Bacitracin is a peptide antibiotic nonribosomally produced by Bacillus licheniformis. The bcrABC genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells. Bacillus subtilis 168, which has no bacitracin synthesizing operon, has several genes homologous to bcrABC. It was found that the disruption of Bacitracin is a dodecapeptide antibiotic produced by some strains of Bacillus licheniformis and Bacillus subtilis (2, 11). The synthesis is nonribosomally catalyzed by a multienzyme complex composed of three subunits, BacA, BacB, and BacC, whose genes have been cloned and sequenced (6,9,12,18,22). Bacitracin has potent antibiotic activity against gram-positive bacteria (30). The inhibition of peptidoglycan biosynthesis is the best-characterized bactericidal effect of bacitracin (27). It forms a complex mediated by a metal ion (Zn 2ϩ ) with the lipid C 55 -isoprenyl pyrophosphate (IPP) (24, 26), which is a carrier of a peptidoglycan unit or a disaccharide with pentapeptide across the membrane. Bacitracin, by binding to IPP, inhibits the conversion of IPP to C 55 -isoprenyl phosphate, which is catalyzed by a membrane associated pyrophosphatase (25).B. licheniformis, a bacitracin producer, has an ABC transporter system which is hypothesized to pump out bacitracin for self-protection (19). The transporter is composed of two membrane proteins, BcrB and BcrC, and two identical ATP-binding subunits, BcrA. Neumüller et al. recently reported that in B. licheniformis, bcrABC genes are localized about 3 kb downstream of the bacitracin biosynthetic operon bacABC (14). Between the bacABC operon and bcrABC genes, they also identified bacR and bacS genes which encode proteins with high homology to response regulator and sensory kinase of two-component regulatory systems and are involved in the regulation of bcrA expression.The B. subtilis genome project determined the entire DNA sequence of strain 168 and found that there are two operons which encode nonribosomal peptide antibiotic synthetase complexes (13). One is a surfactin synthetase operon (4), and the other is a plipastatin (fengycin) synthetase operon (29, 31). There is no bacitracin synthetase operon in B. subtilis 168. B. subtilis 168 is more sensitive to bacitracin than B. licheniformis, a bacitracin producer. Heterologous expression of bcrABC transporter in B. subtilis results in an increase of bacitracin resistance to a level similar to that observed in a bacitracin producer (5,19,20). A homology search reveals that in B. subtilis 168, there are several homologues of BcrA, -B, and -C of B. licheniformis. In this study we showed that the disruption of the ywoA gene, which encodes the BcrC homologue, resulted in a marked decrease of resistance to bacitracin. We also reported that the transcription of the ywoA gene was dependent on extracytoplasmic function (ECF) factors, M and X . MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis 168...

In members of one of the subfamilies of the bacterial ATP binding cassette (ABC) transporters, the two nucleotide binding domains are fused as a single peptide and the proteins have no membrane-spanning domain partners. Most of the ABC efflux transporters of this subfamily have been characterized in actinomycetes, producing macrolide, lincosamide, and streptogramin antibiotics. Among 40 ABC efflux transporters of Bacillus subtilis, five proteins belong to this subfamily. None of these proteins has been functionally characterized. We examined macrolide, lincosamide, and streptogramin antibiotic resistance in insertional disruptants of the genes that encode these proteins. It was found that only a disruptant of vmlR (formerly named expZ) showed hypersensitivity to virginiamycin M and lincomycin. Expression of the vmlR gene was induced by the addition of these antibiotics in growth medium. Primer extension analysis revealed that transcription of the vmlR gene initiates at an adenosine residue located 225 bp upstream of the initiation codon. From the analysis of the vmlR and lacZ fusion genes, a 52-bp deletion from ؉159 to ؉211 resulted in constitutive expression of the vmlR gene. In this region, a typical -independent transcriptional terminator was found. It was suggested that the majority of transcription ends at this termination signal in the absence of antibiotics, whereas under induced conditions, RNA polymerase reads through the terminator, and transcription continues to the downstream vmlR coding region, resulting in an increase in vmlR expression. No stabilization of vmlR mRNA occurred under the induced conditions.

Harmful environmental agents cause nasal inflammation, and chronic nasal inflammation induces a loss of olfactory sensory neurons (OSNs) and reversible atrophy of the olfactory bulb (OB). Here, we investigated the mechanisms underlying this inflammation-induced OB atrophy by histologically and biochemically comparing the OB changes in mouse models of nasal inflammation and odor deprivation. In addition, we examined whether odor stimulation is necessary for OB recovery from atrophy. One group of adult male C57BL/6 mice was administered lipopolysaccharide (LPS) unilaterally for 10 weeks to induce nasal inflammation (control animals received saline), and a second group received unilateral naris closures (NCs) for 10 weeks of odor deprivation. The OBs atrophied in both models, but odor deprivation shrank the glomerular, external plexiform, mitral, and granule cell layers (GCLs), whereas the olfactory nerve, glomerular, and external plexiform layers (EPLs) atrophied as a result of nasal inflammation. Additionally, nasal inflammation, but not odor deprivation, caused neuroinflammation in the OB, inducing glial activation and elevated expression of interleukin-1β (IL-1β) and TNFα. After 10 weeks of nasal inflammation, mice were housed for another 10 weeks with no additional treatment or with unilateral NC. Nasal inflammation and glial activation subsided in both groups, but glomerular and EPLs recovered only in those with no additional treatment. Our findings demonstrate that nasal inflammation and odor deprivation differentially induce layer-specific degeneration in the OB, that loss of OSN activity rather than neuroinflammation is a major cause of inflammation-induced OB atrophy, and that odor stimulation is required for OB recovery from atrophy.

A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase.Bacteria possess pumps of low specificity that can extrude structurally unrelated compounds in an energy-dependent manner (12,16,20). Mutations in the genes encoding these pumps increase the susceptibilities of the mutant bacteria to diverse cytotoxic compounds (11,15,21), and overexpression of these pumps from genes on multicopy plasmids confers multidrug resistance to bacteria (1,13,18,19).Recently, experimental evidence has accumulated showing that some bacteria have multiple drug efflux pumps with overlapping substrate specificities. Escherichia coli has AcrAB and EnvCD efflux pump systems (with more than 65% identical amino acids) and similar substrate specificities (11,(14)(15)(16). In the gram-positive bacterium Bacillus subtilis, two highly homologous (51% identity) multidrug transporters, Bmr and Blt, have been reported (1,18). Overexpression of these transporters was shown to result in increased resistance to the same spectrum of substances. It is not yet clear whether these individual pumps have any specific physiological function other than to protect bacteria from various toxins in the environment.In this article, we report a third multidrug transporter gene of B. subtilis. Overexpression of this gene, bmr3, on a multicopy plasmid resulted in resistance to puromycin, tosufloxacin, and norfloxacin which are also substrates of the two other multidrug transporters of B. subtilis. Bmr3 protein shows moderate homology to EmrB, a multidrug transporter of E. coli (13), and to a lesser extent to Bmr and Blt (1,18). The expression pattern of the bmr3 gene was dependent on the growth phase and different from those of bmr and blt genes. Therefore, these three multidrug transporters might have distinct roles under different physiological conditions.Cloning and sequencing of the bmr3 gene. The cmp gene of E. coli, which is located just downstream of the divE gene, encodes a 23.5-kDa hypothetical membrane protein with unknown function (24). While attempting to clone a homolog of this gene, we obtained a clone, pH233, from a genomic library of B. subtilis 168 cloned into pUC18 that gave a distinct hybridization signal under low-stringency conditions. Sequence analysis showed that the 3.3-kb insert of pH233 contained two open reading frames (ORFs) oriented in opposite directions, as shown in Fig. 1A. Although the 0.7-kb HindIII fragment of ORF 1 (ORF-1) displayed 50% identity with the cmp gene at the level of nucleotide sequence, there was no homology at the level of amino acid sequence. A homology search revealed that the predicted 512 amino acid sequence of ORF-1 showed moderate homology to EmrB (25% identity and 57% similarity), one of the multidrug transporters of E. coli (13), and to a putative protein p...

The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding