SummaryBceA and bceB encode a nucleotide-binding domain (NBD) and membrane-spanning domain (MSD) subunit, respectively, of an ATP-binding cassette (ABC) transporter in Bacillus subtilis . Disruption of these genes resulted in hypersensitivity to bacitracin, a peptide antibiotic that is non-ribosomally synthesized in some strains of Bacillus . Northern hybridization analyses showed that expression of the bceAB operon is induced by bacitracin present in the growth medium. The bceRS genes encoding a twocomponent regulatory system are located immediately upstream of bceAB . Deletion analyses of the bceAB promoter together with DNase I footprinting experiments revealed that a sensor kinase, BceS, responds to extracellular bacitracin either directly or indirectly and transmits a signal to a cognate response regulator, BceR. The regulator binds directly to the upstream region of the bceAB promoter and upregulates the expression of bceAB genes. The bcrC gene product is additionally involved in bacitracin resistance. The expression of bcrC is dependent on the ECF s s s s factors, s s s s M and s s s s X , but not on the BceRS two-component system. In view of these results, possible roles of BceA, BceB and BcrC in bacitracin resistance of B. subtilis 168 are discussed.

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.

A spontaneous mutant isolated in the presence of a high concentration of puromycin acquired a multidrugresistant phenotype. Expression of the bmr3 gene was dramatically increased. A base substitution, T to A at the ؉4 position, detected in the mutant resulted in the stabilization of bmr3 mRNA.

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.