Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae.
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The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine. (+info)
Presence of plasmid pA15 correlates with prevalence of constitutive MLS(B) resistance in group A streptococcal isolates at a university hospital in southern Taiwan.
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OBJECTIVES: To investigate the role of a plasmid bearing the erm(B) gene on the prevalence of the macrolide, lincosamide and group B streptogramin (MLS(B)) phenotype of group A streptococci (GAS) and to characterize the plasmid and determine the clonal relation between the erythromycin-resistant isolates. METHODS: Two hundred and five erythromycin-resistant GAS isolates were collected from 1990 to 2006. Colony hybridization, PCR, plasmid curing and PFGE techniques were used to analyse the mechanisms behind the phenotypes. RESULTS: Among the 56 isolates with constitutive MLS(B) (cMLS(B)) resistance, 53 isolates harboured a plasmid, pA15, of 19 kb. erm(B) was on pA15 and it confered a cMLS(B) resistance phenotype. The prevalence rate of the pA15-containing isolates was 36.3% from 1993 to 1995, but the plasmid could not be detected from 2004 to 2006. To link the high-level resistance to pA15, clinical isolate A15 was selected and pA15 was cured by novobiocin. In the plasmid-cured strain SW503, the erythromycin MIC decreased from 256 to 0.032 mg/L. By electroporation, pA15 was re-introduced into the plasmid-cured erythromycin-susceptible strain, and the high-level erythromycin resistance was restored. Plasmid pA15 was also transferred to group B streptococci and group C streptococci by electroporation. In all the pA15-containing isolates, emm1 type was present and pulse type J was predominant (48 of 54 isolates). CONCLUSIONS: The plasmid pA15 mediated cMLS(B) resistance in the mid-1990s, but pA15 was not detected in the clinical isolates from 2004 onwards, which correlates with the absence of cMLS(B) resistance in this region. (+info)
Differences in potential for selection of clindamycin-resistant mutants between inducible erm(A) and erm(C) Staphylococcus aureus genes.
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In staphylococci, inducible macrolide-lincosamide-streptogramin B (MLS(B)) resistance is conferred by the erm(C) or erm(A) gene. This phenotype is characterized by the erythromycin-clindamycin "D-zone" test. Although clindamycin appears active in vitro, exposure of MLS(B)-inducible Staphylococcus aureus to this antibiotic may result in the selection of clindamycin-resistant mutants, either in vitro or in vivo. We have compared the frequencies of mutation to clindamycin resistance for 28 isolates of S. aureus inducibly resistant to erythromycin and bearing the erm(C) (n = 18) or erm(A) (n = 10) gene. Seven isolates susceptible to erythromycin or bearing the msr(A) gene (efflux) were used as controls. The frequencies of mutation to clindamycin resistance for the erm(A) isolates (mean +/- standard deviation, 3.4 x 10(-8) +/- 2.4 x 10(-8)) were only slightly higher than those for the controls (1.1 x 10(-8) +/- 6.4 x 10(-9)). By contrast, erm(C) isolates displayed a mean frequency of mutation to clindamycin resistance (4.7 x 10(-7) +/- 5.5 x 10(-7)) 14-fold higher than that of the S. aureus isolates with erm(A). The difference was also observed, although to a lower extent, when erm(C) and erm(A) were cloned into S. aureus RN4220. We conclude that erm(C) and erm(A) have different genetic potentials for selection of clindamycin-resistant mutants. By the disk diffusion method, erm(C) and erm(A) isolates could be distinguished on the basis of high- and low-level resistance to oleandomycin, respectively. (+info)
VisG is essential for biosynthesis of virginiamycin S, a streptogramin type B antibiotic, as a provider of the nonproteinogenic amino acid phenylglycine.
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