Chemotaxis in Bacillus subtilis requires either of two functionally redundant CheW homologs.

We have characterized mutants in a novel gene of Bacillus subtilis, cheV, which encodes a protein homologous to both CheW and CheY. A null mutant in cheV is only slightly defective in capillary and tethered cell assays. However, a double mutant lacking both CheV and CheW has a strong tumble bias, does not respond to addition of attractant, and shows essentially no accumulation in capillary assays. Thus, CheV and CheW appear in part to be functionally redundant. A strain lacking CheW and expressing only the CheW domain of CheV is chemotactic, suggesting that the truncated CheV protein retains in vivo function. We speculate that CheV and CheW function together to couple CheA activation to methyl-accepting chemotaxis protein receptor status and that possible CheA-dependent phosphorylation of CheV contributes to adaptation.     

Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis.

Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.     

Complementation and characterization of chemotaxis mutants of Bacillus subtilis.

A set of chemotaxis mutants of Bacillus subtilis was complemented by using SP beta c2 transducing bacteriophage either containing cloned segments of DNA or derived from abnormal excision of SP beta c2 dl2::Tn917 inserted into the chemotaxis region. Representative mutants were characterized in capillary assays for chemotaxis toward four amino acids and mannitol and in tethered-cell experiments for addition and removal of two attractants and two repellents. Twenty complementation groups were identified, in addition to the cheR previously characterized. All were found to be defective in chemotaxis toward all chemoeffectors. They were assigned the names cheA through cheU. The large number of general chemotaxis genes in B. subtilis, in contrast to the six in Escherichia coli, suggests fundamental differences in the mechanism of chemotaxis in the two species.     

Isolation and characterization of four plasmids from Bacillus subtilis.

Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6). The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.     

Influence of attractants and repellents on methyl group turnover on methyl-accepting chemotaxis proteins of Bacillus subtilis and role of CheW.

The ability of attractants and repellents to affect the turnover of methyl groups on the methyl-accepting chemotaxis proteins (MCPs) was examined for Bacillus subtilis. Attractants were found to cause an increase in the turnover of methyl groups esterified to the MCPs, while repellents caused a decrease. These reactions do not require CheW. However, a cheW null mutant exhibits enhanced turnover in unstimulated cells. Assuming that the turnover of methyl groups on the MCPs reflects a change in the activity of CheA, these results suggest that the activation of CheA via chemoeffector binding at the receptor does not require CheW.     

Sequence of a PAL-related lipoprotein from Bacillus subtilis

The sequence of a small Bacillus subtilis lipoprotein is reported. The gene encodes a protein of 124 amino acids, which shows a low but statistically significant homology to the peptidoglycan-associated lipoproteins (PAL) of Escherichia coli and Haemophilus influenzae. Although the functions of these proteins have not been confirmed, they are obviously structural proteins. In E. coli the gene for the peptidoglycan-associated lipoprotein appears to be essential.     

Cloning and characterization of the polC region of Bacillus subtilis.

The polC gene of Bacillus subtilis is defined by five temperature-sensitive mutations and the 6-(p-hydroxyphenylazo)-uracil (HPUra) resistance mutation azp-12. Biochemical evidence suggests that polC codes for the 160-kilodalton DNA polymerase III. A recombinant plasmid, p154t, was isolated and found to contain the azp-12 marker and one end of the polC gene (N. C. Brown and M. H. Barnes, J. Cell. Biochem. 78 [Suppl.]: 116, 1983). The azp-12 marker was localized to a 1-kilobase DNA segment which was used as a probe to isolate recombinant lambda phages containing polC region sequences. A complete polC gene was constructed by in vitro ligation of DNA segments derived from two of the recombinant phages. The resulting plasmid, pRO10, directed the synthesis of four proteins of 160, 76, 39, and 32 kilodaltons in Escherichia coli maxicells. Recombination-deficient (recE) B. subtilis PSL1 containing pRO10 produced an HPUra-resistant polymerase III activity which was lost when the strain was cured of pRO10. In vivo, the HPUra resistance of the plasmid-encoded polymerase III appeared to be recessive to the resident HPUra-sensitive polymerase III enzyme.     

Isolation and characterization of Bacillus subtilis mutants altered in competence.

We isolated and characterized four Bacillus subtilis competence-deficient mutants. The mutants were obtained by nitrosoguanidine mutagenesis and by screening for mutants unable to be transformed both on solid and in liquid medium. Most of the mutants obtained in this way were tested for their sensitivity to the DNA-damaging agents methyl methanesulfonate, mitomycin C, and UV light. Among the mutants which did not show an increased sensitivity to these agents, four were chosen for further characterization. Data were obtained which indicate that the mutants are reduced in chromosomal and plasmid transformation and in transfection, whereas they are not altered in transduction and in protoplast transformation. Transformation experiments carried out by mixing a culture of a mutant with a culture of a wild-type strain gave some complementation for competence with one of the strains. The mutants were also characterized for their capacity to bind, take up, and break down transforming DNA; furthermore, the four competence mutations were mapped, and the results indicate that they belong to four different genes.     

Identification and characterization of sporulation gene spoVS from Bacillus subtilis.

We report the identification and characterization of an additional sporulation gene from Bacillus subtilis called spoVS, which is induced early in sporulation under the control of sigma H. We show that spoVS is an 86-codon-long open reading frame and is capable of encoding a protein of 8,796 Da which exhibits little similarity to other proteins in the databases. Null mutations in spoVS have two contrasting phenotypes. In otherwise wild-type cells they block sporulation at stage V, impairing the development of heat resistance and coat assembly. However, the presence of a spoVS mutation in a spoIIB spoVG double mutant (which is blocked at the stage [II] of polar septation) acts as a partial suppressor, allowing sporulation to advance to a late stage. The implications of the contrasting phenotypes are discussed in the context of the formation and maturation of the polar septum.     

Sequence features of the replication terminus of the Bacillus subtilis chromosome.

The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtilis 168 chromosome has been determined. The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence. The arrest site occurs in an A + T-rich region between two open reading frames and very close to one of two imperfect inverted repeats (47-48 nucleotides each) which are separated by 59 nucleotides. The closeness of approach of the arrested clockwise fork to the first imperfect inverted repeat encountered in this region raises the possibility of a role for the inverted repeats in the mechanism of fork arrest.     

Sequence specificity of Bacillus subtilis DNA gyrase in vivo

Linearization of pBG0 (a hydrid between Escherichia coli plasmid pBR322 and Staphylococcus aureus plasmid pUB110) was performed by lysis of the oxolinic acid treated Bacillus subtilis protoplasts with sodium dodecyl sulfate. This plasmid DNA linearization was used both for a detailed mapping of DNA gyrase cleavage sites of various strength and for the nucleotide sequence determinations at the points of gyrase-mediated scission by introducing the XhoI linker DNA. A total of 40 plasmids carrying inserted XhoI linker were sequenced by labeling 3' termini of XhoI sites; 38 of them were found to contain a duplication of four base-pairs of the plasmid sequence flanking the linker, which were characteristic of the oxolinic acid-induced DNA cleavage by E. coli DNA gyrase in vitro and in vivo. The relative strength of these sequenced sites was established by comparing their positions to the sites mapped on the appropriate plasmid genome. This allowed us to propose a consensus sequence of B. subtilis DNA gyrase in vivo cleavage site:GNAT GATCATNC% MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D% aebbfv3ySLgzGueE0jxyaibaiiYdd9qrFfea0dXdf9vqai-hEir8Ve% ea0de9qq-hbrpepeea0db9q8as0-LqLs-Jirpepeea0-as0Fb9pgea% 0lrP0xe9Fve9Fve9qapdbaqaaeGacaGaaiaabeqaamaabaabcaGcba% GaaeikaiaabsfacaqGPaGaaeiiaiaabccacaqGGaGaaeiiaiaabcca% caqGOaGaae4raiaabMcacaqGGaGaaeiiaiaabccacaqGGaGaaeiiai% aabccacaqGGaGaaeiiaiaabccacaqGGaGaaeiiaiaabccacaqGGaGa% aeiiaiaabccacaqGOaGaaeyqaiaabMcaaaa!4E92!\[{\rm{(T) (G) (A)}}\]where N is any nucleotide. The bases in parentheses were preferred secondarily. The involvement of DNA gyrase in illegitimate recombination events in Bacillus subtilis is discussed.