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...

From the analysis of the measured radii of gyration of the RNA (R, = 6.6 0.3 nm) and protein (R, = 10.2 f 0.5 nm) components of the 50-S subparticle of Eschevichia coli ribosomes it is concluded that proteins containing a large amount of hydrodynamically bound water are located on the periphery of the tightly packed RNA. We found that the common features of the measured X-ray scattering curves of the E. coli 70-S ribosome, its 30-S and 50-S subparticles and wheat 80-S ribosomes in the region of scattering angles corresponding to scattering vectors p from 1 to 5 nm-' reflect features of the RNA compact packing. A hypothesis is proposed that the compact packing of RNA helices in the range of Bragg distances of 4.5-2.0 nm is a general structural feature of all ribosomal particles.One of the main elements of the proposed models of ribosomal subparticles (40-S rabbit reticulocytes [2], 50-S and 30-S Eschevichia coli [3,4]) is a uniform distribution of RNA and protein in them. This assertion is based on the following facts: (a) a slight change occurs in the radius of gyration of the 50-S subparticle of E. coli ribosomes at high sucrose concentrations [3]; (b) the X-ray scattering curve for an homogeneous body of a simple shape can well account for a 1000-fold drop in the intensity of scattering from the 50-S subparticle of E. coli [3,4] and a 500-fold drop in the intensity of scattering from the 30-S subparticle of E. coli [4,5].In 1970 we suggested that the RNA and protein are not uniformly distributed in ribosomes, since the radius of gyration of the 50-S ribosomal subparticle measured by X-ray scattering (7.5 nm) was too small in comparison with its hydrodynamic radius (9.0 nm) obtained from measurements of the diffusion coefficient [6]. To explain the divergences between the electronic and hydrodynamic radii of gyration, an hypothesis was proposed on the existence of a dense RNA region within the 50-S subparticle of E. coli ribosomes [6].The main purpose of the first part of this work is to obtain quantitative parameters characterizing the RNA and protein distribution in the 5 0 3 sub-A part of this work has been mentioned previously [I].particle of E. coli ribosomes. With this in view we applied the method of joint use of light, X-ray and neutron scattering [7,8] ; this differs in principle from the 'method of a contrasting agent' in X-ray scattering and 'contrast variation method' in neutron scattering since it does not involve a solvent change and therefore guarantees the integrity of the particle structure. As a result a considerable dependence of the radius of gyration of the 50-S subparticle on the type of emission was found which could indicate a nonuniform distribution of the RNA and protein within it. Analysis of the RNA and protein radii of gyration leads to the conclusion that proteins containing a large amount of hydrodynamically bound water are located on the periphery of the tightly packed RNA within the 50-S subparticle. These data suggested that the existence of weak maxima on the curves of X-ray...