Showing papers by "George M. Weinstock published in 2001"

Initial sequencing and analysis of the human genome.

Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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

Abstract: 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.

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

Abstract: 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).

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

Genetic Organization of Plasmid ColJs, Encoding Colicin Js Activity, Immunity, and Release Genes

Abstract: Colicins are plasmid-encoded toxic exoproteins that are produced by colicinogenic strains of Escherichia coli and some related species of the family Enterobacteriaceae (28, 29). To date, at least 23 colicin types have been described in detail (19, 27, 30, 34). They exert an inhibitory effect on sensitive bacteria of the same family and preferably on strains of the same species. The molecular masses of colicins range between 29,000 and 75,000 Da (7). Colicin polypeptide chains can be divided into separate functional domains, each of which is responsible for one step in the interaction between the colicin and a sensitive bacterium. The central domain of colicins is involved in the attachment of the colicin molecule to a specific outer membrane receptor protein, the N-terminal domain mediates translocation through the cell envelope, and the C-terminal domain exerts the lethal effect (4, 6, 7). At least 12 different outer membrane proteins have been shown to be colicin receptors, two different translocation systems (Ton and Tol) used by colicins have been identified, and six different modes of molecular lethal action of colicins have been described (7, 10, 22, 34). Some molecules initially described as colicins were later reclassified as microcins, e.g., colicin V as microcin V and colicin X as microcin B19. In contrast to these oligopeptide microcins (3), colicins are larger proteins. Moreover, colicins are not posttranslationally modified, are usually inducible by the SOS response, and also differ from microcins by the mode of export from the producer bacteria. Colicin Js was originally described as a bacteriocin of Shigella sonnei colicinotype 7 (1, 2). In 1987, colicin type 7 was reclassified in accordance with Fredericq's original classification scheme (14) and designated colicin Js. Its particular physicochemical and biological characteristics were published (33). For a number of reasons, colicin Js appeared to be a rather exceptional colicin type: producer bacteria, as well as the indicator strain S. sonnei 17 (colicin type 6), were involved in outbreaks of epidemic diarrhea (12). Colicin Js showed a unique antimicrobial spectrum, being inactive against standard E. coli colicin indicators. Indirect fluorimetry measurements indicated that the mode of action of colicin Js was not analogous to that of either pore-forming or nuclease-type colicins (33). Colicin Js was shown to be active against enteroinvasive E. coli (EIEC) serotypes (17). The sensitivity to Js was 90% associated with the ability of EIEC strains to produce experimental keratoconjunctivitis in rabbits. Strains belonging to EIEC serotypes that were not sensitive to colicin Js were, as a rule, negative in the enteroinvasiveness test (17). This communication presents a number of new details of the colicin Js system. These include the structure of the colicin Js coding region on plasmid ColJs; identification of genes for colicin activity, immunity, and release; and molecular characterization of the Js polypeptide.

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

Abstract: 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 ∼42,500 genes in the human, slightly more than reported previously.

Genetic organization of plasmid ColJs, encoding colicin Jsactivity,

Abstract: 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.