The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydrophobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the importance of various regions in transport.

Karstic cave systems in Slovenia receive substantial amounts of organic input from adjacent forest and freshwater systems. These caves host microbial communities that consist of distinct small colonies differing in colour and shape. Visible to the naked eye, the colonies cover cave walls and are strewn with light-reflecting water droplets. In this study, the diversity of prokaryotes constituting these unusual microbial communities in Pajsarjeva jama cave was examined. A molecular survey based on small subunit rRNA diversity showed a high diversity within the Bacteria, while members of Archaea were not recovered. A total of eight bacterial phyla were detected. The application of various species richness estimators confirmed the diverse nature of the microbial community sample. Members of Gammaproteobacteria were most abundant in the clone libraries constructed and were followed in abundance by members of Actinobacteria and Nitrospira. In addition, members of Alphaproteobacteria, Betaproteobacteria and Deltaproteobacteria as well as Acidobacteria, Verrucomicrobia, Planctomycetes, Chloroflexi and Gemmatimonadetes were identified in clone libraries. The high number of clones most closely related to environmental 16S rRNA gene clones showed the broad spectrum of unknown and yet to be cultivated microorganisms inhabiting these cave systems.

The bacitracin resistance of Bacillus licheniformis, a producer of bacitracin, is mediated by the ABC transporter Bcr. Bacillus subtilis cells carrying bcr genes on high-copy number plasmids developed collateral detergent sensitivity, as did human cells with overexpressed multidrug resistance P-glycoprotein. Resistance against bacitracin and sensitivity of resistant cells to detergents were shown to be inseparable phenomena associated with the membrane part of Bcr transporter, namely protein BcrC. A fused protein, consisting of ATP-binding protein BcrA and membrane component BcrC was constructed. It resembled a half molecule of P-glycoprotein and was capable of providing a significant degree of antibiotic resistance and detergent sensitivity.

No abstract

Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated. Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex. Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation. Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B. licheniformis genome.