Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.     

Expression of tcaA and mprF and glycopeptide resistance in clinical glycopeptide-intermediate Staphylococcus aureus (GISA) and heteroGISA strains

Two genes recently associated with glycopeptide intermediate resistance in Staphylococcus aureus (GISA) are mprF and tcaA, with inactivation causing shifts in vancomycin resistance. This study reveals that expression levels of both genes are similar in groups of clinical GISA, heteroGISA and glycopeptide susceptible strains, suggesting no association with clinical isolates.     

Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.     

Genetic analysis of Staphylococcus aureus with Tn4001.

Tn4001, a 4.5-kilobase composite transposon with IS256 ends that confers resistance to gentamicin (Gmr), tobramycin, and kanamycin in Staphylococcus aureus, can transpose to diverse chromosomal sites in S. aureus. Chromosomal insertions of Tn4001 were isolated either after UV irradiation of transducing lysates carrying pII147::Tn4001 or by selection for thermoresistant Gmr isolates with strains containing thermosensitive derivatives of plasmids pI258 and pII147 carrying Tn4001. Frequent integration of the entire delivery plasmid occurred under these selective conditions in recombination-proficient hosts. When selection for thermoresistant Gmr isolates was done with these plasmids in recombination-deficient hosts, 99% or more of the Gmr isolates resulted from transposition of Tn4001 in the absence of plasmid integration. Efficient isolation of Tn4001 insertions near markers of interest and the isolation of insertional auxotrophs were achieved. Reversion frequencies of insertional auxotrophs were between 10(-6) and 10(-7) (higher than those observed with Tn551 and Tn917). About 50% of the prototrophic revertants were Gms, and these are attributed to precise excision of Tn4001. The Gmr prototrophic revertants were due to intergenic suppression.     

Insertional inactivation of a gene which controls expression of vancomycin resistance on plasmid pHKK100

Expression of inducible high level vancomycin resistance (Vmr) in enterococci appears to require other plasmid-encoded genes in addition to the previously described structural genes vanA and vanH. Tn917 mutagenesis was used to identify such a region in the Vmr plasmid pHKK100. Insertional inactivation of a 693-bp open reading frame upstream from vanH resulted in complete loss of Vmr. This putative 26,642-Da protein has been designated VanR.     

转座子Tn917在短小芽孢杆菌中的转座作用

Transposition Tn917 was introduced into Bacillus pumilus 289 by protoplast transformation with plasmid pTV32. The temperature-sensitive replication property of pTV32 was maintained in B. pumilus. Tn917 was transposed efficiently in B. pumilus with 4.8 x 10(-4) transposition rate. The yield of auxotrophs was about 0.65% in all insertional mutants. It indicated a prospects for the use of Tn917 as a tool for insertional mutagenesis and genetic manipulation in B. pumilus.     

Modulating activity of vancomycin and daptomycin on the expression of autolysis cell-wall turnover and membrane charge genes in hVISA and VISA strains

Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF.     

Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus.

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.     

Identification of a putative Bacillus subtilis rho gene.

Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product. The insertional site was near the beginning of the open reading frame, which was located in a region of the B. subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented. The predicted B. subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628. The growth of the mutant strain was less than that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30 degrees C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30 degrees C. To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B. subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.     

Isolation of Gln- mutants through transposon, Tn917, mutagenesis in Bacillus brevis.

Transposon, Tn917, carried on pTV1 plasmid has been used successfully to mutagenise Bacillus brevis. The transposon showed preference for insertion at an "aro" site. A second insertional event after elimination of the preferred site with ethidium bromide/acridine orange treatment has permitted isolation of Gln- mutants in B. brevis.     

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.     

Phenotypic and genetic characterization of vancomycin-resistant enterococci from hospitalized humans and from poultry in Korea

Vancomycin resistant enterococci (VRE) isolates from humans (23 isolates) and poultry (20 isolates) were characterized by antibiotic susceptibility, vancomycin resistance transferability, pulsed-field gel electrophoresis (PFGE), and structural analysis of Tn1546-like elements. VRE isolates from humans and poultry showed different resistance patterns, transferability, and transfer rate. In addition to these phenotypic differences between humans and poultry VRE, PFGE and the structure of Tn1546-like elements were also distinct. Most poultry isolates (16/20) were identical to the prototype vanA transposon, Tn1546, while most human isolates (21/23) had multiple integrations of insertion sequence. The transmission of VRE and vancomycin resistance determinant between humans and poultry could not be demonstrated in this study.     

Escherichia coli K-12 mutation that inactivates biodegradative threonine dehydratase by transposon Tn5 insertion.

From a collection of kanamycin-resistant mutants of Escherichia coli K-12 isolated by transposon Tn5 mutagenesis, we have identified a mutant that lacks functional biodegradative threonine dehydratase (EC 4.2.1.16) by direct enzyme assay and by the loss of cross-reacting material with affinity-purified antibodies against the purified enzyme. Aerobic and anaerobic growth of this strain on various carbon sources failed to reveal a phenotype. Evidence for the insertional inactivation of threonine dehydratase by Tn5 was obtained by cloning the DNA segments flanking the Tn5 insertion site into pBR322 and hybridizing the cloned DNA to a synthetic oligodeoxynucleotide probe complementary to the DNA segment coding for a unique hexapeptide at the amino terminus end of the enzyme; the region of homology to the synthetic cDNA sequence appears to be located within about 500 nucleotides from one end of Tn5. Genetic analysis with the transposon element that caused insertional inactivation located the tdc gene at min 67 on the E. coli chromosome.