A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme

Abstract

We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium. This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance. Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression. As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter. Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidodoglycan precursors are wrong.Inducible resistance to vancomycin and related glycopeptide antibiotics in Enterococcus spp. (9,11,13,24,28) has emerged in recent years as a major health problem in the treatment of infectious diseases (22,29,31). The inducible form of resistance can also serve as a transitional step to constitutive resistance, with important consequences for the clinical effectiveness of antibiotics that belong to this group (12,15,25,26).The VanA type of resistance is one of the major forms of inducible resistance to glycopeptide antibiotics. It is specified by the VanA complex operon, which consists of two coordinately expressed operons, vanRS and vanHAX. The vanRS operon specifies the two-component system histidine kinase receptor VanS and its transcriptional response regulator VanR, while vanHAX specifies enzymes that are responsible for the synthesis and biochemical modification of cell wall peptidoglycan precursors which make bacteria resistant to glycopeptide antibiotics (2, 3). The activated VanS receptor phosphorylates the transcriptional regulator, VanR, which in turn activates transcription of the vanHAX operon (for details, see references 17 and 33). The molecular mechanisms of VanS receptor activation and its ligands are not known, and a study of the virulent bacteria or opportunistic pathogens expressing glycopeptide resistance requires special attention to the containment of these strains.To address these problems, we have developed a new cloning tool and used it to construct a model vancomycin-inducible system in the nonpathogenic strain Bacillus subtilis 168. In this strain, the Enterococcus faecium vanRS operon regulates expression of LacZ from the E. faecium vanH promoter. We have tested the system to measure the inducing activities of antibiotics that inhibit peptidoglycan synthesis and cell wall hydrolytic enzymes. MATERIALS AND METHODSPlasmid pAU101. The general methods used in this work to construct recombinant plasmids and to introduce them into Escherichia coli were as described in reference 26. PCR was performed by using the general methods described by Mullis and Faloona (21). A 257-bp vanH cassette bounded by BamHI-compatible cohesive ends, 5ЈGATC, and containing the vanH promoter, toge...

Differential Assay for High-Throughput Screening of Antibacterial Compounds

“…Bacitracin, which appeared to be negative, gave a positive signal when tested earlier at 300 µg/mL. 6 Last, gramicidin D and tyrothricin were highly active. In contrast, a group of 9 protein synthesis inhibitors, the RNA synthesis inhibitor rifampicin, and 2 DNA synthesis inhibitors, trimethoprim and novobiocin, were inactive.…”

“…6 The strain carries 1 chromosomally integrated copy of the Escherichia coli lacZ gene under control of PvanH, the vanH promoter from Enterococcus faecium, as a PvanH:lacZ cassette. The cassette was inserted into B. subtilis amyE by homologous recombination, where it produces undetectable levels of β-gal in the absence of vanRS.…”

“…Multiple reporter strains for the identification of cell wall synthesis inhibitors have been reported previously by Grissom-Arnold et al, 2 Kappner and Metz, 3 O'Neill et al, 4 and Quesada et al 5 In our initial studies, we constructed a reporter strain based on vanRS driving β-galactosidase (β-gal) expression via the vanRS-responsive promoter of vanH, PvanH. 6 The recombinant strain responded to vancomycin but, unexpectedly, to other structurally unrelated cell wall synthesis inhibitors as well. BAU-102 was also responsive to enzymes that degrade the cell wall and, in other preliminary studies, to membrane-active detergent/surfactant compounds.…”

Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion

“…Products for the two PCRs were combined, melted, and re-annealed, phosphorylated, and purified as described (37). Phosphorylated flush inserts were then ligated into the NcoI-and HindIII-digested pBAD-6H plasmid.…”

“…Construction of Recombinant Phy Expression Strains-PCRbased modifications of Phys involved paired amplifications that were subsequently combined, melted, and re-annealed to generate blunt-ended doubled-stranded fragments as described (37). The DNA templates for SyA-Cph1 and SyB-Cph1 fulllength coding sequences were PCR-amplified directly from Synechococcus sp.…”