Gene, 1994 Sep 15, 147(1), 85 - 9 Conservation of the 168 divIB gene in Bacillus subtilis W23 and B . licheniformis, and evidence for homology to ftsQ of Escherichia coli; Harry EJ et al.; The chromosomal regions of Bacillus subtilis (Bs) W23 and Bacillus licheniformis (Bl), which span the sequence encoding the homolog of the division initiation gene, divIB, of Bs168 were cloned and sequenced . The high level of conservation of the amino acid (aa) sequence of the DivIB protein (99 and 68% identity for BsW23 and Bl, respectively) was consistent with a significant role for this protein in the cell cycle of the two species . The hydropathy profile for DivIB of Bl was almost identical to that of Bs168 and consistent with a membrane location, as previously established for the latter . The higher than average level of identity (87%) of the 31-aa N-terminal cytoplasmic domain of DivIB between Bs168 and Bl raised the possibility of a special role for this domain . Database analyses using the Bl DivIB sequence and similarity analyses also strongly suggested that DivIB, of Bl and Bs, is a homolog of FtsQ of Escherichia coli . The flanking sequences extending into the unidentified orfs both upstream and downstream from divIB were highly conserved between Bs168 and Bl at both the nucleotide and aa levels . It was confirmed that orf4 of Bs168 is dispensable. Gene, 1994 Sep 15, 147(1), 13 - 20 A genetic and molecular characterization of the recA gene from Staphylococcus aureus; Bayles KW et al.; Previous studies have identified mutant strains of Staphylococcus aureus that have deficiencies in genetic recombination and DNA repair . Although these phenotypes were tentatively attributed to mutations within the S . aureus recA gene, experimental evidence to confirm this has never been reported . To characterize recA from S . aureus, we first isolated transposon insertion mutations that were in close proximity to the recA-like mutation (uvs-568) in strain 112 UVS-1 . This allowed for the mobilization of the uvs-568 mutation into strain RN4220, the common laboratory strain of S . aureus . Next, using Bacillus subtilis recA as a probe, we cloned S . aureus recA and determined its nucleotide sequence . The deduced amino acid (aa) sequence of RecA contained 347 aa and was 74% identical to B . subtilis RecA . Using a cloned DNA fragment originating from within S . aureus recA, we then constructed a recA null mutant strain, designated KB103, which exhibited the same phenotypic characteristics imposed by the uvs-568 mutation in the same background . Furthermore, genetic and physical mapping of S . aureus recA placed it in the same region as the uvs-568 mutation . These data strongly suggest that these mutations represent different alleles of the same recA gene. EMBO J, 1994 Sep 15, 13(18), 4353 - 60 A new protein domain for binding to DNA through the minor groove; Freire R et al.; Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication . Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding . We have constructed site-directed mutants at the polar side of the putative alpha-helix . DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained . A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant . Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment . This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove . In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA . All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. Gene, 1994 Sep 15, 147(1), 91 - 4 Construction of His6-tagging vectors allowing single-step purification of GroES and other polypeptides produced in Bacillus subtilis; Schon U et al.; Two plasmid expression vectors for Bacillus subtilis have been constructed that direct the synthesis of fusion proteins containing a stretch of six histidine residues (His6) at either the N or C terminus . The His6 tag allows the rapid enrichment of proteins by metal chelate affinity chromatography in a denatured or native state . As an example of the general utility of one of these expression vectors, we produced the GroES protein encoded by the groESL operon of B . stearothermophilus. Nucleic Acids Res, 1994 Sep 11, 22(18), 3715 - 21 The DNA polymerase genes of several HMU-bacteriophages have similar group I introns with highly divergent open reading frames; Goodrich-Blair H et al.; A previous report described the discovery of a group I, self-splicing intron in the DNA polymerase gene of the Bacillus subtilis bacteriophage SPO1 (1) . In this study, the DNA polymerase genes of three close relatives of SPO1: SP82, 2C and phi e, were also found to be interrupted by an intron . All of these introns have group I secondary structures that are extremely similar to one another in primary sequence . Each is interrupted by an open reading frame (ORF) that, unlike the intron core or exon sequences, are highly diverged . Unlike the relatives of Escherichia coli bacteriophage T4, most of which do not have introns (2), this intron seems to be common among the relatives of SPO1. J Bacteriol, 1994 Sep, 176(18), 5831 - 4 Overproduction, isolation, and DNA-binding characteristics of Xre, the repressor protein from the Bacillus subtilis defective prophage PBSX; McDonnell GE et al.; PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168 . Lysogeny is maintained by the PBSX-encoded repressor, Xre . The Xre protein was overproduced in Escherichia coli and isolated by affinity chromatography . Gel retardation and DNase I footprinting studies indicated that Xre binds to four sites close to its own gene . These sites overlap putative promoters for xre and a divergent transcriptional unit, containing the middle genes. J Bacteriol, 1994 Sep, 176(18), 5820 - 30 Genetic control of bacterial suicide: regulation of the induction of PBSX in Bacillus subtilis; McDonnell GE et al.; PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168 . Bacteria carrying the PBSX genome are induced by DNA-damaging agents to lyse and produce PBSX particles . The particles cannot propagate the PBSX genome . The particles produced by this suicidal response kill strains nonlysogenic for PBSX . A 5.2-kb region which controls the induction of PBSX has been sequenced . The genes identified include the previously identified repressor gene xre and a positive control factor gene, pcf . Pcf is similar to known sigma factors and acts at the late promoter PL, which has been located distal to pcf . The first two genes expressed from the late promoter show homology to genes encoding the subunits of phage terminases. J Bacteriol, 1994 Sep, 176(18), 5796 - 801 Effect of alteration of charged residues at the N termini of signal peptides on protein export in Bacillus subtilis; Chen M et al.; The role of positively charged residues at the N termini of signal peptides in protein export has been studied in Bacillus subtilis . Bacillus signal peptides (alkaline protease {Apr} and neutral protease {Npr} from Bacillus amyloliquefaciens) were altered and fused to mature levansucrase (Lvs) . The effects of the various alterations on the export of Lvs in B . subtilis were determined . The replacement of positively charged residues with neutral residues in both Apr and Npr signal peptides resulted in a slight defect in the export of Lvs from B . subtilis . Introduction of a negatively charged residue (aspartic acid) at the N terminus of Npr signal peptide blocked the export of Lvs . However, Apr signal peptide with a net charge of -3 (three aspartic acid residues) was still functional. J Bacteriol, 1994 Sep, 176(18), 5762 - 70 comK acts as an autoregulatory control switch in the signal transduction route to competence in Bacillus subtilis; van Sinderen D et al.; The comK gene is a regulatory transcription unit which is essential for the development of genetic competence in Bacillus subtilis . The transcription of comK is under strict nutritional and growth phase-dependent control and has been shown to depend on the gene products of comA and srfA . In this report, we show that expression of comK is dependent on its own gene product as well as on the gene products of all other tested regulatory genes known to be involved in competence development (abrB, comA, comP, degU, sin, spo0A, spo0H, spo0K, and srfA) . A mecA mutation is able to suppress the competence deficiency of mutations in any of these regulatory loci except for mutations in spo0A and, as we show here, in comK . Furthermore, we show that the presence of comK on a multiple copy plasmid leads to derepression of comK expression, causing an almost constitutive expression of competence in minimal medium as well as permitting competence development in complex medium . We infer from these results that the signals which trigger competence development, after having been received and processed by the various components of the competence signal transduction pathway, all converge at the level of comK expression . As soon as derepression of comK expression occurs, the positive autoregulation rapidly results in accumulation of the comK gene product, which subsequently induces competence. J Bacteriol, 1994 Sep, 176(18), 5753 - 61 The regulation of competence transcription factor synthesis constitutes a critical control point in the regulation of competence in Bacillus subtilis; Hahn J et al.; comK, which encodes the competence transcription factor, is itself transcriptionally activated at the transition from exponential growth to stationary phase in Bacillus subtilis . MecA, a negative regulator of competence, also inhibits comK transcription when overexpressed, and a mecA null mutation results in comK overexpression . Although null mutations in mecA, as well as in another gene, mecB, are known to bypass the requirements for nearly all of the competence regulatory genes, the comK requirement is not suppressed by mecA inactivation . Various competence regulatory genes (comA, srfA, degU, abrB, sin, and spo0A) are shown to be required for the expression of comK . srfA transcription is shown to occur equally in cells destined for competence and those destined not to become competent . In contrast, comK transcription is restricted to the presumptive competent cells . These and other results are combined to describe a regulatory pathway for competence. J Bacteriol, 1994 Sep, 176(18), 5673 - 80 Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis; Ogura M et al.; Bacillus subtilis secretes extracellular proteases whose production is positively regulated by a two-component regulatory system, DegS-DegU, and other regulatory factors including DegR . To identify an additional regulatory gene(s) for exoprotease production, we performed a shotgun cloning in the cell carrying multiple copies of degR and found a transformant producing large amounts of the exoproteases . The plasmid in this transformant, pLC1, showed a synergistic effect with multiple copies of degR on the production of the extracellular proteases, and it required degS for its enhancing effect . The DNA region responsible for the enhancement contained the proB gene, as shown by restriction analyses and sequence determination . The proB gene encoding gamma-glutamyl kinase was followed by the proA gene encoding glutamyl-gamma-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon . pLC1 contained the complete proB gene and a part of proA lacking the proA C-terminal region . It was also found that proB on the chromosome showed a synergistic effect with multiple copies of degR . We consider on the basis of these results that the metabolic intermediate, gamma-glutamyl phosphate, would transmit a signal to DegS, resulting in a higher level of phosphorylated DegU . Possible involvement of DegR in this process is discussed. J Bacteriol, 1994 Sep, 176(17), 5466 - 73 Analysis of Bacillus subtilis hut operon expression indicates that histidine-dependent induction is mediated primarily by transcriptional antitermination and that amino acid repression is mediated by two mechanisms: regulation of transcription initiation and inhibition of histidine transport; Wray LV Jr et al.; Expression of the Bacillus subtilis hut operon is induced by histidine and subject to regulation by carbon catabolite repression and amino acid repression . A set of hut-lacZ transcriptional fusions was constructed and used to identify the cis-acting sites required for histidine induction and amino acid repression . Histidine induction was found to be primarily mediated by transcriptional antitermination at a palindromic sequence located immediately downstream of the first structural gene in the hut operon, hutP . High levels of histidine induction were observed only in hut-lacZ fusions which contained this palindromic sequence . The hutC1 mutation, which results in constitutive expression of the hut operon, was sequenced and found to contain a GC to TA transversion located within the stem-loop structure . Transcription of hut DNA in vitro revealed that the palindromic structure functions as a transcriptional terminator with wild-type hut DNA but not with hutC1 DNA . Two sites were found to be involved in amino acid repression of hut expression: (i) an operator, hutOA, which lies downstream of the hut promoter, and (ii) the hut terminator . The rate of {14C}histidine uptake in amino acid-grown cells was sixfold lower than that seen in cells grown without amino acids . Thus, inhibition of histidine transport in amino acid-grown cells indirectly regulates hut expression by interfering with histidine induction at the hut terminator. J Bacteriol, 1994 Sep, 176(17), 5364 - 71 Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline; Boch J et al.; Exogenously provided glycine betaine functions as an efficient osmoprotectant for Bacillus subtilis in high-osmolarity environments . This gram-positive soil organism is not able to increase the intracellular level of glycine betaine through de novo synthesis in defined medium (A . M . Whatmore, J . A . Chudek, and R . H . Reed, J . Gen . Microbiol . 136:2527-2535, 1990) . We found, however, that B . subtilis can synthesize glycine betaine when its biosynthetic precursor, choline, is present in the growth medium . Uptake studies with radiolabelled {methyl-14C}choline demonstrated that choline transport is osmotically controlled and is mediated by a high-affinity uptake system . Choline transport of cells grown in low- and high-osmolarity media showed Michaelis-Menten kinetics with Km values of 3 and 5 microM and maximum rates of transport (Vmax) of 10 and 36 nmol min-1 mg of protein-1, respectively . The choline transporter exhibited considerable substrate specificity, and the results of competition experiments suggest that the fully methylated quaternary ammonium group is a key feature for substrate recognition . Thin-layer chromatography revealed that the radioactivity from exogenously provided {methyl-14C}choline accumulated intracellularly as {methyl-14C}glycine betaine, demonstrating that B . subtilis possesses enzymes for the oxidative conversion of choline into glycine betaine . Exogenously provided choline significantly increased the growth rate of B . subtilis in high-osmolarity media and permitted its proliferation under conditions that are otherwise strongly inhibitory for its growth . Choline and glycine betaine were not used as sole sources of carbon or nitrogen, consistent with their functional role in the process of adaptation of B . subtilis to high-osmolarity stress. J Bacteriol, 1994 Sep, 176(17), 5357 - 63 Properties of Bacillus subtilis small, acid-soluble spore proteins with changes in the sequence recognized by their specific protease; Carrillo-Martinez Y et al.; Alpha/beta-type small, acid-soluble proteins (SASP) of dormant spores of Bacillus subtilis bind to DNA and increase its resistance to a variety of damaging agents both in vivo and in vitro . When spores germinate, degradation of alpha/beta-type SASP is rapidly initiated by a sequence-specific protease, which is termed GPR . Three mutations have been introduced into the B . subtilis sspC gene, which codes for the wild-type alpha/beta-type SASP SspCwt; all three mutations change residues in the highly conserved sequence recognized by GPR . In one mutant protein (SspCV), residue 33 (Ser) was changed to Val; in the second (SspCDL), residues 30 and 31 (Glu and Ile) were changed to Asp and Leu, respectively; and in the third mutant protein (SspCDLV), residues 30, 31, and 33 were changed to Asp, Leu, and Val . All three mutant proteins were rapidly degraded by GPR during spore germination, and SspCDL and SspCDLV were degraded by GPR in vitro at rates 8 to 9% of that for SspCwt, although not exclusively at the single site cleaved by GPR in SspCwt . These results indicate (i) that the sequence specificity of GPR is broader than originally imagined and (ii) that GPR can cleave the sequence in SspCDLV . Since the latter sequence is identical to that cleaved during the proteolytic activation of GPR, this result further supports an autoprocessing model for GPR activation during sporulation . The properties of these mutant proteins were also examined, both in vivo in B . subtilis spores and in Escherichia coli and in vitro with purified protein . SspC(v) interacted with DNA similarly to SspC(wt) in vivo, resorting UV and heat resistance to spores lacking major alpha/beta-type SASP to the same extent as SspC(wt) . In contrasst, SspC(DL) had much less effect on DNA properties in vivo and bound strongly only to poly(dG) . poly(dC) in vitro; SspC(DLV) exhibited only weak binding to poly(dG).poly(dC) in vitro . These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the binding of these proteins to spore DNA and alteration of DNA properties and show further that the GRP recognition region in alpha/beta-type SASP plays some role in DNA binding. J Bacteriol, 1994 Sep, 176(17), 5320 - 9 spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis; Ireton K et al.; The spo0J gene of Bacillus subtilis is required for the initiation of sporulation . We show that the sporulation defect caused by null mutations in spo0J is suppressed by a null mutation in the gene located directly upstream from spo0J, soj (suppressor of spo0J) . These results indicate that Soj inhibits the initiation of sporulation and that Spo0J antagonizes that inhibition . Further genetic experiments indicated that Soj ultimately affects sporulation by inhibiting the activation (phosphorylation) of the developmental transcription factor encoded by spo0A . In addition, the temperature-sensitive sporulation phenotype caused by the ftsA279 (spoIIN279) mutation was partly suppressed by the soj null mutation, indicating that FtsA might also affect the activity of Soj . Soj and Spo0J are known to be similar in sequence to a family of proteins involved in plasmid partitioning, including ParA and ParB of prophage P1, SopA and SopB of F, and IncC and KorB of RK2, spo0J was found to be required for normal chromosome partitioning as well as for sporulation . spo0J null mutants produced a significant proportion of anucleate cells during vegetative growth . The dual functions of Spo0J could provide a mechanism for regulating the initiation of sporulation in response to activity of the chromosome partition machinery. J Bacteriol, 1994 Sep, 176(17), 5218 - 24 Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting; Rong JC et al.; The Bacillus subtilis sigA gene encodes the primary sigma factor of RNA polymerase and is essential for cell growth . We have mutated conserved region 2.3 of the sigma A protein to substitute each of seven aromatic amino acids with alanine . Several of these aromatic amino acids are proposed to form a melting motif which facilitates the strand separation step of initiation . Holoenzymes containing mutant sigma factors recognize promoters, but some are defective for DNA melting in vitro . We have studied the ability of each mutant sigma factor to support cell growth by gene replacement and complementation . The two region 2.3 mutants least impaired in promoter melting in vitro (Y180A and Y184A) support cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures . A strain expressing only the Y189A variant of the sigma A protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis . Only the wild-type and Y180A sigma A proteins efficiently complement a temperature-sensitive allele of sigA . Overexpression of three of the sigma A proteins defective for promoter melting in vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death . These results indicate that mutations which specifically impair DNA melting in vitro also impair sigma function in vivo and therefore support the hypothesis that sigma plays an essential role in both DNA melting and promoter recognition. Microbiol Res, 1994 Sep, 149(3), 241 - 6 Effects of the antimycotic molecule Iturin A2, secreted by Bacillus subtilis strain M51, on arbuscular mycorrhizal fungi; Citernesi AS et al.; A new system, devised for the study of early stages of arbuscular mycorrhizal infection, was used to test the effect of the biological control agent Iturin A2, secreted by the strain M51 of Bacillus subtilis, on the development of arbuscular mycorrhizal fungi . The saprophytic growth of the fungus Glomus mosseae was inhibited by Iturin A2 concentrations higher than 100 micrograms/g of sand; whereas, in the presence of the tomato host plant, both, pre-infection events and intraradical growth were not negatively influenced by the antifungal compound; furthermore, the development of arbuscular mycorrhizal symbiosis was not impeded by the biocontrol agent in field conditions, while Fusarium oxysporum f . sp . lycopersici infection was hindered. Berl Munch Tierarztl Wochenschr, 1994 Sep, 107(9), 308 - 13 {The identification of Bacillus cereus, Bacillus lichenformis and Bacillus subtilis strains using the coagglutination reaction}; Hellmann E et al.; Polyclonal rabbit antisera against 4 B . cereus strains were selected from a total of 9 B . cereus antisera and pooled . This serum agglutinated all available B . cereus strains (n = 63) at a titre > or = 1:64 when tested by a slide co-agglutination reaction . One hundred and thirty-six Bacillus strains belonging to 17 other species reacted with higher serum concentrations only (titres mainly < or = 1:16) . A pooled antiserum comprised of two B . licheniformis antisera and two B . subtilis antisera agglutinated all 43 B . licheniformis strains and all 38 B . subtilis strains at a titre > or = 1:128, but one strain each of the species B . pumilus, B . macerans and B . fastidiosus was also agglutinated at a titre 1:128. Microbiology, 1994 Sep, 140 ( Pt 9), 2299 - 304 Identification of a control region for expression of the forespore-specific Bacillus subtilis locus spoVA; Moldover B et al.; The role of a 20 bp conserved region located 45-64 nucleotides 5' of the spoVA transcription start point in Bacillus subtilis and Bacillus licheniformis was investigated by deletion analysis and by mobility shift assay . Deletions 5' of this conserved sequence had little effect on expression of a spoVA-lacZ fusion, whereas deletions extending into the sequence reduced expression of the spoVA-lacZ fusion by 85% . The timing of expression of spoVA was not affected by deletion of the sequence . The region was shown by mobility shift assays to bind specifically to a protein . Binding activity was detected in protein extracts prepared from bacteria 1 h or more after they had started to sporulate, but not in extracts prepared from vegetative bacteria . Mutations in all known spoO loci were screened but none prevented appearance of the binding activity; nor did mutations in any of the stage II and III loci tested . It is concluded that the 20 bp conserved region is the binding site of an activator that is subject to temporal regulation independent of known spo loci. Microbiology, 1994 Sep, 140 ( Pt 9), 2289 - 98 Cloning and nucleotide sequencing of a 15 kb region of the Bacillus subtilis genome containing the iol operon; Yoshida K et al.; Within the framework of an international project on the sequencing of the whole Bacillus subtilis genome, a 15 kb chromosome segment, which contains the iol operon involved in inositol utilization, has been cloned and sequenced . This region (14,974 bp) contains 12 complete open reading frames (ORFs; genes) and two partial ones; the seventh gene (E83G) is the idh gene encoding inositol dehydrogenase . All the genes identified are transcribed in the same direction as that of the movement of the replication fork . A homology search for their products deduced from the 12 complete ORFs revealed that eight of them exhibit significant homology to known proteins such as fructokinase, acetolactate synthase, fructose-1,6-bisphosphate aldolase (B . subtilis), and PhoB and FtsE proteins (Escherichia coli) . It also implied that two genes (B65D and B65E) might encode a set of two-component regulatory proteins and that the B65F gene might encode a protein belonging to the ATP-binding cassette (ABC) family . Based on the features of the nucleotide sequence determined and the results of the homology search, the primary structure of the iol operon is predicted. Microbiology, 1994 Sep, 140 ( Pt 9), 2279 - 88 Analysis of Bacillus subtilis tag gene expression using transcriptional fusions; Mauel C et al.; Five of the genes known to encode the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF . To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions . Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA . No influence of the chromosomal context was observed . Phosphate limitation was accompanied by reduced tag gene expression . Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II . During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth . In contrast to tagC (dinC), the expression of which is DNA-damage-inducible, the induction of SOS functions had no effect on tagA and tagD gene expression . The biological significance of these results is discussed. Appl Environ Microbiol, 1994 Sep, 60(9), 3425 - 8 Purification and some properties of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6; Kaneko S et al.; alpha-L-Arabinofuranosidase (EC 3.2.1.55) was purified from culture supernatant of Bacillus subtilis 3-6 . The enzyme had a molecular weight of 61,000 and displayed maximum activity at pH 7.0 and 60 degrees C . It released arabinose from O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranos e (A1X2), O-beta-D-xylopyranosyl-(1-->4)-{O-alpha-L-arabinofuranosyl-(1-->3)}- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (A1X3), and arabinan, but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L- arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyr anosyl- (1-->4)-D-xylopyranose (A1X4), arabinoxylan, gum arabic, or arabinogalactan. Appl Environ Microbiol, 1994 Sep, 60(9), 3381 - 9 The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis; Hofemeister B et al.; We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1) . A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis . The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T . vulgaris 94-2A culture . The amino acid sequence was aligned with several known alpha-amylase sequences . We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T . vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47 . The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B . subtilis . Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1 . The expression levels in B . subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene. J Dent Res, 1994 Sep, 73(9), 1560 - 7 Sterilization of teeth by gamma radiation; White JM et al.; Clinical simulations and restorative materials research and development conducted in vitro require the use of large numbers of extracted teeth . The simultaneous need for infection control procedures and minimal alterations of structure and properties of the tissue prompted this study of gamma irradiation as a method to eliminate microbes associated with extracted teeth and their storage solutions . Evaluations of potential change in structure of dentin were conducted in terms of permeability, Fourier transform infrared spectroscopy (FTIR), and optical properties . The dose required for sterilization by gamma irradiation was established by means of a tooth model inoculated with Bacillus subtilis (10(8) organisms/mL) . Sterilization occurred at a dose above 173 krad with use of a Cesium (Cs137) radiation source . Gamma irradiation did not affect permeability of crown segments of dentin . A comparative evaluation of the effects of four sterilization methods on dentin disks was based on FTIR and ultraviolet-visible-near infrared (UV/VIS/NIR) spectra before and after sterilization by (1) gamma irradiation; (2) ethylene oxide; (3) dry heat; and (4) autoclaving . No detectable changes were found with gamma irradiation, but all other methods introduced some detectable change in the spectra . This suggests that common methods of sterilization alter the structure of the dentin, but gamma irradiation shows promise as a method which both is effective and introduces no detectable changes as measured by FTIR, UV/VIS/NIR, or permeability. FEMS Microbiol Lett, 1994 Sep 1, 121(3), 315 - 20 Molecular genetical and phenotypical analysis of the gerM spore germination gene of Bacillus subtilis 168; Slynn GM et al.; The gerM gene, encoding a single product of 22.5 kDa, has been identified by subcloning and sequencing of DNA recovered from adjacent to a Tn917 insertion . The gene product has a potential lipoprotein signal sequence, but otherwise has no homology to known sequences . Spores of the gerM mutant were more heat sensitive than wild-type, but their dipicolinic acid content was normal . The level of cortical peptidoglycan in mutant spores is also normal but release at germination of hexosamine-containing fragments, the breakdown products of cortex degradation, is less complete than wild-type . The sporulation, resistance and germination phenotypes of the gerM mutant would be consistent with the gene product having a role, either directly or indirectly, in peptidoglycan synthesis during sporulation. Eur J Biochem, 1994 Sep 1, 224(2), 519 - 24 Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases; Janecek S; Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions . These sequence regions were examined from structure/function and evolutionary perspectives . An unrooted evolutionary tree of alpha-amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions . The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctis alpha-amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous alpha-amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch-saccharifying Bacillus subtilis alpha-amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an alpha-amylase with an unusually large polypeptide chain (943 residues in the mature enzyme) . Due to a very high degree of similarity, the whole amino acid sequences of three groups of alpha-amylases, namely (a) fungi and yeasts, (b) plants, and (c) A . haloplanctis, streptomycetes, T . curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments . Possible rooting of the trees was also discussed . Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic alpha-amylase, the possible disulfide bridges were established for each of these groups of homologous alpha-amylases. Mikrobiologiia, 1994 Sep-Oct, 63(5), 812 - 20 {Growth and spore formation of Bacillus subtilis in periodic and dialysis cultured: experiment and modeling}; Evdokimova NV et al.; The transition of bacilli to maintenance state in dialysis culture was revealed . The maintenance state is characterized by the lack of growth and spore formation . The experimental results are satisfactorily described by a modification of the synthetic chemostat model . As a results of model realization it was supposed that maintenance state of bacilli was connected with synthesis of autoinhibitor and initiation of spore formation. J Biochem (Tokyo), 1994 Sep, 116(3), 554 - 9 Substrate-dependent change in the pH-activity profile of alkaline endo-1,4-beta-glucanase from an alkaline Bacillus sp; Hitomi J et al.; A neutral cellulase (BSC) from Bacillus subtilis and an alkaline cellulase (NK1) from alkalophilic Bacillus sp . N-4 show significant amino acid sequence homology . Despite the high homology, the pH-activity profiles of the two enzymes for carboxymethyl cellulose (CMC) hydrolysis are quite different; BSC shows a sharp optimum pH at 6, whereas NK1 shows its full activity in a broad range, from pH 6 to 10.5 . For elucidation of the reasons for the difference in their pH-activity profiles, their activities were examined at various pHs using a series of cellooligosaccharides and their derivatives, cellotetraose (G4), cellopentaose (G5), cellohexaose (G6), cellopentaitol (G5OH), and cellohexaitol (G6OH), as substrates . The optimum pH of BSC was around 6 for all the cellooligosaccharides examined . On the other hand, the optimum pH of NK1 varied depending on the substrate, i.e., a sharp optimum at pH 6 with G4 and G5OH, and a broad optimum of pH 6 to 10.5 with G5, G6, and G6OH . Comparison of the kinetic parameters of the two cellulases at pH 7 and 9 using G6OH as a substrate revealed that NK1 showed similar values at both pHs, while BSC showed a greatly increased Km value for this substrate at pH 9 . In addition, NK1 showed a greatly increased Km value for G5OH hydrolysis at pH 9 . Both enzymes cleaved these substrates at the same position, which suggests the same productive binding mode of these substrates with both enzymes . All these observations suggest that the reduced enzyme activity of BSC in the alkaline pH range can be attributed to a decrease in the affinity of a subsite for the third glucose moiety from the scissile site of these substrates. J Nat Prod, 1994 Sep, 57(9), 1245 - 50 Antibacterial triterpenoid acids from Dillenia papuana; Nick A et al.; Three new oleanene-type triterpenoids, dillenic acids A {1}, B {2}, and C {3}, and two known compounds, 3-oxoolean-1,12-dien-30-oic acid {4} (a new natural product) and the lupene derivative betulinaldehyde, have been isolated from the Papua New Guinean medicinal plant Dillenia papuana . The structures of the new compounds were elucidated as 2 alpha-hydroxy-3-oxoolean-12-en-30-oic acid, 2-oxo-3 beta-hydroxyolean-12-en-30-oic acid and 1 alpha-hydroxy-3-oxoolean-12-en-30-oic acid . The 1H- and 13C-nmr data of all new compounds were assigned unambiguously using a variety of 2D nmr experiments including 1H-1H-COSY, HMQC, HMBC, and NOESY experiments . Of these compounds, 1-4 showed antibacterial activities against Bacillus subtilis, Escherichia coli, and Micrococcus luteus. Nucleic Acids Res, 1994 Sep, 22(17), 3660 - 2 The Ribonuclease P database; Brown JW et al.; The Ribonuclease P Sequence database is a compilation of RNase P sequences, sequence alignments, secondary structures, three-dimensional models, and accessory information . In its initial form, the database contains information on RNase P RNA in bacteria and archaea, and RNase P protein in bacteria . The sequences themselves are presented phylogenetically ordered and aligned . The database also contains secondary structures of bacterial and archaeal RNAs, including specially annotated 'reference' secondary structures of Escherichia coli and Bacillus subtilis RNase P RNAs, a minimum phylogenetic consensus structure, and coordinates for models of three-dimensional structure. EMBO J, 1994 Sep 1, 13(17), 3945 - 52 Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA; LaGrandeur TE et al.; Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs . The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA . RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension . Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA . Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis . Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected . One protection among all three RNAs was attributed to the precursor segment of pre-tRNA . Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection . Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure . Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop. Biochemistry, 1994 Aug 30, 33(34), 10401 - 7 Metal-metal bonding in biology: EXAFS evidence for a 2.5 A copper-copper bond in the CuA center of cytochrome oxidase; Blackburn NJ et al.; Evidence for a direct Cu-Cu bond in the CuA center of cytochrome oxidase is reported . Simulation of the X-ray absorption spectrum of a recombinant CuA-binding domain of Bacillus subtilis cytochrome oxidase, and comparison with a structurally characterized directly-bonding Cu(1.5) .. . Cu(1.5) inorganic complex, suggests that a Cu-Cu interaction of 2.5 +/- 0.1 A together with a short 2.2 A Cu-S interaction may be present in the CuA site . In light of these data, previous interpretations of the EXAFS of a number of cytochrome oxidase and nitrous oxide reductase enzymes which modeled the 2.6 A interaction as a long Cu-S(methionine) bond are possibly incorrect . A structural model based on the new data is presented which suggests that the CuA sites in cytochrome oxidase and N2O reductase are likely composed of a pair of modified type 1 copper centers with one histidine, one cysteine, and one weakly bound ligand (Met and/or Gln) joined by a Cu-Cu bond. Biochemistry, 1994 Aug 30, 33(34), 10294 - 304 A kinetic mechanism for cleavage of precursor tRNA(Asp) catalyzed by the RNA component of Bacillus subtilis ribonuclease P; Beebe JA et al.; A kinetic mechanism is presented for the cleavage of Bacillus subtilis precursor tRNA(Asp) catalyzed by the RNA component of B . subtilis ribonuclease P (RNase P) under optimal conditions (50 mM Tris Cl (pH 8.0), 100 mM MgCl2, and 800 mM NH4Cl, 37 degrees C) . This kinetic mechanism was derived from measuring pre-steady-state, steady-state, single-turnover, and binding kinetics using a combination of quench-flow, gel filtration, and gel shift techniques . A minimal kinetic description involves the following: (1) binding of pre-tRNA(Asp) to RNase P RNA rapidly (6.3 x 10(6) M-1 s-1), but slower than the diffusion-controlled limit; (2) cleavage of the phosphodiester bond with a rate constant of 6 s-1; (3) dissociation of products in a kinetically preferred pathway, with the 5' RNA fragment dissociating first (> or = 0.2 s-1) followed by rate-limiting tRNA dissociation (0.02 s-1); and (4) formation of a second conformer of RNase P RNA during the catalytic cycle that is less stable and binds pre-tRNA(Asp) significantly more slowly (7 x 10(4) M-1 s-1) . This scheme involves the isolation of individual steps in the reaction sequence, is consistent with steady-state data, and pinpoints the rate-determining step under a variety of conditions . This kinetic mechanism will facilitate a more accurate definition of the role of metals, pH, and the protein component in each step of the reaction and provide an essential background for understanding the influence of structural changes on the catalytic activity. Nucleic Acids Res, 1994 Aug 25, 22(16), 3392 - 6 tRNA-like structures in 10Sa RNAs of Mycoplasma capricolum and Bacillus subtilis; Ushida C et al.; The stable RNAs, whose sequences are homologous to 10Sa RNA of Escherichia coli, have been isolated from Mycoplasma capricolum and Bacillus subtilis, both belonging to the Gram-positive bacterial group . The total nucleotide sequences of the RNAs have been determined by partial RNA sequencing and DNA sequencing of their genes . A comparison of the sequences, together with those of other bacterial 10Sa RNAs so far known, has shown that the 5'- and 3'-end sequences are well conserved among species, while the central parts reveal little homologies . Unexpectedly, the conserved 5'- and 3'-regions can be folded in a common tRNA-like structure containing an amino acid-acceptor stem and a T phi C-stem/loop . The 3'-terminal CCA sequence of B.subtilis 10Sa RNA is not encoded on the DNA, but is added after transcription . Furthermore, the RNA is aminoacylatable with alanine in vitro, and binds to the 70S ribosome in vivo. Biochemistry, 1994 Aug 23, 33(33), 9960 - 7 Zinc chelation and structural stability of adenylate kinase from Bacillus subtilis; Perrier V et al.; Adenylate kinase from Bacillus subtilis, like the enzyme from Bacillus stearothermophilus, contains a structural zinc atom . Cys153 in the enzyme from B . stearothermophilus, which is involved in the zinc coordination, is replaced in the adenylate kinase from B . subtilis by an aspartic acid residue . Therefore, we were interested in establishing whether this difference has an impact on the structure, the metal chelation, and the overall stability of these proteins . We also were interested in determining whether His138, which is conserved in many adenylate kinases, can act as a fourth partner in the metal chelation and, in general, whether His can successfully replace Cys or Asp in coordinating zinc in the adenylate kinase from B . subtilis . The adk gene from B . subtilis was cloned by polymerase chain reaction . The wild-type protein, together with several variants obtained by site-directed mutagenesis, were expressed in Escherichia coli and analyzed by biochemical and physicochemical methods . The H138N and D153C mutants of adenylate kinase from B . subtilis exhibited properties similar to those of the wild-type protein, indicating that His138 is not involved in metal coordination and that Asp153, just like Cys in the analogous position in the enzyme from B . stearothermophilus, can participate in zinc chelation . This is the first experimental evidence indicating that aspartic acid can be involved in the coordination of a structural zinc atom . On the other hand, the D153H and D153T variants showed significant changes in their zinc-binding properties . Dialysis of the latter proteins against buffer (in both the presence and the absence of 2 mM EDTA) resulted in removal of the metal ion and loss of enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 23, 33(33), 9953 - 9 Monofunctional chorismate mutase from Bacillus subtilis: FTIR studies and the mechanism of action of the enzyme; Gray JV et al.; The Fourier transform infrared (FTIR) spectrum of the complex between prephenate and the monofunctional chorismate mutase from Bacillus subtilis displays one prominent band at 1714 cm-1 . Using isotopically-labeled ligand, we have shown that this band corresponds to the ketonic carbonyl stretching vibration of enzyme-bound prephenate . The frequency of this carbonyl vibration of prephenate does not change significantly on binding to the protein . These data indicate that chorismate mutase does not use electrophilic catalysis in the rearrangement of chorismate . A comparison of the resolution-enhanced FTIR spectra of the unliganded mutase and of the protein complexed with its ligands reveals marked differences in the amide I' vibration band . These changes suggest that structural alterations in the protein occur upon binding prephenate . When combined with information from the crystal structure of the enzyme and its complexes, it appears that significant ordering of the C-terminal region occurs upon ligand binding . These changes at the active site may be important for efficient catalysis and likely influence the association and dissociation rates of the enzyme and its ligands . The enzymic rearrangement of chorismate evidently proceeds via a pericyclic process, and much, if not all, of the rate acceleration derives from the selective binding of the appropriate conformer of the substrate, with some additional contribution possible from electrostatic stabilization of the transition state. J Mol Biol, 1994 Aug 19, 241(3), 335 - 40 The minimal sequence needed to define a functional DNA terminator in Bacillus subtilis; Smith MT et al.; The 47 bp DNA replication terminator (IRI) of Bacillus subtilis, contains two binding sites, A and B, for the replication terminator protein (RTP) . Each site binds a dimer of RTP . Removal of the first two base-pairs (bp 1-2) from IRI completely destroyed in vivo terminator (fork arrest) function and was accompanied by loss of RTP binding to the A site, which is distal to the approaching fork that is arrested . Removal of base-pairs 34 to 47 from the other end, proximal to the approaching fork, lowered in vivo function to approximately 50% of the complete IRI . RTP binding appeared to be largely unaffected . Terminator function remained at the approximately 50% level with further deletions that proceeded as far as to include base-pair 28; and RTP binding remained largely unaffected . Removal of more of the sequence beyond base-pair 27 and into the region that makes extensive contact with RTP resulted in a further impairment to in vivo function, and caused altered RTP binding . The base-pairs 1 to 24 segment retained only 16% fork arrest activity and the effect on RTP binding was largely evidenced by an elimination of the ability of this extensively truncated sequence to fill the B site alone . The behaviour of the various terminator deletions emphasize the importance of the previously defined RTP-DNA contacts which allow the binding of RTP to the two overlapping sites, A and B, of IRI for terminator function . A comparison of the affinities of selected truncated terminators for RTP raises the possibility that the overall affinity of RTP for its DNA terminator is not the sole determinant of terminator function. Biochim Biophys Acta, 1994 Aug 17, 1207(2), 159 - 64 Site-directed mutagenesis of the putative active site of endoglucanase K from Bacillus sp . KSM-330; Ozaki K et al.; The roles of one Glu and four Asp residues of endoglucanase K from Bacillus sp . KSM-330, which are conserved in all the endo-beta-glucanases in the family D, were analyzed by site-directed mutagenesis . The gene for endoglucanase K was mutated to replace Asp-154, Asp-191, Asp-193 or Asp-300 by Asn, or to replace Glu-130 by Gln in the encoded enzyme . Mutant and wild-type genes were separately expressed in Bacillus subtilis and the resultant enzymes were purified from the culture broth . All mutant enzymes exhibited the same mobility on SDS-polyacrylamide gel electrophoresis as the wild-type enzyme and gave similar circular dichroism spectra to that of the wild-type enzyme . Substitution of Glu-130, Asp-191, Asp-193 or Asp-300 significantly decreased the specific activity of the enzyme toward CM-cellulose . Kinetic analysis of the abilities of these mutant enzymes to liberate p-nitrophenol from p-nitrophenylcellotrioside revealed that all the mutant enzymes had very much lower kcat values than that of the wild-type enzyme, while the Km values of these mutant enzymes were almost the same as that of the wild-type enzyme . Of these Glu and Asp residues, Glu-130 and Asp-191 seem to be most likely to be catalytic residues because substitutions of these residues resulted in the lowest kcat values of the mutant enzymes. Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8224 - 8 Electron microscopic studies of the interaction between a Bacillus subtilis alpha/beta-type small, acid-soluble spore protein with DNA: protein binding is cooperative, stiffens the DNA, and induces negative supercoiling; Griffith J et al.; DNA within spores of Bacillus subtilis is complexed with a group of alpha/beta-type small acid-soluble spore proteins (alpha/beta-type SASPs), which have almost identical primary sequences and DNA binding properties . Here electron microscopic and cyclization studies were carried out on alpha/beta-type SASP-DNA complexes . When an alpha/beta-type SASP was incubated with linear DNA, the protein bound cooperatively, forming a helical coating 6.6 +/- 0.4 nm wide with a 2.9 +/- 0.3 nm periodicity . alpha/beta-Type SASP binding to an 890-bp DNA was weakest at an (A+T)-rich region that was highly bent, but binding eliminated the bending . alpha/beta-Type SASP binding did not alter the rise per bp in DNA but greatly increased the DNA stiffness as measured by both electron microscopic and cyclization assays . Addition of alpha/beta-type SASPs to negatively supertwisted DNA led to protein binding without significant alteration of the plectonemically interwound appearance of the DNA . Addition of alpha/beta-type SASPs to relaxed or nicked circular DNA led to molecules that by electron microscopy appeared similar to supertwisted DNA . The introduction of negative supertwists in nicked circular DNA by alpha/beta-type SASPs was confirmed by ligation of these molecules followed by topoisomer analyses using agarose gel electrophoresis. Eur J Biochem, 1994 Aug 15, 224(1), 89 - 96 {Ala4}surfactin, a novel isoform from Bacillus subtilis studied by mass and NMR spectroscopies; Peypoux F et al.; When Bacillus subtilis S 499 was grown on a culture medium containing L-alanine as nitrogen source, a mixture of surfactins was obtained . Suitable chromatographic conditions allowed the separation of isoforms . Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry . It contains a peptide sequence which differs from that of standard surfactin by the replacement of the L-valine residue by L-alanine residue in position 4 . The folding mode of {Ala4}surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed. Anal Biochem, 1994 Aug 15, 221(1), 61 - 5 High-pressure liquid chromatography assay for quantitatively monitoring spore photoproduct repair mediated by spore photoproduct lyase during germination of uv-irradiated Bacillus subtilis spores; Sun Y et al.; The major DNA photoproduct formed upon uv irradiation of Bacillus subtilis spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, informally referred to as spore photoproduct (SP) . A rapid separation technique for detecting and quantitating SP by HPLC was developed to replace traditional paper chromatography . Tritiated thymine and thymine-containing photoproducts from trifluoroacetic acid-hydrolyzed DNA purified from uv-irradiated cells or spores of B . subtilis were identified and isolated from paper chromatograms, subjected to HPLC on a Microsorb phenyl 5-microns column using 100% water as the mobile phase, and detected by scintillation counting of collected fractions . At a flow rate of 1.8 ml per minute, thymine-containing compounds eluted in the order thymine (T; 5.5 min), cis-syn cyclobutyl thymine-thymine dimers (TT; 7.5 min), cyclobutyl uracil-thymine dimers (UT, the acid breakdown product of cytosine-thymine (CT) dimers; 9.5 min), and SP (14.5 min) . The method was used to quantitate the amount of SP produced upon irradiation of B . subtilis spores and to monitor repair of SP in vivo by the enzyme SP lyase during spore germination. J Mol Biol, 1994 Aug 12, 241(2), 178 - 92 Interactions of wild-type and truncated LevR of Bacillus subtilis with the upstream activating sequence of the levanase operon; Martin-Verstraete I et al.; Transcription of the levanase operon of Bacillus subtilis is controlled by LevR, an activator of the NifA/NtrC family of regulators . An upstream activating sequence (UAS) located in a 16 bp palindromic structure has previously been characterized . LevR was overproduced in B . subtilis and interaction between the activator and the UAS was demonstrated by gel shift and footprint experiments . The LevR protein specifically binds to the two-halves of the palindromic structure centered at -125 bases upstream from the transcriptional start site . In addition, footprint analysis suggests that LevR interacts with a third DNA region located at positions -90 to -80 . To investigate the function of the different domains of the LevR activator, stop codons were introduced at various positions in the levR gene . The ability of the truncated LevR polypeptides to activate transcription, to respond to the inducer or to interact with the UAS was tested . The results obtained suggest that LevR is a multidomain protein . The amino-terminal part of the protein is required for DNA binding whereas the central domain allows the activation of transcription . The carboxy-terminal region is involved in the modulation of the LevR activity by the inducer. Appl Environ Microbiol, 1994 Aug, 60(8), 2793 - 801 Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633; Klein C et al.; Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633 . Recently, we reported regarding genes spaB, spaT, and spaC (C . Klein, C . Kaletta, N . Schnell, and K.-D . Entian, Appl . Environ . Microbiol . 58:132-142, 1992) which are involved in the biosynthesis of subtilin, and genes spaR and spaK (C . Klein, C . Kaletta, and K.-D . Entian, Appl . Environ . Microbiol . 59:296-303, 1993), which regulate subtilin biosynthesis via a histidine kinase/response regulator system . Further sequence analysis revealed the presence of three additional open reading frames, spaI, spaF, and spaG, downstream of the structural gene spaS . The spaI gene encodes a hydrophilic 19.3-kDa lipoprotein containing a consensus signal sequence, indicating that this protein might be membrane anchored . A similar gene, nisI, has been identified in the nisin producer . SpaF shows strong homology to members of the family of ABC transporters . spaG encodes a hydrophobic protein which might form the active transporter together with SpaF . Gene disruption mutants in all three genes were still able to produce subtilin; however, these mutants were more sensitive to subtilin than the wild-type strain . These results show that these genes are involved in the immunity mechanism of the producer strain . A similar involvement of an ABC transporter in the self-protection mechanism has been described for the McbE and McbF transporter, which confers immunity against microcin B17 in Escherichia coli . Mutants containing mutations in the genes spaR and spaK, which are responsible for regulation of subtilin biosynthesis, also became more sensitive to subtilin.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1994 Aug 1, 13(15), 3456 - 63 Cloning and expression of the MEP1 gene encoding an ammonium transporter in Saccharomyces cerevisiae; Marini AM et al.; In Saccharomyces cerevisiae, the transport of ammonium across the plasma membrane for use as a nitrogen source is mediated by at least two functionally distinct transport systems whose respective encoding genes are called MEP1 and MEP2 . Mutations in the MEP2 gene affect high affinity, low capacity ammonium transport while mutations in the MEP1 gene disrupt a lower affinity, higher capacity system . In this work, the MEP1 gene has been cloned and sequenced and its expression analyzed . The predicted amino acid sequence reveals a highly hydrophobic, 54 kDa protein with 10 or 11 putative membrane-spanning regions . The predicted Mep1p protein shares high sequence similarity with several bacterial proteins of unknown function, notably the product of the nitrogen-regulated nrgA gene of Bacillus subtilis, and with that of a partial cDNA sequence derived from Caenorhabditis elegans . The Mep1p and related proteins appear to define a new family of transmembrane proteins evolutionarily conserved in at least bacteria, fungi and animals . The MEP1 gene is most highly expressed when the cells are grown on low concentrations of ammonium or on 'poor' nitrogen sources like urea or proline . It is down-regulated, on the other hand, when the concentration of ammonium is high or when other 'good' nitrogen sources like glutamine or asparagine are supplied in the culture medium . The overall properties of Mep1p indicate that it is a transporter of ammonium . Its main function appears to be to enable cells grown under nitrogen-limiting conditions to incorporate ammonium present at relatively low concentrations in the growth medium. J Bacteriol, 1994 Aug, 176(16), 4914 - 23 Analysis of the SOS inducing signal in Bacillus subtilis using Escherichia coli LexA as a probe; Lovett CM Jr et al.; We analyzed the Bacillus subtilis SOS response using Escherichia coli LexA protein as a probe to measure the kinetics of SOS activation and DNA repair in wild-type and DNA repair-deficient strains . By examining the effects of DNA-damaging agents that produce the SOS inducing signal in E . coli by three distinct pathways, we obtained evidence that the nature of the SOS inducing signal has been conserved in B . subtilis . In particular, we used the B . subtilis DNA polymerase III inhibitor, 6-(p-hydroxyphenylazo)-uracil, to show that DNA replication is required to generate the SOS inducing signal following UV irradiation . We also present evidence that single-stranded gaps, generated by excision repair, serve as part of the UV inducing signal . By assaying the SOS response in B . subtilis dinA, dinB, and dinC mutants, we identified distinct deficiencies in SOS activation and DNA repair that suggest roles for the corresponding gene products in the SOS response. J Bacteriol, 1994 Aug, 176(15), 4742 - 9 Overexpression of Bacillus thuringiensis HknA, a histidine protein kinase homology, bypasses early Spo mutations that result in CryIIIA overproduction; Malvar T et al.; The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed protein that is toxic to coleopteran insect larvae . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the asporogenous B . thuringiensis subsp . morrisoni strain EG1351, which harbors the native cryIIIA-encoding 88-MDa plasmid, showed a 2.5-fold overproduction of the CryIIIA protein compared with that of an isogenic wild-type strain . Further studies showed that neither CryIIIA protein synthesis nor CryIIIA protein processing was affected in strain EG1351 during vegetative growth . In an attempt to characterize the EG1351 mutation by complementation of function, the hknA gene was identified and cloned from a B . thuringiensis cosmid library . Primer extension analysis of hknA mRNA in wild-type B . thuringiensis demonstrated that the hknA gene is transcribed during vegetative growth from a sigma A-like promoter . Multiple copies of either the hknA gene or the Bacillus subtilis kinA (spoIIJ) gene were shown to bypass the sporulation defect in strain EG1351 as well as a spo0F mutation in B . thuringiensis EG1634 . Additional studies showed that the hknA gene was not defective in strain EG1351 . The results of this study suggest that hknA encodes a novel histidine protein kinase involved in B . thuringiensis sporulation . We also propose that the CryIIIA-overproducing phenotype of strain EG1351 is most likely due to a defect in the phosphorylation of Spo0A and confirm that CryIIIA production is not dependent on sporulation. J Bacteriol, 1994 Aug, 176(15), 4734 - 41 Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant; Agaisse H et al.; Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase . cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B . thuringiensis . Activation of cryIIIA expression was not altered in B . subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L . This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase . Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B . subtilis wild-type strain grown on nutrient broth sporulation medium . However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains . Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain . beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant . However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A . When the cryIIIA gene is expressed in a B . subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B . thuringiensis strains. J Bacteriol, 1994 Aug, 176(15), 4680 - 90 Transcriptional regulation of Bacillus subtilis citrate synthase genes; Jin S et al.; The Bacillus subtilis citrate synthase genes citA and citZ were repressed during early exponential growth phase in nutrient broth medium and were induced as cells reached the end of exponential phase . Both genes were also induced by treatment of cells with the drug decoyinine . After induction, the steady-state level of citZ mRNA was about five times higher than that of citA mRNA . At least some of the citZ transcripts read through into the isocitrate dehydrogenase (citC) gene . Transcription from an apparent promoter site located near the 3' end of the citZ gene also contributed to expression of citC . In minimal medium, citA transcription was about 6-fold lower when glucose was the sole carbon source than it was when succinate was the carbon source . Expression of the citZ gene was repressed 2-fold by glucose and 10-fold when glucose and glutamate were present simultaneously . This latter synergistic repression is similar to the effect of glucose and glutamate on steady-state citrate synthase enzyme activity . CitR, a protein of the LysR family, appeared to be a repressor of citA but not of citZ. J Bacteriol, 1994 Aug, 176(15), 4669 - 79 Identification of two distinct Bacillus subtilis citrate synthase genes; Jin S et al.; Two distinct Bacillus subtilis genes (citA and citZ) were found to encode citrate synthase isozymes that catalyze the first step of the Krebs cycle . The citA gene was cloned by genetic complementation of an Escherichia coli citrate synthase mutant strain (W620) and was in a monocistronic transcriptional unit . A divergently transcribed gene, citR, could encode a protein with strong similarity to the bacterial LysR family of regulatory proteins . A null mutation in citA had little effect on citrate synthase enzyme activity or sporulation . The residual citrate synthase activity was purified from a citA null mutant strain, and the partial amino acid sequence for the purified protein (CitZ) was determined . The citZ gene was cloned from B . subtilis chromosomal DNA by using a PCR-generated probe synthesized with oligonucleotide primers derived from the partial amino acid sequence of purified CitZ . The citZ gene proved to be the first gene in a tricistronic cluster that also included citC (coding for isocitrate dehydrogenase) and citH (coding for malate dehydrogenase) . A mutation in citZ caused a substantial loss of citrate synthase enzyme activity, glutamate auxotrophy, and a defect in sporulation. J Bacteriol, 1994 Aug, 176(15), 4642 - 5 Mutation of the putative nucleotide binding site of the Bacillus subtilis membrane protein ComFA abolishes the uptake of DNA during transformation; Londono-Vallejo JA et al.; ComFA is a membrane protein required for the uptake of transforming DNA following its binding to the Bacillus subtilis competent-cell surface . ComFA, which resembles members of the DEAD family of ATP-driven helicases, contains sequences similar to those found in many ATP-binding proteins and thought to represent the ATP-binding sites of these proteins . We have suggested that ComFA may function as a DNA translocase and/or helicase, using the energy of ATP hydrolysis to mediate the uptake of DNA . As a partial test of this hypothesis, we have introduced mutations into highly conserved glycyl and lysyl residues of the putative ATP-binding site, located, respectively, at positions 151 and 152, and determined the effects of these alterations on in vivo function . A substitution of the conserved lysyl by a glutamyl residue (K152E) and a double G151R-K152N mutation each resulted in a nearly 1,000-fold decrease in transformability, equivalent to that observed in a ComFA null mutant . A K152N mutation caused a partial loss-of-function phenotype . These effects were manifested at the level of DNA uptake; no marked effects on the final levels of DNA binding were noted . When either the K152E mutant allele or the G151R-K152N double mutant allele was combined in single copy with wild-type comFA, a dominant negative phenotype expressed on the level of DNA uptake was observed, suggesting that ComFA acts in a complex with other proteins, with additional molecules of ComFA, or with both. J Bacteriol, 1994 Aug, 176(15), 4558 - 64 Coupling of flagellin gene transcription to flagellar assembly in Bacillus subtilis; Barilla D et al.; The regulation of flagellin gene expression in Bacillus subtilis was examined in vivo by means of a lacZ translational fusion to the flagellin structural gene (hag) . We have tested the effects of two known mutations (flaA4 and flaA15) in the major flagellar operon and of three deletions . One deletion was in frame in the fliI cistron, one was out of frame in the fliK cistron, and the last spanned about 21 kb of the flaA operon . In all instances, the expression of the flagellin gene was defective . Flagellin gene expression was restored in the strain with the 21-kb deletion by overexpression of the sigD gene under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible spac promoter . These results indicate that transcription of the flagellin gene is dependent on the formation of the flagellar basal body but that such a requirement can be bypassed by overexpression of sigD . Lack of expression of hag was observed in the presence of flaD1, flaD2, and delta sin mutations as well. J Bacteriol, 1994 Aug, 176(15), 4518 - 26 Interaction between the acceptor end of tRNA and the T box stimulates antitermination in the Bacillus subtilis tyrS gene: a new role for the discriminator base; Grundy FJ et al.; The Bacillus subtilis tyrS gene is a member of a group of gram-positive aminoacyl-tRNA synthetase and amino acid biosynthesis genes which are regulated by transcription antitermination . Each gene in the group is specifically induced by limitation for the appropriate amino acid . This response is mediated by interaction of the cognate tRNA with the mRNA leader region to promote formation of an antiterminator structure . The tRNA interacts with the leader by codon-anticodon pairing at a position designated the specifier sequence which is upstream of the antiterminator . In this study, an additional site of possible contact between the tRNA and the leader was identified through covariation of leader mRNA and tRNA sequences . Mutations in the acceptor end of tRNA(Tyr) could suppress mutations in the side bulge of the antiterminator, in a pattern consistent with base pairing . This base pairing may thereby directly affect the formation and/or function of the antiterminator . The discriminator position of the tRNA, an important identity determinant for a number of tRNAs, including tRNA(Tyr), was shown to act as a second specificity determinant for assuring response to the appropriate tRNA . Furthermore, overproduction of an unchargeable variant of tRNA(Tyr) resulted in antitermination in the absence of limitation for tyrosine, supporting the proposal that uncharged tRNA is the effector in this system. Virology, 1994 Aug 1, 202(2), 930 - 9 Analysis of the Bacillus subtilis bacteriophages SPP1 and SF6 gene 1 product: a protein involved in the initiation of headful packaging; Chai S et al.; Gene 1 product (G1P) of the related Bacillus subtilis bacteriophages SPP1, SF6, and rho 15 is essential for DNA maturation and packaging . A DNA segment containing gene 1 of phage SF6 or rho 15 origin was cloned and sequenced . SF6 and rho 15 G1P (both with predicted molecular mass of 16.7 kDa) share 71% identity with G1P of SPP1 . The G1P of all three phages contains three conserved segments (I, II, and III) . Within segments I and II helix-turn-helix DNA binding and nucleotide binding motifs were predicted . G1P of both SPP1 and SF6 origin was purified . SPP1 G1P protein (20.7 kDa), purified from cells overexpressing the cloned gene, purifies together with another polypeptide, having a molecular mass of about 13 kDa . The 13-kDa polypeptide results from a translation start signal within gene 1, and hence was termed SPP1 G1P* . G1P of both SPP1 and SF6 binds specifically to a pac-containing DNA fragment, whereas G1P*, which lacks segment I, does not . Chimeric G1P proteins were obtained by domain swapping between gene 1 of SPP1 and SF6 . The results presented here suggest that the G1P DNA binding motif lies in segment I and the major determinant for G1P::G1P interaction might lie in segment II . Segment III and the extended C-terminal part of SPP1 G1P are dispensable . The G1P::G2P interacting region remains uncharacterized. Virology, 1994 Aug 1, 202(2), 1046 - 9 Nucleotide sequence and complementation studies of the gene 35 region of the Bacillus subtilis bacteriophage SPP1; Weise F et al.; Genetic analysis of the Bacillus subtilis bacteriophage SPP1 defective in gene 35 shows that the gene 35 product (G35P) is essential for SPP1 growth . The defect in growth of SPP1tsl17 and SPP1tsl20F at nonpermissive temperature is overcome by wild-type gene 35 expressed from a plasmid . The region where gene 35 maps was cloned and sequenced . Analysis of the nucleotide sequence (5884-bp) around gene 35 revealed 13 open reading frames (orfs) . We have assigned the term gene to three of these orfs; gene 35, gene 36, the product of which shares homology with SSB proteins, and gene 38, given the gene order orf 34-orf 34.1-orf 34.2-orf34.3-orf34.4-gene 35-gene 36-orf 36.1-orf 37-orf 37.1-orf 37.2-orf 37.3-gene 38 . Gene 35 encodes a protein of 32.0 kDa . By using the T7 promoter-expression system for gene 35 a radioactive band of the expected molecular mass was detected. Virology, 1994 Aug 1, 202(2), 1039 - 42 A highly sensitive system for the in vitro assembly of bacteriophage phi 29 of Bacillus subtilis; Lee CS et al.; A sensitive system for the assay of bacteriophage phi 29 assembly in vitro was developed using 12 recombinant proteins and synthetic pRNA . This system detected in vitro assembled infectious phages up to 10(7) plaque forming units (PFU) per milliliter without any background . phi 29 DNA-gp3 concentration dependence in phage assembly was found to be first order, while the DNA-packaging protein gp16 dependence was higher order . The requirement for specific phi 29 pRNA for phi 29 DNA packaging was confirmed by the finding that no plaques were formed when only Escherichia coli RNAs were present . The activity of a mutant pRNA, with 10(5)-fold reduction in DNA packaging efficiency, was also demonstrated . Additionally, the tail proteins were found to have dual roles, one acting as phage tails and the other stabilizing the DNA-gp3 filled capsids. Mol Microbiol, 1994 Aug, 13(3), 417 - 26 Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation; Koide A et al.; Sporulation in Bacillus subtilis depends on an intact oligopeptide transport system, the Opp system . Mutants in opp sporulate poorly but second-site revertants can be found that restore sporulation and peptide transport . These second-site mutations were found in a second oligopeptide transport system, app, in which the peptide-binding protein, AppA, is mutant owing to a frame-shift mutation, and the revertants restore the original frame . The AppA mutation is present in the 168 strain of B . subtilis . The app operon consists of five genes in the order appD-appF-appA-appB-appC, with the locus designations corresponding to their homologue in the opp operon . Homology between the app and opp proteins ranges from 54% identity for AppF and OppF, to 22% identity for AppA and OppA . Both the App and Opp permease systems can transport tetra- and pentapeptides, but tripeptides are not transported by the App system . Strains of the genotype app+ opp- are resistant to the tripeptide antibiotic bialaphos . The repaired App system can substitute completely for the Opp system in both sporulation and competence for genetic transformation . The phenotypes raised some speculation about the subunit configuration of the Opp system. Am J Dent, 1994 Aug, 7(4), 220 - 2 Sterilization efficacy of a forced-air, dry heat sterilizer; Hohlt WF et al.; PURPOSE: To evaluate forced-air, dry heat sterilizer and test its ability to kill spores of Bacillus subtilis during both available cycles using packaged and unpackaged instruments . MATERIALS AND METHODS: A standard set of six orthodontic instruments were processed unpackaged through replicate 6-minute cycles with spore-strips on the top and bottom of each instrument . The spore strips were analyzed by culturing in Trypticase-soy broth for 7 days at 37 degrees C . RESULTS: No sterilization failures from properly placed spore-strips were detected in any of the 18 replicate cycles . Measurements of temperatures at three chamber sites during these cycles yielded only two of 36 instances when any portion of the chamber was at a temperature below the set temperature; however, no sterilization failures occurred during either of the two cycles involved . Also, a set of four orthodontic pliers were packaged and processed through replicate 12-minute cycles with spore-strips placed inside and attached to the outside of each package . None of the total 144 spore-strips processed through six replicate cycles showed growth after culture analysis . The study demonstrated sterilization efficacy for both the wrapped and unwrapped instrument cycles using biological indicators typically used to monitor dental office sterilizers. Mycopathologia, 1994 Aug, 127(2), 123 - 7 Iturin A: a potential new fungicide for stored grains; Klich MA et al.; The removal of many synthetic fungicides from the market has created a demand for new, environmentally safe fungicides . Iturin A, a cyclic lipopeptide produced by Bacillus subtilis, has strong antifungal properties and low mammalian toxicity . To determine the efficacy of this compound as a potential fungicide on stored feed grains, lots of corn, peanuts and cottonseed were treated with varying concentrations of iturin A . The mycoflora of treated seed was assayed along with that of untreated seed and seed treated with fungicides used commercially for planting seed . Fungal species varied considerably in their sensitivity to iturin A . Significant reductions in total mycoflora occurred in most seed lots tested at iturin A concentrations of 50 to 100 ppm. J Nat Prod, 1994 Aug, 57(8), 1178 - 82 Antibiotic activity and absolute configuation of 8S-heptadeca-2(Z),9(Z)-diene-4,6-diyne-1,8-diol from Bupleurum salicifolium; Estevez-Braun A et al.; A polyacetylene has been isolated from Bupleurum salicifolium . Its structure and absolute configuration were determined to be 8S-heptadeca-2(Z),9(Z)-diene-4,6-diyne-1,8-diol {1} by means of 1H- and 13C-nmr spectroscopic studies, including 1H-13C heteronuclear correlation (HMQC) and long-range correlation spectra with inverse detection (HMBC) . Its absolute configuration was determined by application of the Horeau method . This compound exhibited significant antibiotic activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis . Also isolated during this investigation were the known compounds; betulin, herniarin, 6,7,8-trimethoxycoumarin, p-hydroxyphenethyl alcohol, pluviatolide, guamaroline, bursehernin, guayadequiol, kaerophyllin, and matairesinol dimethyl ether. Curr Biol, 1994 Aug 1, 4(8), 734 - 5 Intercellular signalling . Knowing that you're among friends; Gomer RH; Factors that are simultaneously secreted and sensed are used by cells to monitor local cell density; a recently discovered factor of this type controls the transformation-competence of Bacillus subtilis. Can J Microbiol, 1994 Aug, 40(8), 651 - 7 Cloning and sequence analysis of the hemB gene of Staphylococcus aureus; Kafala B et al.; The hemB gene is a member of the family of genes encoding enzymes of the porphyrin biosynthetic pathway and codes for the enzyme porphobilinogen synthase, which is responsible for the conversion of delta-aminolevulinic acid to porphobilinogen . To clone the hemB gene of Staphylococcus aureus we used Tn917-mediated transposon mutagenesis . Tn917 confers resistance to erythromycin and is carried by plasmid pTV1ts, which has thermosensitive replication . Hem mutants were selected by growth in the presence of kanamycin and erythromycin at 43 degrees C . Preliminary identification of the hem mutants was based on their dwarf colony growth, which could be restored to normal by hemin . DNA extracted from one of the hem mutants was digested with several restriction endonucleases and hybridized to a probe representing the XbaI-AvaI end of Tn917 . A BglII-EcoRI fragment of 4.5 kb gave a positive signal and was cloned into pUC18 . Transformants were identified by colony hybridization with the Tn917 probe . The positive clones were sequenced, starting from the transposon end . The results allowed us to identify an open reading frame whose nucleotide sequence presented a homology of 63% to the sequence of the hemB gene of Bacillus subtilis and of 55% to the sequence of the hemB gene of Escherichia coli K12 . No other nucleotide sequences, except those belonging to known hemB genes, presented significant homologies to our sequence . The cloning of the hemB gene of S . aureus was confirmed by the ability of the gene to complement a hemB mutant of E . coli K12 . To our knowledge, this is the first report of the cloning of a hem gene in S . aureus. Microbiology, 1994 Aug, 140 ( Pt 8), 2173 - 7 Osmoresistance of spores from Bacillus subtilis and the effect of ssp mutations; Ruzal SM et al.; Spores of Bacillus subtilis show similar plating efficiency on media with or without 1.5 M NaCl . In contrast, vegetative cells are osmosensitive unless the stationary phase has been reached . In the present work, loss of heat and osmotic resistance during germination was studied . Their kinetics and sensitivity to protein synthesis inhibition were different: heat resistance was lost first and even in the presence of chloramphenicol, whereas loss of osmotolerance occurred later and was inhibited in the presence of this antibiotic . The influence of spore-associated small acid-soluble proteins (SASPs) on spore osmotolerance was investigated using ssp mutants: all produced spores which germinated poorly and were sensitive to osmotic strength . SASP-E deficiency was particularly significant . Spore osmotolerance was largely restored in complementation assays performed with cloned ssp genes . It is possible that germination-associated degradation of SASP proteins provides osmotically significant levels of amino acids (especially glutamate). Microbiology, 1994 Aug, 140 ( Pt 8), 1855 - 67 Lytic enzymes associated with defective prophages of Bacillus subtilis: sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX; Longchamp PF et al.; Prophage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins . The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX . The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis . Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced . Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin . The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein . The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide . The presence on the B . subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed. Microbiology, 1994 Aug, 140 ( Pt 8), 1847 - 54 Identification of TlpC, a novel 62 kDa MCP-like protein from Bacillus subtilis; Hanlon DW et al.; We report the sequence and characterization of the Bacillus subtilis tlpC gene . tlpC encodes a 61.8 kDa polypeptide (TlpC) which exhibits 30% amino acid identity with the Escherichia coli methyl-accepting chemotaxis proteins (MCPs) and 38% identity with B . subtilis MCPs within the C-terminal domain . The putative methylation sites parallel those of the B . subtilis MCPs, rather than those of the E . coli receptors . TlpC is methylated both in vivo and in vitro although the level of methylation is poor . In addition, the E . coli anti-Trg antibody is shown to cross-react with this membrane protein . Inactivation of the tlpC gene confirms that TlpC is not one of the previously characterized MCPs from B . subtilis . Capillary assays were performed using a variety of chemoeffectors, which included all 20 amino acids, several sugars, and several compounds previously classified as repellents . However, no chemotactic defect was observed for any of the chemoeffectors tested . We suggest that TlpC is similar to an evolutionary intermediate from which the major chemotactic transducers from B . subtilis arose. Microbiology, 1994 Aug, 140 ( Pt 8), 1829 - 38 Analysis of a ribose transport operon from Bacillus subtilis; Woodson K et al.; The csa-15 locus of Bacillus subtilis corresponds to an operon encoding proteins which display features characteristic of the ABC group of transporters . Sequence analysis reveals a very high level of identity to the ribose transport operon of Escherichia coli . This hypothesis is supported by the observation that strains carrying mutagenic insertions in this operon are unable to grow on ribose as sole carbon source . Expression of this operon is directed by a single SigA-type promoter which is negatively regulated by Spo0A during the late-exponential/transition state of the growth cycle . Expression is also subject to catabolite repression and this mode of regulation is dominant to control of expression by Spo0A. J Bacteriol, 1994 Aug, 176(16), 5177 - 80 Effect of degS-degU mutations on the expression of sigD, encoding an alternative sigma factor, and autolysin operon of Bacillus subtilis; Tokunaga T et al.; Primer extension analysis of transcripts of the Bacillus subtilis autolysin (cwlB) operon indicated that SigD-dependent transcripts from the Pd promoter are missing in the degU32(Hy) and degS200 (Hy) mutants . The degU32(Hy) mutation caused a 99% reduction in the expression of a sigD-lacZ translational fusion gene constructed in the B . subtilis chromosome . The phosphorylated form of the DegU protein seems to be a regulator for expression of the sigD gene. J Bacteriol, 1994 Aug, 176(16), 4937 - 40 Expression of Escherichia coli SecB in Bacillus subtilis facilitates secretion of the SecB-dependent maltose-binding protein of E . coli; Collier DN; Less than 20% of the Escherichia coli maltose-binding protein (MBP) synthesized in Bacillus subtilis is exported . However, a portion of the secreted MBP was processed cotranslationally . Coexpression of SecB, a secretion-related chaperone of E . coli, stimulated posttranslational export of MBP in B . subtilis but inhibited its cotranslational processing . Export of a SecB-independent MBP-ribose-binding protein hybrid precursor was not enhanced by SecB . A slowly folding MBP derivative (MBP-Y283D) was more efficiently secreted than wild-type MBP, suggesting that the antifolding activity of SecB promotes posttranslational secretion of MBP in B . subtilis. J Bacteriol, 1994 Aug, 176(15), 4527 - 33 Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA; Grundy FJ et al.; The Bacillus subtilis acsA (acetyl coenzyme A synthetase) and acuABC (acetoin utilization) genes were previously identified in the region downstream from the ccpA gene, which encodes a protein required for catabolite repression of the amyE (alpha-amylase) gene . The acsA and acuABC genes are divergently transcribed, with only 20 bp separating the -35 sequences of their promoters . Expression of these genes was maximal in stationary phase and was repressed by the addition of glucose to the growth medium . Two sites resembling amyO, the cis-acting regulatory target site for amyE, were identified in the acsA and acuABC promoter regions . Glucose repression of acsA and acuABC transcription was dependent on both CcpA and the amyO-like sequences. Biol Chem Hoppe Seyler, 1994 Aug, 375(8), 551 - 9 The mannose transporter of Escherichia coli K12: oligomeric structure, and function of two conserved cysteines; Rhiel E et al.; The mannose transporter of E . coli is a member of the phosphotransferase system . It consists of two membrane spanning subunits, IICMan (27.64 kDa) and IIDMan (31.02 kDa) and a peripheral subunit IIABMan (35.02 kDa) . It acts by a mechanism that couples vectorial translocation to phosphorylation of the substrate . The subunit ratio determined from densitometric scans of polyacrylamide gels is close to IIABMan2 IICMan1 IIDMan2 . A molecular mass of 100 +/- 20 kDa was calculated from electronmicrographs of freeze fractured proteoliposomes containing particles of the IICMan/IIDMan subcomplex with a mean diameter of 6.3 +/- 1.1 nm . This is most compatible with IICMan:IIDMan subunit compositions of 1:2 (89.7 kDa) . Fusion proteins between IICMan and IIDMan were generated, with the subunits connected either by a two-residue linker or a 20 residue Ala Pro rich hinge . The fusion proteins had 5%-15% of control phosphotransferase activity . The one with the Ala Pro rich linker could be cleaved with trypsin resulting in a 7 fold increase of activity while the fusion with the two residue linker was resistant to limited trypsinolysis . Taking into account the inside-out orientation of the membrane vesicles the C-terminus of IICMan and the N-terminus of IIDMan are both predicted to be on the cytoplasmic side of the membrane . Two cysteines in IICMan and IIDMan which are conserved in the homologous subunits of the fructose transporter of Bacillus subtilis and of sorbose transporter of Klebsiella pneumoniae are not necessary for phosphotransferase function. Mol Microbiol, 1994 Aug, 13(3), 381 - 7 tRNA-directed transcription antitermination; Henkin TM; At least 18 aminoacyl-tRNA synthetase and amino acid biosynthesis genes in several Gram-positive genera appear to be regulated by a common transcription antitermination mechanism . Each gene is induced by limitation for the appropriate amino acid, and not by general amino acid limitation . The mRNA leader regions of these genes exhibit extensive structural conservation . Characterization of the Bacillus subtilis tyrS gene revealed that uncharged tyrosyl-tRNA promotes readthrough of a leader-region terminator; a conformational switch in the leader mRNA between a terminator structure and an antiterminator structure is postulated to mediate antitermination . Two sites of interaction between the tRNA and the leader have been identified by genetic analysis: the tRNA anticodon interacts with a single codon displayed at a precise position in the leader-region structure, and the acceptor end of the tRNA interacts with a side-bulge on the antiterminator. J Mol Biol, 1994 Jul 29, 240(5), 476 - 500 The monofunctional chorismate mutase from Bacillus subtilis . Structure determination of chorismate mutase and its complexes with a transition state analog and prephenate, and implications for the mechanism of the enzymatic reaction; Chook YM et al.; Structures have been determined for chorismate mutase from Bacillus subtilis and of complexes of this enzyme with product and an endo-oxabicyclic transition state analog using multiple isomorphous replacement plus partial structure phase combination and non-crystallographic averaging . In addition to 522 water molecules, the model includes 1380 of the 1524 amino acid residues of the four trimers (each containing 3 x 127 amino acid residues) in the asymmetric unit . Refinement to 1.9 A resolution yields 0.194 for R and r.m.s . deviations from ideal values of 0.014 A for bond lengths and 2.92 degrees for bond angles . The trimer resembles a beta-barrel structure in which a core beta-sheet is surrounded by helices . The structures of the two complexes locate the active sites which are at the interfaces of adjacent pairs of monomers in the trimer . These structures have been refined at 2.2 A to a crystallographic R value of 0.18 and show r.m.s . deviations from ideal values of 0.013 A for bond lengths and 2.84 degrees or 3.05 degrees for bond angles, respectively . The final models have 1398 amino acid residues, nine prephenate molecules and 503 water molecules in the product complex, and 1403 amino acid residues, 12 inhibitor molecules and 530 water molecules in the transition state complex . The active sites of all three of these structures are very similar and provide a structural basis for the biochemical studies that indicate a pericyclic mechanism for conversion of chorismate to prephenate . The absence of reactive catalytic residues on the enzyme, the selective binding of the single reactive conformation of chorismate, the stabilization of the polar transition state, and the possible role of the C-terminal region in "capping" the active site are factors which relate these structures to the million-fold rate enhancement of this reaction. J Mol Biol, 1994 Jul 29, 240(5), 405 - 15 Regulation of the transcription of a cluster of Bacillus subtilis spore coat genes; Zhang J et al.; The pattern of transcription has been examined for a cluster of genes encoding polypeptides some or all of which are assembled into a cross-linked component of the Bacillus subtilis spore coat . Three promoters, designated PVWX, PX and PYZ, were indicated by reverse transcriptase mapping . On the basis of Northern hybridization, it appeared that the cotV, W and X genes were transcribed as a polycistronic mRNA from PVWX as well as a monocistronic cotX mRNA from Px . The cotY and cotZ genes are cotranscribed from the PYZ promoter with a smaller cotY mRNA resulting from premature termination or RNA processing . All four transcripts were synthesized late during sporulation and were not produced in mutants lacking sigma K, which directs RNA polymerase to transcribe genes in the mother-cell compartment of sporulating cells . The DNA-binding protein GerE, which affects transcription of many genes in the mother cell during the late stages of sporulation, was also shown to be involved . There was essentially no cotX mRNA in a gerE mutant and the amounts of cotVWX, cotYZ and cotY mRNAs were somewhat reduced . In vitro run-off transcription studies with sigma K RNA polymerase and GerE confirmed the presence of the three promoters, and directly showed that GerE was necessary for transcription from PX as well as enhanced transcription from the PVWX and PYZ promoters . The DNase I footprints of GerE for all three promoters were immediately upstream of the -35 regions . These GerE binding sites were compared to those in other GerE-responsive promoters and a larger consensus sequence for GerE binding was recognized . This complex transcriptional pattern of the cotVWXYZ cluster is probably necessary to ensure that an optimal amount of each protein is made for the assembly of the spore coat. Biochemistry, 1994 Jul 26, 33(29), 8842 - 52 Proton and nitrogen NMR sequence-specific assignments and secondary structure determination of the Bacillus subtilis SPO1-encoded transcription factor 1; Jia X et al.; Sequence-specific 1H and 15N NMR1 assignments are reported for the transcription factor 1 (TF1), a 22-kDa type II DNA-binding protein (DBPII) that consists of two 99-residue monomers . An assignment strategy is employed that uses six complementary selectively deuterium-labeled TF1 variants and an uniformly 15N-labeled TF1 variant . Two-dimensional and three-dimensional homonuclear and heteronuclear NMR correlated spectra are analyzed and yield nearly complete assignments for the 1H and 15N resonances . Discrete protein secondary structure domains are also defined; in each monomer, three alpha-helices, an antiparallel beta-sheet, and an antiparallel beta-ribbon are identified . Analyses of two dimers formed from two distinct selectively deuteriated monomers serve to identify a number of interproton contacts as either intermonomeric or intramonomeric . An analysis of amide proton exchange reveals that the carboxy-terminal alpha-helix is less stable than the other two alpha-helices in each monomer . A previously proposed working structural model of the TF1 dimer {Geiduschek et al . (1990) J . Struct . Biol . 104, 84-90}, based on the crystal structure of a highly homologous DBPII, the Bacillus stearothermophilus-encoded HU protein, is generally supported by our results . Several departures from this model, however, are noted . Most notably, the carboxy-terminal tail of TF1 adopts an alpha-helical conformation with a backbone distortion at Lys93. Gene, 1994 Jul 22, 145(1), 57 - 63 Another putative heat-shock gene and aminoacyl-tRNA synthetase gene are located upstream from the grpE-like and dnaK-like genes in Chlamydia trachomatis; Schmiel DH et al.; The 4.1-kb sequence of genomic DNA located upstream from the Chlamydia trachomatis grpE-like and dnaK-like heat shock (HS) genes was determined . Another putative HS gene was located just 5' to grpE along with an inverted repeat (IR) sequence proposed to be involved in HS regulation . The overall organization of this locus in Chlamydia resembles that of Bacillus subtilis, rather than Escherichia coli . Two other open reading frames (ORFs) were found in the sequence, one of which has homology to aminoacyl-tRNA synthetases . The other ORF has no significant homology to reported genes . We also examined the codon usage bias for these newly identified chlamydial ORFs and for previously reported chlamydial genes, and found them to be different from E . coli. J Mol Biol, 1994 Jul 22, 240(4), 275 - 80 The Bacillus subtilis replication terminator system functions in Escherichia coli; Young PA et al.; The Bacillus subtilis DNA terminators, IRI + IRII, were inserted into the Escherichia coli plasmid pACYC184 such that the IRI terminator would be in its active orientation with respect to the approaching unidirectionally moving replication fork . When this new plasmid was transferred into E . coli, harbouring an expression plasmid producing the B . subtilis terminator protein RTP, fork arrest was observed to occur at the position of the inserted terminator region . Thus, the B . subtilis replication terminator system can function in E . coli . It was shown that the B . subtilis system operated with approximately 30% of the efficiency of the E . coli system utilizing the R6K TerR2 DNA terminator and the E . coli Tus terminator protein . Assuming that RTP and Tus have quite different folded structures these results suggest that fork arrest in B . subtilis is not dependent upon a highly specific recognition and interaction between RTP positioned on the DNA terminator and a component(s) of the approaching replisome. Gene, 1994 Jul 22, 145(1), 151 - 2 A series of integrative plasmids for Bacillus subtilis containing unique cloning sites in all three open reading frames for translational lacZ fusions; Dahl MK et al.; Two sets of three plasmids each were constructed based on integrative plasmids for Bacillus subtilis . Each set encodes resistance to either chloramphenicol or kanamycin . The plasmids contain a cassette consisting of the resistance gene, BamHI and SmaI cloning sites, and the promoterless lacZ gene . The cassette is flanked by the 3' and 5' ends of the amyE gene (encoding amylase) allowing integration of the cassette into that locus in the B . subtilis chromosome . For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of replication and the beta-lactamase-encoding gene . Within a set, each of the three plasmids carries the unique SmaI and BamHI restriction sites in a different reading frame relative to the 'lacZ gene . These sets of plasmids allow the easy construction of translational fusions with lacZ and single-copy integration at the amyE locus of B . subtilis. J Biol Chem, 1994 Jul 15, 269(28), 18656 - 61 Probing the structure of bacteriophage phi 29 prohead RNA with specific mutations; Reid RJ et al.; Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded DNA genome into a preformed prohead in an ATP-dependent reaction . A 174-residue phi 29-encoded RNA molecule (pRNA) is a structural component of the prohead and is essential for DNA packaging . The secondary and tertiary structures of the prohead binding site on pRNA have been probed using a series of specific mutant pRNAs and by measuring binding to RNA-free proheads and in vitro packaging of the DNA-gene product 3 (DNA.gp3) complex . A pseudoknot in pRNA inferred from phylogenetic studies was confirmed with specific mutations, and this pseudoknot was necessary for DNA.gp3 packaging activity . pRNA was truncated progressively from the 5' and 3' ends to isolate the prohead binding site, and three truncated pRNAs of 79, 71, and 62 residues retained prohead binding activity but could not reconstitute proheads for DNA.gp3 packaging . Mutation resulting in changes of the D hairpin loop and its connecting residues within the prohead binding site of pRNA and DNA packaging studies demonstrated that some alteration of secondary structure in this helix was permissible . The analyses provided further confirmation of a discrete prohead binding domain in pRNA and further delineated specific structural requirements for DNA.gp3 packaging activity which may not be required for prohead binding. Mol Gen Genet, 1994 Jul 8, 244(1), 97 - 103 Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis; Munakata N et al.; We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis . A 163 bp DNA segment from a 5' portion of the gyrA gene was amplified from the DNA of each mutant strain . After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis) . Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains . The patterns were classified into 13 types, and the DNA sequence of each type was determined . A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles . Eight were single base pair substitutions, four were substitutions of two consecutive base pairs and one was a substitution of three consecutive base pairs . Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes . We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait. Genetics, 1994 Jul, 137(3), 627 - 36 Mutations in the gene for a tRNA that functions as a regulator of a transcriptional attenuator in Bacillus subtilis; Garrity DB et al.; It has been proposed that uncharged tRNA molecules may act as positive regulatory factors to control the expression of a number of operons in Bacillus subtilis and related bacteria by interacting with leader sequences to cause antitermination . In this study we report the isolation and characterization of regulatory mutations that modify one of the tRNA molecules predicted to have such a regulatory role . Three different alleles of the B . subtilis leucine tRNA gene leuG were found that resulted in higher expression of the ilv-leu biosynthetic operon . Each resulted in a base change in the D-loop of the leucine tRNA molecule with the anticodon 5'-GAG-3' (leucine tRNAGAG) . Experiments with strains that are diploid for mutant and wild-type alleles suggested that both charged and uncharged tRNA molecules may interact with leader sequences to control expression of the operon. Microbiology, 1994 Jul, 140 ( Pt 7), 1613 - 7 A rapid and simple method for Bacillus subtilis transformation on solid media; Hauser PM et al.; Cells of Bacillus subtilis strains 168 and W23 deprived of an amino acid or a base on a given solid medium were found to develop competence . We describe a rapid and simple method of genetic transformation of this organism consisting in spreading a sample containing 1 microgram DNA and 10(7) exponentially growing cells of an auxotrophic mutant onto plates devoid of the required amino acid or base . After overnight incubation, about 100-200 prototrophic transformants per plate were obtained, i.e . a frequency of about 10(-5), as compared to 10(-4) routinely obtained by the method of transformation in liquid medium with frozen competent cells . Plasmids and other chromosomal or plasmid-borne markers, which cannot be directly selected for, were transferred by congression . The dependence of the transformation efficiency on cell density, medium richness, incubation time and the nature of transforming DNA was investigated . We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiological conditions. Microbiology, 1994 Jul, 140 ( Pt 7), 1605 - 11 Characterization of the chromosomes of Bacillus subtilis merodiploid strains by quantitative DNA-DNA hybridization; Hauser PM et al.; The position of junctions and the extent of the duplicated chromosomal regions in Bacillus subtilis merodiploid strains were studied by quantitative DNA-DNA hybridization . We describe a method which allows (i) the identification of genes present in two copies per chromosome and (ii) the measurement of the amount of additional DNA in chromosomes with relatively large duplicated regions (about 10% or more) . Analysis of previously described B . subtilis merodiploid strains GSY1127, GSY1800 and GSY1835 revealed that the duplicated segments represent 29 +/- 2%, 7 +/- 2% and 13 +/- 2% of the chromosome, respectively . Small discrepancies between these and previous genetic linkage data are discussed . Support for a role of prophage SP beta in the formation of merodiploid GSY1835 is provided . In conclusion, the described method confirmed the genetic maps of the merodiploids previously obtained by transduction and transformation crosses and showed that a duplication of a segment is not accompanied by large deletions of other chromosomal regions, providing direct evidence that a cell can accommodate genomes of substantially increased size. Appl Environ Microbiol, 1994 Jul, 60(7), 2647 - 9 Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from being killed by freeze-drying; Fairhead H et al.; Wild-type spores of Bacillus subtilis were resistant to eight cycles of freeze-drying, whereas about 90% of spores lacking the two major DNA-binding proteins (small, acid-soluble proteins alpha and beta) were killed by three to four cycles of freeze-dryings, with significant mutagenesis and DNA damage accompanying the killing . This role for alpha/beta-type small, acid-soluble proteins in spore resistance to freeze-drying may be important in spore survival in the environment. Biopolymers, 1994 Jul, 34(7), 975 - 86 Solution three-dimensional structure of surfactin: a cyclic lipopeptide studied by 1H-NMR, distance geometry, and molecular dynamics; Bonmatin JM et al.; The solution three-dimensional structure of the protonated {Leu7}-surfactin, an heptapeptide extracted from Bacillus subtilis, has been determined from two-dimensional 1H-nmr performed in 2H6-dimethylsulfoxide and combined with molecular modeling . Experimental data included 9 coupling constants, 61 nuclear Overhauser effect derived distances, NH temperature coefficients, and 13C relaxation times . Two distance geometry (DISMAN) protocols converged toward models of the structure and the best of them were refined by restrained and unrestrained molecular dynamics (GROMOS) . Two structures in accord with the set of experimental constraints are presented . Both are characterized by a "horse saddle" topology for ring atoms on which are attached the two polar Glu and Asp side chains showing an orientation clearly opposite to that of the C11-13 aliphatic chain . Amphipathic and surface properties of surfactin are certainly related to the existence of such minor polar and a major hydrophobic domains . The particular "claw" configuration of acidic residues observed in surfactin gives important clues for the understanding of its cation binding and transporting ability. J Bacteriol, 1994 Jul, 176(13), 4192 - 5 Reconstitution of energy-linked activities of the solubilized F1F0 ATP synthase from Bacillus subtilis; Hicks DB et al.; The F1F0 ATP synthases from wild-type Bacillus subtilis and an uncoupler-resistant mutant have comparable subunit structures . In accord with an earlier hypothesis, ATP hydrolysis and ATP-Pi exchange by the two synthases were equally stimulated and inhibited by protonophores, respectively, when reconstituted alone in either wild-type or mutant lipids. J Bacteriol, 1994 Jul, 176(13), 4104 - 10 The dacF-spoIIA operon of Bacillus subtilis, encoding sigma F, is autoregulated; Schuch R et al.; The spoIIA operon of Bacillus subtilis encodes sigma F and two proteins that may regulate sigma factor activity . High level induction of the tricistronic spoIIA operon occurs early during spore formation . At later times, the locus is cotranscribed with the upstream gene dacF, which encodes a putative DD-carboxypeptidase . In this study, the regulation of dacF-spoIIA transcription has been analyzed . Expression of a dacF-lacZ transcriptional fusion during sporulation required sigma F but not the later-expressed sporulation-associated sigma factors . Induction of sigma F