimicrobials, diagnostics, and vaccines. While antimicrobial development is a more difficult, long-term prospect, new diagnostics and vaccines are likely to be the first products of microbial genomics. To take advantage of whole genome sequences, methods for production of gene products in surrogate hosts (heterologous expression) are required that will work for large-scale, high-throughput gene expression. This will allow genomic information from even the most experimentally difficult pathogens to be mined for applications. In addition, screening methods to test gene products for their potential as vaccine candidates are needed for large-scale screening. These areas for technological development should be stimulated by the potential for converting genomic sequence information into applications.

Animals↗
PubMed2000

Analysis of a gene cluster of Enterococcus faecalis involved in polysaccharide biosynthesis.

Previously, we described a gene cluster of Enterococcus faecalis OG1RF that produced an antigenic polysaccharide when cloned in Escherichia coli. The polysaccharide antigen was not detectable in E. faecalis strains, however. Here, we show by reverse transcriptase-PCR that the 16 genes in this region are transcribed in OG1RF. Gene disruption of orfde4, encoding a putative glycosyl transferase, and orfde6, a putative dTDP-rhamnose biosynthesis gene, generated two OG1RF mutants. The mutants showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. In addition, two mucoid E. faecalis isolates from patients with chronic urinary tract infections were found to produce the polysaccharide antigen.

Animals↗
PubMed2000

Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence.

Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene regulator system in the agr locus), FsrB shows 23% identity and 41% similarity to S. aureus AgrB, and FsrC shows 23% identity and 36% similarity to S. aureus AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelE and sprE are cotranscribed and that fsrB and fsrC are also cotranscribed in OG1RF. Northern blot analysis of fsrA, fsrB, fsrC, gelE, and sprE insertion mutants showed that fsrB, fsrC, gelE, and sprE are not expressed in fsrA, fsrB, and fsrC mutants, while insertion in an open reading frame further upstream of fsrA did not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelE and sprE expression, as further confirmed by complementation of fsr gene mutations with a 6-kb fragment which contains all three fsr genes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalis showed that 62% produced gelatinase (Gel(+)), while 91% (including all Gel(+) strains) hybridized to a gelE probe; 71% (including all Gel(+) strains) hybridized to an fsr probe, corroborating the conclusion that both gelE and fsr are necessary for gelatinase production. Testing of fsrA, fsrB, and sprE mutants in a mouse peritonitis model showed that sprE and agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a gelE mutant. These results suggest that sprE and agr-like genes contribute to the virulence of E. faecalis OG1RF in this model.

Amino Acid Sequence↗
PubMed2000

Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of ace during human infections.

Our previous work reported that most Enterococcus faecalis strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46 degrees C, but not 37 degrees C, and we subsequently identified an E. faecalis sequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific ace gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalis strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5' region of ace that encodes the A domain was sequenced, and these sequences showed > or =97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived from E. faecalis strain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalis strains after growth at 46 degrees C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients with E. faecalis endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalis antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalis endocarditis patient serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to collagen types I and IV and laminin. In conclusion, these results show that ace is highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.

Amino Acid Sequence↗
PubMed2000

Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I.

Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46 degrees C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46 degrees C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single approximately 105-kDa band of mutanolysin extracts from OG1RF grown at 46 degrees C, while no band was detected in extracts from OG1RF grown at 37 degrees C, nor from the OG1RF ace mutant grown at 37 or 46 degrees C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 microg/ml, and also inhibited the 46 degrees C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.

Animals↗
PubMed1999

Ace is a collagen-binding MSCRAMM from Enterococcus faecalis.

A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174-319) suggested a structure very similar to that reported for residues 151-318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a beta-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833-838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.

Adhesins, Bacterial↗