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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.


Extracted ion chromatograms (negative mode) of the PG precursors isolated from cells grown in R5 medium without antibiotic or with 25 mg/ml balhimycin (bal25). M600 ΔvanRSSc: S. coelicolor M600 ΔvanRSSc; 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 ∼14 min.
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f6: Extracted ion chromatograms (negative mode) of the PG precursors isolated from cells grown in R5 medium without antibiotic or with 25 mg/ml balhimycin (bal25). M600 ΔvanRSSc: S. coelicolor M600 ΔvanRSSc; 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 ∼14 min.

Mentions: To investigate whether transcription of the vanHAXSc genes indeed resulted in the formation of glycopeptide-resistant PG precursors, which caused the resistant phenotype, we used HPLC/MS to analyze the PG precursor composition of S. coelicolor M600 ΔvanRSSc and of S. coelicolor M600 ΔvanRSSc complemented either with vnlRSAb or vnlRAb. Complementing S. coelicolor M600 ΔvanRSSc with vnlRSAb or with vnlRAb restored the synthesis of resistant PG precursors. In the presence of balhimycin exclusively, PG precursors ending with d-Ala-d-Lac were synthesized (Fig. 6C, D). The PG precursor composition of the glycopeptide-sensitive S. coelicolor M600 ΔvanRSSc mutant was analyzed after growing the strain in the absence of balhimycin. In this mutant, only sensitive cell wall precursors ending on d-Ala-d-Ala (1193 m/z) eluting at a retention time of 10–11 min were detected (Fig. 6A).


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
Extracted ion chromatograms (negative mode) of the PG precursors isolated from cells grown in R5 medium without antibiotic or with 25 mg/ml balhimycin (bal25). M600 ΔvanRSSc: S. coelicolor M600 ΔvanRSSc; 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 ∼14 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036315&req=5

f6: Extracted ion chromatograms (negative mode) of the PG precursors isolated from cells grown in R5 medium without antibiotic or with 25 mg/ml balhimycin (bal25). M600 ΔvanRSSc: S. coelicolor M600 ΔvanRSSc; 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 ∼14 min.
Mentions: To investigate whether transcription of the vanHAXSc genes indeed resulted in the formation of glycopeptide-resistant PG precursors, which caused the resistant phenotype, we used HPLC/MS to analyze the PG precursor composition of S. coelicolor M600 ΔvanRSSc and of S. coelicolor M600 ΔvanRSSc complemented either with vnlRSAb or vnlRAb. Complementing S. coelicolor M600 ΔvanRSSc with vnlRSAb or with vnlRAb restored the synthesis of resistant PG precursors. In the presence of balhimycin exclusively, PG precursors ending with d-Ala-d-Lac were synthesized (Fig. 6C, D). The PG precursor composition of the glycopeptide-sensitive S. coelicolor M600 ΔvanRSSc mutant was analyzed after growing the strain in the absence of balhimycin. In this mutant, only sensitive cell wall precursors ending on d-Ala-d-Ala (1193 m/z) eluting at a retention time of 10–11 min were detected (Fig. 6A).

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.