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PubMed2002

Identification of Salmonella enterica serovar Typhimurium genes associated with growth suppression in stationary-phase nutrient broth cultures and in the chicken intestine.

Over 2,800 Tn 5 insertion mutants of Salmonella enterica sv. Typhimurium were screened for the loss of ability to suppress the multiplication of a spectinomycin-resistant (Spc(r)) but otherwise isogenic S. enterica sv. Typhimurium strain, when the Spc(r) mutant was added to 24-h LB broth cultures of the mutants. Selected "growth non-suppressive" (GNS) mutants were defective in respiration (insertions in arcA and fnr), amino acid biosynthesis (aroA and aroD), nutrient uptake and its regulation (tdcC and crp), and chemotaxis (fliD). In the last GNS mutant, the transposon inactivated yhjH, an ORF with unknown function which shows sequence similarity to di-guanylate cyclase and to novel two-component signal transduction proteins. In newly hatched chickens, all of the mutants, with the exception of the fliDmutant, were also unable to suppress colonization of the alimentary tract by the parent strain inoculated 1 day later. Defined mutations in luxS or sdiA,genes which contribute to quorum sensing in S. enterica sv. Typhimurium, had no effect on the stationary-phase growth suppression. Analysis of a transcriptional fusion construct indicated that yhjH was moderately expressed in the exponential phase of growth and up-regulated upon entry into stationary phase. Expression of yhjH was also considerably suppressed by the addition of supernatant from a 24-h stationary-phase S. enterica sv. Typhimurium culture, suggesting that the gene belongs to a new sensing and signaling regulatory pathway in S. enterica sv. Typhimurium.

Amino Acid Sequence↗
PubMed1988

Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer.

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.

Alkaline Phosphatase↗
PubMed1989

Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA.

Southern blot analysis of 15 proU transposon insertions in Salmonella typhimurium indicated that this operon is at least 3 kilobase pairs in length. The nucleotide sequence of 1.5-kilobase-pair fragment that contains the transcriptional control region of the proU operon and the coding sequences specifying 290 amino acids of the first structural gene of the operon was determined. The predicted amino acid sequence of the product of this gene shows extensive similarity to the HisP, MalK, and other proteins that are inner membrane-associated components of binding protein-dependent transport systems. S1 mapping and primer extension analysis of the proU mRNAs revealed several species with different 5' ends. Two of these endpoints are sufficiently close to sequences that have weak similarities to the consensus -35 and -10 promoter sequences that they are likely to define two transcription start sites. However, we cannot rule out the possibility that some or all of the 5' endpoints detected arose as a result of the degradation of a longer mRNA. The expression of proU-lacZ operon fusions located on plasmids was normal in S. typhimurium regardless of the plasmid copy number. The sequences mediating normal, osmoregulated expression of the proU operon were shown by subcloning to be contained on an 815-base-pair fragment. A 350-base-pair subclone of this fragment placed onto a lacZ expression vector directed a high-level constitutive expression of beta-galactosidase, suggesting that there is a site for negative regulation in the proU transcriptional control region which has been deleted in the construction of this plasmid.

Amino Acid Sequence↗
PubMed2003

Identification of genes involved in the switch between the smooth and rugose phenotypes of Vibrio cholerae.

Vibrio cholerae can switch to a 'rugose' phenotype characterized by an exopolysaccharide (EPS) matrix, wrinkled colony morphology, increased biofilm formation and increased survival under specific conditions. The vps gene cluster responsible for the biosynthesis of the rugose EPS (rEPS) is positively regulated by VpsR. We recently identified media (APW#3) promoting EPS production and the rugose phenotype and found epidemic strains switch at a higher frequency than non-pathogenic strains, suggesting this switch and the rugose phenotype are important in cholera epidemiology. In this study, transposon mutagenesis on a smooth V. cholerae strain was used to identify mutants that were unable to shift to the rugose phenotype under inducing conditions to better understand the molecular basis of the switch. We identified vpsR, galE and vps previously associated with the rugose phenotype, and also identified genes not previously associated with the phenotype, including rfbD and rfbE having roles in LPS (lipopolysaccharide) synthesis and aroB and aroK with roles in aromatic amino acid synthesis. Additionally, a mutation in amiB encoding N-acetylmuramoyl-L-alanine amidase caused defects in the switch, motility and cell morphology. We also found that a gene encoding a novel regulatory protein we termed RocS (regulation of cell signaling) containing a GGDEF and EAL domains and associated with c-di-GMP levels is important for the rugose phenotype, EPS, biofilm formation and motility. We propose that modulation of cyclic dinucleotide (e.g. c-di-GMP) levels might have application in regulating various phenotypes of prokaryotes. Our study shows the molecular complexity of the switch between the smooth and rugose phenotypes of V. cholerae and may be relevant to similar phenotypes in other species.

Biofilms↗
PubMed1994

Identification of genes affecting production of the adhesive holdfast of a marine caulobacter.

Caulobacters are stalked bacteria that produce a structure termed a holdfast which enables firm attachment to surfaces. Tn5 insertion mutagenesis was used to identify genes affecting holdfast production or function in the marine strain MCS6. Twelve thousand Tn5 insertion mutants were screened for adhesion defects by an assay involving the attachment of cells to polystyrene microtiter dish wells. Among adhesion-defective mutants, those with multiple polar (pleiotropic) defects were excluded and the remainder were examined for the presence of holdfast. Forty-one mutants that produced no detectable holdfast or a significantly reduced amount were found. Southern blot and pulsed-field gel electrophoresis analyses indicated that 11 unique Tn5 insertions were clustered in three regions of the genome. In addition, 71 mutants that adhered poorly or not at all to polystyrene, yet still produced a holdfast, were found. Southern blot and pulsed-field gel electrophoresis analyses of 15 of these mutants showed eight unique Tn5 insertion sites clustered in two additional regions of the genome. An assay involving attachment to glass treated with siloxane chemicals (producing surfaces with varying degrees of hydrophobicity or hydrophilicity) was used to attempt characterization of this phenotype. Unexpectedly, no simple pattern of differences in binding between the mutants and wild-type caulobacters was found. In particular, no reduction in the ability of the mutants to bind to hydrophobic surfaces was noted. Complementation with cosmid clones was successful in nearly all cases and confirmed the designation of five genomic regions of holdfast-related genes. No detectable cross-hybridization was observed with several holdfast-related gene regions from a freshwater caulobacter, providing further evidence that the marine and freshwater caulobacters are genetically distinct.

Bacterial Adhesion↗
PubMed2006

High-throughput identification of conditionally essential genes in bacteria: from STM to TSM.

Signature-tagged mutagenesis (STM) provided the first widely applicable high-throughput method for detecting conditionally essential genes in bacteria by using negative selection to screen large pools of transposon (Tn) mutants. STM requires no prior knowledge of the bacterium's genome sequence, and has been used to study a large number of Gram-positive and Gram-negative species, greatly expanding the repertoires of known virulence factors for these organisms. Originally, hybridization of radiolabelled probes to colony or dot blots was used to detect differences in populations of tagged mutants before and after growth under a selective condition. Modifications of the tag detection method involving polymerase chain reaction (PCR) amplification and visualisation by gel electrophoresis have been developed and can be automated through the use of robotics. Genetic footprinting is another negative selection technique that uses PCR amplification to detect loss of mutants from a pool. Unlike PCR-STM, this technique allows direct amplification of Tn-flanking sequences. However, it requires the bacterium's whole genome sequence in order to design specific primers for every gene of interest. More recently, a number of techniques have been described that combine the negative-selection principle of STM and genetic footprinting with the genome-wide screening power of DNA microarrays. These techniques, although also requiring whole genome sequences, use either a form of linker-mediated or semi-random PCR to amplify and label Tn-flanking regions for hybridization to microarrays. The superior sensitivity microarray detection allows greater numbers of mutants to be screened per pool, as well as determination of the coverage/distribution of insertions in the library prior to screening, two significant advantages over STM.

Bacteria↗
PubMed2001

Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358.

The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomonas putida WCS358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the beta-ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA expression in response to PHB. In this study the identification and characterization of the pobC-pobA locus of P. putida WCS358 is presented. The p-hydroxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high identity to other PobR proteins studied and belonged to the AraC family of regulatory proteins, hence it has been designated POBC: Using the pobA promoter transcriptionally fused to a promoterless lacZ gene it was observed that induction via PobC occurred very efficiently when PHB was present and to a lesser but still significant level also in the presence of PCA. This PobC-PCA response was genetically demonstrated by making use of pobC::Tn5 and pcaH::Tn5 mutants of strain WCS358 constructed in this study. In pobC mutants both the p-hydroxybenzoic and PCA response were not observed, whereas in the pcaH mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent pobA activation was still observed. Finally, the activation of pobA by PHB varied according to the concentration and it was observed that in the pcaR::Tn5 regulatory mutant of strain WCS358 the pobA promoter activity was reduced. PcaR is a regulator involved in the regulation of several loci of the beta-ketoadipate pathway, one of which is pcaK. It was postulated that the reduction of pobA activation in pcaR::Tn5 mutants was because there was no expression of the pcaK gene encoding the PHB transport protein resulting in lower levels of PHB present inside the cell.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1995

Identification of mycoplasma membrane proteins by systematic Tn phoA mutagenesis of a recombinant library.

Wall-less prokaryotes in the genus Mycoplasma include over 90 species of infectious agents whose pathogenicity for humans and other animals is currently being assessed. Molecular characterization of surface proteins is critical in this regard but is hampered by the lack of genetic systems in these organisms. We used TnphoA transposition to systematically mutagenize, in Escherichia coli, a genomic plasmid library constructed from Mycoplasma fermentans, a potential human pathogen. The strategy circumvented problems of expressing mycoplasma genes containing UGA (Trp) codons and relied on the construction of the vector pG7ZCW, designed to reduce TnphoA transposition into vector sequences. Functional phoA gene fusions directly identified genes encoding 19 putative membrane-associated proteins of M. fermentans. Sequences of fusion constructs defined three types of export sequence: (1) non-cleavable, membrane-spanning sequences, (2) signal peptides with signal peptidase (SPase) I-like cleavage sites, and (3) signal peptides with SPase II-like lipoprotein-cleavage sites which, like most other mycoplasmal lipoprotein signals analysed to date, differed from those in several Gram-negative and Gram-positive eubacteria in their lack of a Leu residue at the -3 position. Antibodies to synthetic peptides that were deduced from two fusions to predicted lipoproteins, identified corresponding amphiphilic membrane proteins of 57 kDa and 78 kDa expressed in the mycoplasma. The P57 sequence contained a proline-rich N-terminal region analogous to an adhesin of Mycoplasma gallisepticum. The P78 protein was identical to a serologically defined phase-variant surface lipoprotein. TnphoA mutagenesis provides an efficient means of systematically characterizing functionally diverse lipoproteins and other exported proteins in mycoplasmas.

Alkaline Phosphatase↗
PubMed1990

Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions.

Twenty-four independent phosphate starvation-inducible (psi) transcriptional fusions made with Mu d1(lacZbla) were analyzed by sequencing the psi::lacZ(Mu d1) chromosomal junctions by using DNAs amplified with the polymerase chain reaction or mini-Mu cloning. Our DNA sequence analysis showed that the MuR DNA in Mu d1 has an unexpected structure that is comprised of 104 bases of MuR DNA in the form of a large inverted repeat, which we denoted Mu d1-R. Also, Mu d1s in the phoA and phn (psiD) loci of the phosphate regulon showed regional specificities for the insertion sites despite the randomness of Mu d1 insertions into the genome as a whole. Gene products or open reading frames were identified for seven unknown psi::lacZ(Mu d1) transcriptional fusions by searching DNA data bases with the sequences adjacent and upstream of the Mu d1s. One psiC::lacZ(Mu d1) lies in the ugpB gene of the ugpBAEC operon, which encodes a periplasmic sn-glycerol-3-phosphate-binding protein; two psiQ::lacZ(Mu d1)s lie in the gltB gene, and one psiQ::lacZ(Mu d1) lies in the gltD gene of the gltBDF operon, encoding the large and small subunits of glutamate synthase, respectively; and the psi-51::lacZ(Mu d1) lies in the glpB gene of the glpABC operon, which codes for the anaerobically regulated glycerol-3-phosphate dehydrogenase. psiE and psiF::lacZ(Mu d1)s lie in uncharacterized open reading frames near the xylE and phoA genes, respectively. Six other psi::lacZ(Mu d1)s lie in yet unreported Escherichia coli sequences.

Bacteriophage mu↗
PubMed1994

Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli.

The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, which stimulates the RF-1 and RF-2 activities, was originally identified in Escherichia coli, but it has received little attention since the 1970s. To search for the gene encoding RF-3, we selected nonsense-suppressor mutations by random insertion mutagenesis on the assumption that a loss of function of RF-3 would lead to misreading of stop signals. One of these mutations, named tos-1 (for transposon-induced opal suppressor), mapped to the 99.2 min region on the E. coli chromosome and suppressed all three stop codons. Complementation studies and analyses of the DNA and protein sequences revealed that the tos gene encodes a 59,442-Da protein, with sequence homology to elongation factor EF-G, including G-domain motifs, and that the tos-1 insertion eliminated the C-terminal one-fifth of the protein. Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codon-dependent in vitro termination assay. The stimulation was significantly reduced by GTP, GDP, and the beta,gamma-methylene analog of GTP, but not by GMP. These results fit perfectly with those described in the original publications on RF-3, and the tos gene has therefore been designated prfC. A completely null prfC mutation made by reverse genetics affected the cell growth under the limited set of physiological and strain conditions.

Amino Acid Sequence↗
PubMed1999

Identification of a gene in Staphylococcus xylosus encoding a novel glucose uptake protein.

By transposon Tn917 mutagenesis, two mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designated glcU, encoding a protein involved in glucose uptake in S. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of beta-galactosidase, alpha-glucosidase, and beta-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons into glcU reduced glucose dehydrogenase activity, suggesting cotranscription of glcU and gdh. Insertional inactivation of the gdh gene and deletion of the glcU gene without affecting gdh expression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in the glcU mutant strains. With cloned glcU, an Escherichia coli mutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized by glcU in nonphosphorylated form. A gene from Bacillus subtilis, ycxE, that is homologous to glcU, could substitute for glcU in the E. coli glucose growth experiments and restored glucose repression in the S. xylosus glcU mutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined.

Bacillus subtilis↗
PubMed1988

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene.

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.

Agrobacterium tumefaciens↗
PubMed2001

Identification of a new plasmid-encoded sec-dependent bacteriocin produced by Listeria innocua 743.

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.

Amino Acid Sequence↗
PubMed2000

Usefulness of the I/D angiotensin-converting enzyme genotype for detecting the risk of left ventricular hypertrophy in pharmacologically treated hypertensive men.

The insertion/deletion polymorphism (I/D) of the angiotensin-converting enzyme (ACE) gene has been associated in some studies with a higher prevalence of left ventricular hypertrophy (LVH), but few of them were performed on pharmacologically treated hypertensive patients. The present study was undertaken to determine whether ACE genotype determination could help in the identification of pharmacologically treated hypertensive patients at a higher risk of LVH. Ninety-six consecutive men with essential hypertension were selected for the study. Left ventricular mass (LVM) was assessed by echocardiography and indexed by body surface area and 82 patients were considered suitable for the study. Three groups of patients were defined on the basis of their I/D ACE genotype: DD (n = 39), ID (n = 33) and II (n = 10). There were no statistically significant differences between the three groups regarding to the severity of hypertension at diagnosis, degree of control of blood pressure or type of antihypertensive drug therapy used. No statistically significant differences were found between the three groups regarding to LVM index (total 124 +/- 31, DD 121 +/- 29, ID 127 +/- 35 and II 122 +/- 18 g/m2), relative wall thickness (total 0.5 +/- 0. 2, DD 0.5 +/- 0.3, ID 0.48 +/- 0.07 and II 0.47 +/- 0.04) or prevalence of LVH (total 34%, DD 31%, ID 39% and II 30% by Cornell criteria and total 39%, DD 33%, ID 45% and II 40% by Framingham criteria). Furthermore, the I and D allele frequency distribution was similar in the whole group of patients, in patients with LVH, and in a control group of healthy volunteers. Our data do not support that the I/D ACE genotype determination helps in identifying treated hypertensive patients at higher risk of LVH. Journal of Human Hypertension (2000) 14, 327-331

Adult↗
PubMed1995

Identification of a gene encoding a bacteriophage-related integrase in a vap region of the Dichelobacter nodosus genome.

Dichelobacter nodosus is the principal causative agent of ovine footrot. Nucleotide (nt) sequences from the D. nodosus genome have been isolated and a series of overlapping lambda clones defining vap (virulence-associated protein) regions 1, 2 and 3 have been reported [Katz et al., J. Bacteriol. 176 (1994) 2663-2669]. In the present study, the limits of the virulence-associated (va) DNA around vap regions 1 and 3 were determined by dot-blot hybridization experiments using plasmid subclones to probe genomic DNA from the D. nodosus virulent strain A198 and the benign strain C305. This va region was found to be approx. 11.9 kb in length, and to be interrupted by a short DNA segment which is also found in the benign D. nodosus strain. Sequence analysis of the entire region revealed an ORF, intA, which is very similar to the integrases of bacteriophages phi R73, P4 and Sf6. Bacteriophages phi R73 and P4 integrate into the 3' ends of tRNA genes, with the integrase genes adjacent to the tRNA genes. A similar arrangement was found in the D. nodosus va region. A 19-bp nt sequence was found to be repeated at the ends of the va region, and may represent the bacteriphage attachment site. These findings suggest that D. nodosus may have acquired these DNA sequences by the integration of a bacteriophage, or an integrative plasmid that contains a bacteriophage-related integrase gene. The high similarity of the D. nodosus integrase to integrases from coliphages suggests that these va sequences may be transferred between distantly related bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.

Amino Acid Sequence↗
PubMed2004

Large-scale identification of genes required for full virulence of Staphylococcus aureus.

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Animals↗
PubMed1999

Methicillin-resistant Staphylococcus aureus clonal types in the Czech Republic.

Molecular surveillance studies have documented the extensive spread of methicillin-resistant Staphylococcus aureus (MRSA) clones. Studies carried out by Centro de Epidemiologia Molecular-Network for Tracking Gram-Positive Pathogenic Bacteria (CEM/NET) led to the identification of two international multidrug-resistant strains, which were designated as the Iberian and Brazilian MRSA clones and which were defined by multiple genomic typing methods; these included ClaI restriction digests hybridized with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis (PFGE). The genotypic characteristics of these clones are distinct: the Iberian clone is defined as mecA type I, Tn554 type E (or its variants), and PFGE pattern A (I:E:A), whereas the Brazilian clone is defined as mecA type XI (or its variants), Tn554 type B, and PFGE pattern B (XI:B:B). In this study, we characterized 59 single-patient isolates of MRSA collected during 1996 and 1997 at seven hospitals located in Prague and five other cities in the Czech Republic by using the methodologies mentioned above and by using ribotyping of EcoRI and HindIII digests hybridized with a 16S-23S DNA probe. The Brazilian MRSA clone (XI:B:B) was the major clone (80%) spread in two hospitals located in Prague and one located in Brno; the Iberian MRSA clone (I:E:A or its variant I:DD:A), although less representative (12%), was detected in two hospitals, one in Prague and the other in Plzen. Almost all the strains belonging to clone XI:B:B (45 of 47) corresponded to a unique ribotype, E1H1, whereas most strains of the I:E:A and I:DD:A clonal types (6 of 7) corresponded to ribotype E2H2.

Bacterial Proteins↗