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1.
Science ; 299(5615): 2071-4, 2003 Mar 28.
Article in English | MEDLINE | ID: mdl-12663927

ABSTRACT

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.


Subject(s)
Biological Evolution , Enterococcus faecalis/genetics , Genome, Bacterial , Interspersed Repetitive Sequences , Sequence Analysis, DNA , Vancomycin Resistance/genetics , Adhesins, Bacterial/genetics , Bacterial Adhesion , Bacterial Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chromosomes, Bacterial/genetics , Conjugation, Genetic , Conserved Sequence , DNA Transposable Elements , Digestive System/microbiology , Drug Resistance, Multiple, Bacterial , Enterococcus faecalis/drug effects , Enterococcus faecalis/pathogenicity , Enterococcus faecalis/physiology , Gene Transfer, Horizontal , Gram-Positive Bacterial Infections/microbiology , Humans , Lysogeny , Open Reading Frames , Oxidative Stress , Plasmids , Synteny , Virulence/genetics , Virulence Factors/genetics
2.
Drug Discov Today ; 6(17): 887-892, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11522517

ABSTRACT

In this era of genomic science, knowledge about biological function is integrated increasingly with DNA sequence data. One area that has been significantly impacted by this accumulation of information is the discovery of drugs to treat microbial infections. Genome sequencing and bioinformatics is driving the discovery and development of novel classes of broad-spectrum antimicrobial compounds, and could enable medical science to keep pace with the increasing resistance of bacteria, fungi and parasites to current antimicrobials. This review discusses the use of genomic information in the rapid identification of target genes for antimicrobial drug discovery.

3.
Science ; 293(5529): 498-506, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11463916

ABSTRACT

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.


Subject(s)
Genome, Bacterial , Sequence Analysis, DNA , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/pathogenicity , Antigens, Bacterial , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Bacterial Vaccines , Base Composition , Carbohydrate Metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chromosomes, Bacterial/genetics , Computational Biology , DNA Transposable Elements , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Duplication , Genes, Bacterial , Hexosamines/metabolism , Oligonucleotide Array Sequence Analysis , Recombination, Genetic , Repetitive Sequences, Nucleic Acid , Species Specificity , Streptococcus pneumoniae/immunology , Streptococcus pneumoniae/metabolism , Virulence , rRNA Operon
4.
Expert Opin Investig Drugs ; 10(8): 1409-22, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11772259

ABSTRACT

Efflux is the process in which bacteria transport compounds outside the cell which are potentially toxic, such as drugs or chemicals or compounds. Efflux pumps can be identified not only by biochemical, microbiological, or molecular means but with the availability of microbial genomic sequences, by the application of bioinformatics analysis of DNA sequences for key conserved structure motifs. Efflux has been identified as a relevant contributor to bacterial resistance in the clinic and is now recognised as one of the most important causes of intrinsic antibiotic resistance in bacteria, especially in Pseudomonas aeruginosa. With the recognition of efflux as a major factor in bacterial resistance, several companies have invested in the identification and development of bacterial efflux pump inhibitors. Among those, Microcide, Pfizer, Paratek and several academic laboratories are in the process of exploring efflux pump inhibitors from synthetic, natural products and peptidomimetics. Inhibiting bacterial efflux with a non-antibiotic inhibitor would restore activity of an antibiotic subject to efflux (similar to the use of beta-lactamase inhibitors to combat beta-lactamase production by bacteria). The feasibility of such an approach has been experimentally demonstrated in vitro and in vivo for efflux reversal of levofloxacin.


Subject(s)
Bacteria/metabolism , Bacterial Toxins/metabolism , Bacteria/drug effects , Biological Transport, Active/drug effects , Drug Resistance
5.
Science ; 287(5459): 1809-15, 2000 Mar 10.
Article in English | MEDLINE | ID: mdl-10710307

ABSTRACT

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.


Subject(s)
Genome, Bacterial , Neisseria meningitidis/genetics , Neisseria meningitidis/pathogenicity , Sequence Analysis, DNA , Antigenic Variation , Antigens, Bacterial/immunology , Bacteremia/microbiology , Bacterial Capsules/genetics , Bacterial Proteins/genetics , Bacterial Proteins/physiology , DNA Transposable Elements , Evolution, Molecular , Fimbriae, Bacterial/genetics , Humans , Meningitis, Meningococcal/microbiology , Meningococcal Infections/microbiology , Molecular Sequence Data , Mutation , Neisseria meningitidis/classification , Neisseria meningitidis/physiology , Open Reading Frames , Operon , Phylogeny , Recombination, Genetic , Serotyping , Transformation, Bacterial , Virulence/genetics
6.
Appl Environ Microbiol ; 65(5): 1883-90, 1999 May.
Article in English | MEDLINE | ID: mdl-10223974

ABSTRACT

To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of Streptococcus pneumoniae, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in Escherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the Bacillus subtilis competence gene comFA. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless lacZ reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.


Subject(s)
Genes, Bacterial , Mutagenesis, Insertional , Streptococcus pneumoniae/genetics , Bacillus subtilis/genetics , Base Sequence , DNA Primers/genetics , Escherichia coli/genetics , Gene Library , Mutation , Plasmids/genetics , Transformation, Genetic
7.
Mol Microbiol ; 29(4): 1029-38, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9767571

ABSTRACT

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60,232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.


Subject(s)
Bacteriocins/biosynthesis , Bacteriocins/genetics , Lactococcus lactis/genetics , Lactococcus lactis/metabolism , Plasmids/genetics , Bacteriophages/pathogenicity , Base Sequence , Chromosome Mapping , Conjugation, Genetic , DNA Transposable Elements/genetics , DNA, Bacterial/genetics , Genes, Bacterial , Lactococcus lactis/virology , Molecular Sequence Data , Open Reading Frames , Phenotype , Sequence Homology, Nucleic Acid
8.
Nature ; 390(6658): 364-70, 1997 Nov 27.
Article in English | MEDLINE | ID: mdl-9389475

ABSTRACT

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.


Subject(s)
Archaeoglobus fulgidus/genetics , Genes, Archaeal , Genome , Archaeoglobus fulgidus/metabolism , Archaeoglobus fulgidus/physiology , Base Sequence , Cell Division , DNA, Bacterial/genetics , Energy Metabolism , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Protein Biosynthesis , Transcription, Genetic
9.
Nature ; 388(6642): 539-47, 1997 Aug 07.
Article in English | MEDLINE | ID: mdl-9252185

ABSTRACT

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.


Subject(s)
Genome, Bacterial , Helicobacter pylori/genetics , Antigenic Variation , Bacterial Adhesion , Bacterial Proteins/metabolism , Base Sequence , Biological Evolution , Cell Division , DNA Repair , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Helicobacter pylori/metabolism , Helicobacter pylori/pathogenicity , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Biosynthesis , Recombination, Genetic , Transcription, Genetic , Virulence
10.
Microb Comp Genomics ; 2(4): 313-21, 1997.
Article in English | MEDLINE | ID: mdl-9689229

ABSTRACT

The availability of completely sequenced genomes has created an opportunity for high throughput mutational studies. Using the conjugative transposon Tn916, a pilot project was initiated to determine the efficiency of gene disruption in the first completely sequenced bacterium, Haemophilus influenzae Rd strain KW20. DNA was isolated from Tn916-mutagenized cells, and the point of transposon insertion was determined by inverse PCR, DNA sequencing, and mapping to the wild-type genome sequence. Analysis of the insertion sites at the nucleotide level demonstrated a biased pattern of insertion into regions rich in stretches of A's and T's. Although Tn916 integrated at multiple dispersed positions throughout the chromosome, 9 of 10 insertion events occurred in noncoding, intergenic DNA. It was determined that the intergenic DNA was over 5% more A + T-rich than that of protein coding sequences. This suggests that A + T-rich sequences similar to the Tn916 insertion site would be more likely to reside in the intergenic DNA. In an effort to identify other likely sites for transposon integration, a hidden Markov model of the consensus target insertion site was derived from the Tn916-H. influenzae junction fragments and searched against the entire genome. Eighty percent of the 30 highest-scoring predicted Tn916 target sites were from intergenic, nonprotein-coding regions of the genome. These data support the hypothesis that Tn916 has a marked preference for insertion into noncoding DNA for H. influenzae, suggesting that this mobile element has evolved to minimize disruption of host cell function on integration.


Subject(s)
DNA Transposable Elements/genetics , DNA, Bacterial/genetics , Haemophilus influenzae/genetics , Base Sequence , DNA, Bacterial/analysis , Markov Chains , Molecular Sequence Data , Mutagenesis , Pilot Projects , Sequence Alignment
11.
Science ; 273(5278): 1058-73, 1996 Aug 23.
Article in English | MEDLINE | ID: mdl-8688087

ABSTRACT

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.


Subject(s)
Bacterial Proteins/genetics , DNA, Bacterial/genetics , Genome, Bacterial , Methanococcus/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Composition , Base Sequence , Biological Transport/genetics , Carbon Dioxide/metabolism , Chromosome Mapping , Chromosomes, Bacterial/genetics , DNA Replication , Databases, Factual , Energy Metabolism/genetics , Genes, Bacterial , Hydrogen/metabolism , Methane/metabolism , Methanococcus/physiology , Molecular Sequence Data , Protein Biosynthesis , Sequence Analysis, DNA , Transcription, Genetic
12.
J Biol Chem ; 270(48): 28676-80, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-7499387

ABSTRACT

Hyaluronic acid is a high molecular weight glycosaminoglycan composed of repeating subunits of glucuronic acid and N-acetylglucosamine. It is synthesized by the group A streptococcal membrane-associated enzyme hyaluronate synthase. In previous reports, the locus required for expression of hyaluronic acid, the has operon, was identified and found to consist of two genes, hasA and hasB encoding hyaluronate synthase and UDP-glucose dehydrogenase, respectively. Since a transcription terminator was not found at the end of hasB, it was the aim of this study to identify the remaining gene(s) in the has operon. By utilizing the Tn1000 method of DNA sequencing and inverse polymerase chain reaction, hasC, the third gene in the has operon was shown to be 915 base pairs in length (304 amino acids) and located 192 base pairs downstream of hasB. Sequence similarities to other genes suggested that hasC encodes UDP-glucose pyrophosphorylase. Overexpression of hasC using isopropyl-1-thio-beta-D-galactopyranoside induction of the T7 promoter in the pET translation system allowed for the production of bacterial extracts from Escherichia coli that possessed increased UDP-glucose pyrophosphorylase activity as compared to nondetectable levels in extracts with vector alone. In addition, expression of HasC resulted in a protein of approximately 36 kDa as shown by SDS-polyacrylamide gel electrophoresis. These data as well as complementation analysis of hasC in an E. coli galU mutant confirmed that hasC encodes UDP-glucose pyrophosphorylase. Finally, since sequence analysis identified a potential rho-independent transcription terminator at the 3-prime terminus of the gene, hasC is the third and probably the final gene in the has operon.


Subject(s)
Hyaluronic Acid/biosynthesis , Operon , Streptococcus pyogenes/metabolism , UTP-Glucose-1-Phosphate Uridylyltransferase/genetics , Amino Acid Sequence , Bacteriophage T7/genetics , Base Sequence , Catalysis , Cloning, Molecular , DNA, Recombinant , Escherichia coli/genetics , Genetic Complementation Test , Glucosephosphates/metabolism , Molecular Sequence Data , Mutation , Sequence Homology, Amino Acid , UTP-Glucose-1-Phosphate Uridylyltransferase/chemistry , UTP-Glucose-1-Phosphate Uridylyltransferase/metabolism , Uridine Diphosphate Glucose/biosynthesis , Uridine Triphosphate/metabolism
13.
Science ; 270(5235): 397-403, 1995 Oct 20.
Article in English | MEDLINE | ID: mdl-7569993

ABSTRACT

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.


Subject(s)
Genome, Bacterial , Mycoplasma/genetics , Sequence Analysis, DNA , Antigenic Variation/genetics , Bacterial Proteins/genetics , Biological Transport/genetics , DNA Repair/genetics , DNA Replication/genetics , DNA, Bacterial/genetics , Databases, Factual , Energy Metabolism/genetics , Genes, Bacterial , Haemophilus influenzae/genetics , Molecular Sequence Data , Mycoplasma/immunology , Mycoplasma/metabolism , Open Reading Frames , Protein Biosynthesis , Transcription, Genetic
14.
Science ; 269(5223): 496-512, 1995 Jul 28.
Article in English | MEDLINE | ID: mdl-7542800

ABSTRACT

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.


Subject(s)
Chromosome Mapping , DNA, Bacterial/genetics , Genome, Bacterial , Haemophilus influenzae/genetics , Sequence Analysis, DNA , Bacterial Proteins/genetics , Base Composition , Base Sequence , Chromosome Mapping/methods , Chromosomes, Bacterial , Cloning, Molecular , Costs and Cost Analysis , Databases, Factual , Genes, Bacterial , Haemophilus influenzae/physiology , Molecular Sequence Data , Operon , RNA, Bacterial/genetics , RNA, Ribosomal/genetics , Repetitive Sequences, Nucleic Acid , Sequence Analysis, DNA/methods , Software
15.
Science ; 269(5223): 538-40, 1995 Jul 28.
Article in English | MEDLINE | ID: mdl-7542802

ABSTRACT

The naturally transformable, Gram-negative bacterium Haemophilus influenzae Rd preferentially takes up DNA of its own species by recognizing a 9-base pair sequence, 5'-AAGTGCGGT, carried in multiple copies in its chromosome. With the availability of the complete genome sequence, 1465 copies of the 9-base pair uptake site have been identified. Alignment of these sites unexpectedly reveals an extended consensus region of 29 base pairs containing the core 9-base pair region and two downstream 6-base pair A/T-rich regions, each spaced about one helix turn apart. Seventeen percent of the sites are in inverted repeat pairs, many of which are located downstream to gene termini and are capable of forming stem-loop structures in messenger RNA that might function as signals for transcription termination.


Subject(s)
DNA, Bacterial/genetics , Genome, Bacterial , Haemophilus influenzae/genetics , Transformation, Bacterial , Base Composition , Base Sequence , Chromosome Mapping , Consensus Sequence , Conserved Sequence , DNA, Bacterial/chemistry , Escherichia coli/genetics , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotide Probes , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , Repetitive Sequences, Nucleic Acid , Transcription, Genetic
16.
J Biol Chem ; 269(1): 169-75, 1994 Jan 07.
Article in English | MEDLINE | ID: mdl-8276791

ABSTRACT

The mechanism by which group A streptococci produce the antiphagocytic hyaluronate (hyaluronic acid) capsule is incompletely understood. Enzymes known to be essential for synthesis of this polysaccharide include the membrane-associated hyaluronate synthase as well as those required for production of the substrate sugars UDP-N-acetylglucosamine and UDP-glucuronic acid. In this study, a Tn916 insertion that inactivates hyaluronate synthetic activity was localized to a gene designated hasA in the hyaluronic acid synthesis operon. This gene has recently been preliminarily identified as the group A streptococcal hyaluronate synthase. The DNA sequence and transcription start site of hasA were determined, and the predicted HasA protein was shown to have characteristics of a membrane protein. Amino acid sequence homology suggests that HasA is related to a family of proteins involved in polysaccharide production and cell differentiation. Finally, in addition to the loss of hyaluronate synthase activity, the hasA::Tn916 insertion was demonstrated to correlate with a loss of UDP-glucuronic acid dehydrogenase activity. These results suggest that the genes required for hyaluronate synthase activity and production of the UDP-glucuronic acid substrate are transcribed as a unit in group A streptococci.


Subject(s)
Glucuronosyltransferase/genetics , Glycosyltransferases , Hyaluronic Acid/biosynthesis , Hyaluronic Acid/genetics , Membrane Proteins , Operon , Streptococcus pyogenes/metabolism , Transferases , Xenopus Proteins , Amino Acid Sequence , Base Sequence , DNA Transposable Elements , DNA, Bacterial , Glucuronosyltransferase/metabolism , Hyaluronan Synthases , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Transcription, Genetic
17.
J Biol Chem ; 268(10): 7118-24, 1993 Apr 05.
Article in English | MEDLINE | ID: mdl-8463246

ABSTRACT

The membrane-associated hyaluronate synthase produces capsular hyaluronate in group A streptococci by the alternate addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. Previous studies identified a locus required for hyaluronate synthase activity and suggested that a gene involved in the production of UDP-glucuronic acid (UDP-glucose dehydrogenase) also mapped to the locus. In the present study the putative UDP-glucose dehydrogenase gene (hasB) was cloned and the DNA sequence determined. The hasB gene product was shown to have global similarity with AlgD, a dehydrogenase, which catalyzes the production of GDP-mannuronic acid for the alginate capsule of Pseudomonas aeruginosa. Regions of local homology have been identified which apparently correspond to the NAD-binding and enzyme active sites of HasB and AlgD. In order to show that hasB expression correlated with UDP-glucose dehydrogenase activity, the hasB gene was cloned under control of the T7 promoter. Hyperexpression of hasB resulted in a protein of approximately 47 kDa and high levels of UDP-glucose dehydrogenase activity were observed. These data demonstrate that hasB encodes the UDP-glucose dehydrogenase of group A streptococci.


Subject(s)
Hyaluronic Acid/biosynthesis , Operon , Streptococcus/genetics , Uridine Diphosphate Glucose Dehydrogenase/genetics , Amino Acid Sequence , Base Sequence , Carbohydrate Dehydrogenases/genetics , Cloning, Molecular , DNA, Bacterial , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Molecular Sequence Data , Mutagenesis, Insertional , Restriction Mapping , Sequence Homology, Amino Acid , Streptococcus/enzymology , Uridine Diphosphate Glucose Dehydrogenase/metabolism
18.
J Exp Med ; 175(5): 1291-9, 1992 May 01.
Article in English | MEDLINE | ID: mdl-1569398

ABSTRACT

To characterize the production of hyaluronate capsule by the membrane-associated enzyme hyaluronate synthase (HAS), group A streptococci from a recent outbreak of acute rheumatic fever were mutagenized via Tn916 insertion. Acapsular transconjugants harboring multiple, nontandem copies of the transposon were identified and found to lack HAS activity (less than 1% of wild-type levels). Generalized transduction was then performed to determine which Tn916 insertion was responsible for the HAS- phenotype. These marker exchange experiments resulted in the isolation of two distinct classes of acapsular transductants, designated WF61 and WF62. Both transductants also lacked significant HAS activity, and excision of the transposon from WF62 restored capsular hyaluronate production. Southern analysis of WF61 DNA demonstrated a large deletion of genomic DNA adjacent to the Tn916 insertion. This deletion event is presumably responsible for the observed stability of the acapsular phenotype of WF61. Further analyses of transductant whole-cell DNA indicated that the transposon insertions of WF61 and WF62 were separated by 2.5 kb. These studies define a locus required for hyaluronate capsule production in group A streptococci. Further genetic analysis of this locus has identified a gene required for HAS activity which wasd inactivated by TN916 in WF62 and deleted in WF61.


Subject(s)
Bacterial Capsules/metabolism , Glucuronosyltransferase/genetics , Glycosyltransferases , Hyaluronic Acid/genetics , Membrane Proteins , Streptococcus pyogenes/genetics , Transferases , Xenopus Proteins , Base Sequence , Blotting, Southern , Conjugation, Genetic , DNA, Bacterial , Genes, Bacterial , Glucuronosyltransferase/metabolism , Hyaluronan Synthases , Hyaluronic Acid/biosynthesis , Molecular Sequence Data , Mutagenesis, Insertional , Polymerase Chain Reaction , Restriction Mapping , Streptococcus pyogenes/enzymology
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