Acetoin is a major extracellular product of Bacillus subtilis grown on glucose and other fermentable carbon sources. The enzymes responsible for the formation of acetoin, acetolactate synthase, and acetolactate decarboxylase are synthesized in detectable amounts only in cells that have reached stationary phase. We have cloned and sequenced the genes encoding these enzymes, alsS and alsD, as well as a gene, alsR, that regulates their expression. alsS and alsD appear to compose a single operon, while alsR is transcribed divergently from the alsSD operon. AlsR shows significant homology to the LysR family of bacterial activator proteins, and when alsR is disrupted the alsSD operon is not expressed. Transcriptional fusions to alsS and alsR revealed that AlsR is required for the transcription of the alsSD operon, which increases during stationary phase. Two mutations that cause increased expression of the alsSD operon have been isolated, cloned, and sequenced. They each change an amino acid in the AlsR protein.
We used primer extension and mutational analysis to identify a promoter upstream of iIvB, the first gene in the ilv-keu operon of Bacilus subtilis. Between the promoter and ilvB, there is a 482-bp leader region which contains a sequence that resembles a factor-independent transcription terminator. In in vitro transcription experiments, 90%o of transcripts initiated at the ilvB promoter ended at a site near this terminator. Primer extension analysis of RNA synthesized in vivo showed that the steady-state level of mRNA upstream of the terminator was twofold higher from cells limited for leucine than it was from cells grown with excess leucine. mRNA downstream of the terminator was 14-fold higher in cells limited for leucine than in cells grown with excess leucine. Measurement of mRNA degradation rates showed that the half-life of ilv-leu mRNA was the same when the cells were grown with or without leucine. These data demonstrate that the ilv-Iu operon is regulated by transcription attenuation.In Bacillus subtilis, 7 of the 10 genes required for synthesis of leucine, isoleucine, and valine form a single operon called the ilv-leu operon (15). The operon was cloned, and a putative promoter, a leader region, and the genes ilvB, ilvN, and ilvC were sequenced (14). By using transcriptional fusions of the ilv-leu regulatory region to the lacZ gene of Escherichia coli, Vandeyar et al. found that ilv-leu promoter expression was 10-fold higher in cells starved for leucine than in cells grown with excess leucine. Starvation for isoleucine and valine had no such effect (14). In this article, we identify the ilv-leu operon promoter and show that, between the promoter and the first structural gene, there is a 482-bp leader region with a sequence resembling a factorindependent transcription termination site. We demonstrate that most transcription terminates at or near this site in vitro and also in vivo when cells are grown in a medium containing excess leucine. MATERIALS AND METHODSBacterial strains and growth conditions. B. subtilis CU4609 (trpC2 leuB16 ilvN::Tn917) was used for leucine limitation experiments. B. subtilis CU1065 (trpC2) was a recipient strain for promoter insertion mutagenesis. E. coli XL1 Blue [recA4 lac endAI gyrA96 thi hsdRl7supE44 relAI (F'proAB lacIq lacZAM15 TnlO)] (Stratagene) was used for plasmid construction. Complex medium was LB broth containing 50 jig of ampicillin per ml when required. Minimal medium contained 0.5% glucose, 1 ,ug of biotin per ml, 20 ,ug of tryptophan per ml, 35 ,ug of isoleucine per ml, 70 ,ug of valine per ml, and a salts mixture to produce final concentrations of, per liter, 2 g of (NH4)2SO4, 13.8 g of K2HPO4 3H20, 6 g of KH2PO4, 0.2 mg of MnCl2 .4H20, 1 g of sodium citrate, and 0.2 g of MgSO4. Cultures were incubated at 37°C and aerated by shaking in baffled flasks.Plasmid construction. A 791-bp polymerase chain reaction product which contained sequences 240 bp upstream and 544
The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcusfaecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J.Christie and G. M. Dunny, Plasmid 15:230-241, 1986 from the chromosome to various sites on the streptococcal plasmid pAD1 (5,14).Recent work in one of our laboratories resulted in the generation of a physical and genetic map of the pheromoneinducible conjugative S. faecalis tetracycline resistance plasmid pCF10 (4). The major features of this plasmid include a 25-kb region (designated TRA) that determines plasmid transfer and pheromone response functions. A distinct 16-kb region containing the tetracycline resistance determinant (Tet9) showed a high degree of sequence-homology with the conjugative transposon Tn916 (14, 15). However, we were unable to ascribe any of the genetic functions that would be predicted for a conjugative transposon to this region of pCF10. Of particular significance is the stability of the Tetr determinant and its continued association with pCF10 through multiple rounds of conjugative transfer. In contrast, when Tn916 and related elements are inserted into conjugative plasmids, they commonly excise and segregate from these plasmids at high frequency (by a proposed zygotic induction mechanism) (16)
The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon. Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.
Duramycin is a polypeptide antibiotic (molecular weight 2012) obtained from culture filtrates of Streptomyces cinnamomeus forma azacoluta. In this work, we show that low concentrations of duramycin induced aggregation of lipid vesicles containing unsaturated phosphatidylethanolamine and unsaturated monogalactosyl diglyceride, and of sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Furthermore, duramycin inhibited the ATP-dependent Ca2+ uptake in sarcoplasmic reticulum vesicles without affecting the hydrolysis of ATP or the permeability of Ca2+. Also, duramycin only inhibited the bacteriorhodopsin proton pump reconstituted into phospholipid vesicles containing phosphatidylethanolamine. We have isolated a duramycin-resistant strain of Bacillus subtilis and have mapped the location of duramycin resistance. In this strain, the secretion of protons and influx of calcium were resistant to duramycin, and its lipid composition was profoundly different from that of the parent strain. No phosphatidylethanolamine was detected in the resistant strain. Our findings are consistent with the idea that duramycin recognizes a particular membrane conformation determined by the presence of phosphatidylethanolamine or monogalactosyl diglyceride.
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