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The VanRS Homologous Two-Component System VnlRS Ab of the Glycopeptide Producer Amycolatopsis balhimycina Activates Transcription of the vanHAX Sc Genes in Streptomyces coelicolor , but not in A. balhimycina

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ABSTRACT

In enterococci and in Streptomyces coelicolor, a glycopeptide nonproducer, the glycopeptide resistance genes vanHAX are colocalized with vanRS. The two-component system (TCS) VanRS activates vanHAX transcription upon sensing the presence of glycopeptides. Amycolatopsis balhimycina, the producer of the vancomycin-like glycopeptide balhimycin, also possesses vanHAXAb genes. The genes for the VanRS-like TCS VnlRSAb, together with the carboxypeptidase gene vanYAb, are part of the balhimycin biosynthetic gene cluster, which is located 2 Mb separate from the vanHAXAb. The deletion of vnlRSAb did not affect glycopeptide resistance or balhimycin production. In the A. balhimycina vnlRAb deletion mutant, the vanHAXAb genes were expressed at the same level as in the wild type, and peptidoglycan (PG) analyses proved the synthesis of resistant PG precursors. Whereas vanHAXAb expression in A. balhimycina does not depend on VnlRAb, a VnlRAb-depending regulation of vanYAb was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and RNA-seq analyses. Although VnlRAb does not regulate the vanHAXAb genes in A. balhimycina, its heterologous expression in the glycopeptide-sensitive S. coelicolor ΔvanRSSc deletion mutant restored glycopeptide resistance. VnlRAb activates the vanHAXSc genes even in the absence of VanS. In addition, expression of vnlRAb increases actinorhodin production and influences morphological differentiation in S. coelicolor.

No MeSH data available.


Analysis of balhimycin production, muropeptide composition, and PG precursors in A. balhimycina WT (1), A. balhimycina ΔvnlRAb (2), A. balhimycina [vnlRAb] (3), and A. balhimycina ΔvnlRAb [vnlRAb] (4). (A) Production of balhimycin measured by HPLC (n = 5). (B) HPLC/MS chromatogram of the muropeptides (positive mode). The first bracket embraces the peaks representing muropeptide monomers, the second the muropeptide dimers. (C) Extracted ion chromatograms of the negative mode from the PG precursors isolated from cells grown in R5 (balhimycin production) and in TSB (no balhimycin production). d-Lac, Pentapeptide precursors ending on d-Ala-d-Lac 1194 m/z at retention time ∼18 min. d-Ala, Pentapeptide precursors ending on d-Ala-d-Ala 1193 m/z at retention time ∼12 min. HPLC, high-performance liquid chromatography; MS, mass spectrometry; PG, peptidoglycan; WT, wild type.
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f2: Analysis of balhimycin production, muropeptide composition, and PG precursors in A. balhimycina WT (1), A. balhimycina ΔvnlRAb (2), A. balhimycina [vnlRAb] (3), and A. balhimycina ΔvnlRAb [vnlRAb] (4). (A) Production of balhimycin measured by HPLC (n = 5). (B) HPLC/MS chromatogram of the muropeptides (positive mode). The first bracket embraces the peaks representing muropeptide monomers, the second the muropeptide dimers. (C) Extracted ion chromatograms of the negative mode from the PG precursors isolated from cells grown in R5 (balhimycin production) and in TSB (no balhimycin production). d-Lac, Pentapeptide precursors ending on d-Ala-d-Lac 1194 m/z at retention time ∼18 min. d-Ala, Pentapeptide precursors ending on d-Ala-d-Ala 1193 m/z at retention time ∼12 min. HPLC, high-performance liquid chromatography; MS, mass spectrometry; PG, peptidoglycan; WT, wild type.

Mentions: Since overexpression of RRs of two-component signal transduction systems often modulates multidrug resistance,43,44 we overexpressed vnlRAb in A. balhimycina to analyze the effects on resistance and antibiotic production. vnlRAb was cloned under the control of the constitutive promoter ermE*p into the integrative plasmid pRM4 (pRM4vnlRAb). pRM4vnlRAb was transferred into A. balhimycina WT and into the A. balhimycina ΔvnlRAb mutant,27 resulting in the recombinant strains A. balhimycina [vnlRAb] and A. balhimycina ΔvnlRAb [vnlRAb], respectively. The phenotypes of the recombinant strains overexpressing VnlRAb and (as a control) that of the deletion mutant A. balhimycina ΔvnlRAb were compared with the WT phenotype (Fig. 2). All strains produced balhimycin at the same level (Fig. 2A). No differences in resistance against balhimycin were observed. Using a method optimized for actinomycetes,27 muropeptides from all A. balhimycina strains cultivated under balhimycin production conditions were isolated. HPLC/MS chromatograms showed the similar muropeptide composition pattern for all strains (Fig. 2B).


The VanRS Homologous Two-Component System VnlRS Ab of the Glycopeptide Producer Amycolatopsis balhimycina Activates Transcription of the vanHAX Sc Genes in Streptomyces coelicolor , but not in A. balhimycina
Analysis of balhimycin production, muropeptide composition, and PG precursors in A. balhimycina WT (1), A. balhimycina ΔvnlRAb (2), A. balhimycina [vnlRAb] (3), and A. balhimycina ΔvnlRAb [vnlRAb] (4). (A) Production of balhimycin measured by HPLC (n = 5). (B) HPLC/MS chromatogram of the muropeptides (positive mode). The first bracket embraces the peaks representing muropeptide monomers, the second the muropeptide dimers. (C) Extracted ion chromatograms of the negative mode from the PG precursors isolated from cells grown in R5 (balhimycin production) and in TSB (no balhimycin production). d-Lac, Pentapeptide precursors ending on d-Ala-d-Lac 1194 m/z at retention time ∼18 min. d-Ala, Pentapeptide precursors ending on d-Ala-d-Ala 1193 m/z at retention time ∼12 min. HPLC, high-performance liquid chromatography; MS, mass spectrometry; PG, peptidoglycan; WT, wild type.
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f2: Analysis of balhimycin production, muropeptide composition, and PG precursors in A. balhimycina WT (1), A. balhimycina ΔvnlRAb (2), A. balhimycina [vnlRAb] (3), and A. balhimycina ΔvnlRAb [vnlRAb] (4). (A) Production of balhimycin measured by HPLC (n = 5). (B) HPLC/MS chromatogram of the muropeptides (positive mode). The first bracket embraces the peaks representing muropeptide monomers, the second the muropeptide dimers. (C) Extracted ion chromatograms of the negative mode from the PG precursors isolated from cells grown in R5 (balhimycin production) and in TSB (no balhimycin production). d-Lac, Pentapeptide precursors ending on d-Ala-d-Lac 1194 m/z at retention time ∼18 min. d-Ala, Pentapeptide precursors ending on d-Ala-d-Ala 1193 m/z at retention time ∼12 min. HPLC, high-performance liquid chromatography; MS, mass spectrometry; PG, peptidoglycan; WT, wild type.
Mentions: Since overexpression of RRs of two-component signal transduction systems often modulates multidrug resistance,43,44 we overexpressed vnlRAb in A. balhimycina to analyze the effects on resistance and antibiotic production. vnlRAb was cloned under the control of the constitutive promoter ermE*p into the integrative plasmid pRM4 (pRM4vnlRAb). pRM4vnlRAb was transferred into A. balhimycina WT and into the A. balhimycina ΔvnlRAb mutant,27 resulting in the recombinant strains A. balhimycina [vnlRAb] and A. balhimycina ΔvnlRAb [vnlRAb], respectively. The phenotypes of the recombinant strains overexpressing VnlRAb and (as a control) that of the deletion mutant A. balhimycina ΔvnlRAb were compared with the WT phenotype (Fig. 2). All strains produced balhimycin at the same level (Fig. 2A). No differences in resistance against balhimycin were observed. Using a method optimized for actinomycetes,27 muropeptides from all A. balhimycina strains cultivated under balhimycin production conditions were isolated. HPLC/MS chromatograms showed the similar muropeptide composition pattern for all strains (Fig. 2B).

View Article: PubMed Central - PubMed

ABSTRACT

In enterococci and in Streptomyces coelicolor, a glycopeptide nonproducer, the glycopeptide resistance genes vanHAX are colocalized with vanRS. The two-component system (TCS) VanRS activates vanHAX transcription upon sensing the presence of glycopeptides. Amycolatopsis balhimycina, the producer of the vancomycin-like glycopeptide balhimycin, also possesses vanHAXAb genes. The genes for the VanRS-like TCS VnlRSAb, together with the carboxypeptidase gene vanYAb, are part of the balhimycin biosynthetic gene cluster, which is located 2 Mb separate from the vanHAXAb. The deletion of vnlRSAb did not affect glycopeptide resistance or balhimycin production. In the A. balhimycina vnlRAb deletion mutant, the vanHAXAb genes were expressed at the same level as in the wild type, and peptidoglycan (PG) analyses proved the synthesis of resistant PG precursors. Whereas vanHAXAb expression in A. balhimycina does not depend on VnlRAb, a VnlRAb-depending regulation of vanYAb was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and RNA-seq analyses. Although VnlRAb does not regulate the vanHAXAb genes in A. balhimycina, its heterologous expression in the glycopeptide-sensitive S. coelicolor ΔvanRSSc deletion mutant restored glycopeptide resistance. VnlRAb activates the vanHAXSc genes even in the absence of VanS. In addition, expression of vnlRAb increases actinorhodin production and influences morphological differentiation in S. coelicolor.

No MeSH data available.