The vancomycin resistance VanRS two‐component signal transduction system of Streptomyces coelicolor

Abstract

SummaryWe took advantage of the vancomycin-dependent phenotype of Streptomyces coelicolor femX null mutants to isolate a collection of spontaneous, drug-independent femX suppressor mutants that expressed the vancomycin-resistance ( van ) genes constitutively. All of the suppressor mutations were in vanS but, unexpectedly, many were predicted to be loss-of-function mutations. Confirming this interpretation, a constructed vanS deletion mutation also resulted in constitutive expression of the van genes, suggesting that VanS negatively regulated VanR function in the absence of drug. In contrast, a vanS pta ackA triple mutant, which should not be able synthesize acetyl phosphate, failed to express the van genes, whereas a pta ackA double mutant showed wild-type, regulated induction of the van genes. These results suggest that in the absence of vancomycin, acetyl phosphate phosphorylates VanR, and VanS acts as a phosphatase to suppress the levels of VanR ∼ ∼ ∼ ∼ P. On exposure to vancomycin, VanS activity switches from a phosphatase to a kinase and vancomycin resistance is induced. In S. coelicolor , the van genes are induced by both vancomycin and the glycopeptide A47934, whereas in Streptomyces toyocaensis (the A47934 producer) resistance is induced by A47934 but not by vancomycin. We exploited this distinction to replace the S. coelicolor vanRS genes with the vanRS genes from S. toyocaensis . The resulting strain acquired the inducer profile of S. toyocaensis , providing circumstantial evidence that the VanS effector ligand is the drug itself, and not an intermediate in cell wall biosynthesis that accumulates as result of drug action. Consistent with this suggestion, we found that non-glycopeptide inhibitors of the late steps in cell wall biosynthesis such as moenomycin A, bacitracin and ramoplanin were not inducers of the S. coelicolor VanRS system, in contrast to results obtained in enterococcal VanRS systems.

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

“…However, in both cases experimental evidence points towards a role of the short extracytoplasmic linker between the two TMR in signal perception. VanS-like HKs interact with vancomycin through their short extracellular sensing domain (25 to 30 aa), presumably by binding the drug directly (104,106). The same is true for PmrB/BasS-like HKs, with a periplasmic linker of 30 to 35 amino acids between the TMR.…”

The σ E Cell Envelope Stress Response of Streptomyces coelicolor Is Influenced by a Novel Lipoprotein, CseA

“…The level of kanamycin resistance conferred on M600 and J2172 by pIJ6880 was assessed on MMCGT, which is agar minimal medium (MM) (21) supplemented with 0.6% (wt/vol) Difco Casamino Acids, 0.75% (vol/vol) Tiger Milk (21), and 0.5% (wt/vol) glucose. Except where described below, other media and culture conditions were as given previously (14,18,21). For the globomycin experiments, wild-type S. coelicolor was grown for 16 h in NMMP liquid medium containing 2 mM MgCl 2 in the presence or absence of the antibiotic (150 g ml Ϫ1 ).…”

In Vivo Studies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d -Ala- d -Ala Termini in the Peptidoglycan Cell Wall

“…However, the explosion in microbial genome sequencing facilitated by recent advances in sequencing technologies has revealed the presence of these genes in many more bacterial sources, interestingly also including many nonpathogenic or non-glycopeptide-producing species (6). This type of organism offers a safe and convenient system for the in vivo study of important aspects of vancomycin resistance, as exemplified by recent work using Streptomyces coelicolor (7)(8)(9)(10). S. coelicolor serves as the model species for Streptomyces, a genus of Gram-positive, mycelial soil bacteria responsible for the production of two-thirds of the commercially important antibiotics.…”