J Bacteriol, 1988 Jan, 170(1), 289 - 95 Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants; Ferrari E et al.; The start point for transcription of the subtilisin (aprE) gene was determined by primer extension analysis and was found to be at a point significantly different from that identified in a previously published report (S . L . Wong, C . W . Price, D . S . Goldfarb, and R . H . Doi, Proc . Natl . Acad . Sci . USA 81:1184-1188, 1984) . An aprE-lacZ fusion was used to analyze expression of the promoter . Deletion analyses of the promoter were performed to determine the extent of the upstream region necessary for activity . This was found to be between -52 and -41 with respect to the transcription start site . Expression of the aprE-lacZ fusion was unimpaired in a mutant deleted for the sigma B subunit of RNA polymerase . Mutations in the gene for the sigma H subunit of RNA polymerase decreased expression of the aprE-lacZ fusion to approximately 25% of that of the wild type . These results leave the identity of the sigma factor responsible for transcription of this gene in question . Mutations in the spo0A gene drastically decreased the activity of the aprE promoter and its upstream deletion derivatives, while the abrB gene, a phenotypic suppressor of spo0 mutations, restored activity of the aprE promoter in all of the deletion derivatives . Thus, inhibition of transcription by the spo0A mutation and its restoration by an abrB mutation could not be separated from the promoter of the aprE gene. J Mol Biol, 1987 Dec 20, 198(4), 609 - 18 Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence; Nicholson WL et al.; The amyR1 locus controls the regulated transcription of amyE, the structural gene encoding alpha-amylase in Bacillus subtilis . Transcription of amyE is activated in early stationary phase cells, and can be repressed by rapidly metabolized carbon sources such as glucose . Transcription of amyE initiates in vitro from a promoter recognized by the major vegetative form of RNA polymerase, E sigma 43 . S1 nuclease mapping of in-vivo amylase transcripts suggests that this promoter is also used in vivo . Two independently isolated cis-acting mutations, gra-5 and gra-10, which abolish glucose-mediated repression of amylase synthesis without altering temporal activation, were determined by DNA sequencing to result from a G.C to A.T transition at a position located five base-pairs downstream from the start site of transcription . While this is the first example of a site involved in catabolite repression of gene expression in a Gram-positive micro-organism, the region surrounding the gra mutations shows considerable homology to certain cis-acting regulatory loci in Escherichia coli, suggesting that such sequences have been evolutionarily conserved. Biochem Biophys Res Commun, 1987 Dec 16, 149(2), 576 - 9 Controlled cell lysis and protoplast formation by enhancement of inhibitors of alanine racemase by glycine; Heaton MP et al.; beta-D-chloroalanine (200-800 micrograms/ml) addition to Bacillus subtilis SB 19 caused a slight inhibition of growth in MLN broth . Inhibitor plus 1% glycine initiated rapid lysis of cells (complete in 2 hours) . D-alanine (500 micrograms/ml) prevented lysis . D-cycloserine behaved similarly . Inhibitors in 0.5 M sucrose and 1% glycine formed stable "primed protoplasts" which were converted to the bacillary form by addition of D-alanine . Characteristics of Dal+ cells with inhibitor are similar to Dal- mutants . Thus Dal+ cells in the presence of alanine racemase inhibitors + glycine can be used for controlled lysis and protoplast formation (suitable for PEG transformation with plasmid DNA) . Inhibitors in 1% glycine exhibit a strong antibacterial action similar to potent antibiotics which inhibit cell wall formation. Biochemistry, 1987 Dec 15, 26(25), 8206 - 13 Effect of the delta subunit of Bacillus subtilis RNA polymerase on initiation of RNA synthesis at two bacteriophage phi 29 promoters; Dobinson KF et al.; Initiation of RNA synthesis by Bacillus subtilis RNA polymerase (sigma-43) has been examined at two early promoters of phage phi 29: the A2 promoter, which is a weak promoter, and the G2 promoter, which is a strong promoter . The delta subunit of the polymerase inhibits the rate of initiation at A2, but not G2 . In addition, formation of stable complexes by the polymerase at A2, but not at G2, requires the presence of the first two nucleotides of the A2 transcript. J Antibiot (Tokyo), 1987 Dec, 40(12), 1682 - 91 Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis . II . Isolation and physico-chemical characterization; Wilson KE et al.; The isolation of difficidin (1) and oxydifficidin (2) from fermentation broth of Bacillus subtilis ATCC 39320 and the physico-chemical characterization of these labile antibiotics are described . The structures of the compounds represent a new class of antibiotics, characterized as highly unsaturated 22-membered macrolide phosphates . Difficidin and oxydifficidin undergo reversible thermal isomerization to 3 and 4 respectively . Biological evaluation of the isomers is presented. J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3313 - 8 Effects of dnats genes on the replication of plasmids in Bacillus subtilis; Forough R et al.; An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis . The resultant plasmid, pKW1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B . subtilis . Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H; pC194 requires only dnaG and H; and pTP5 requires dnaE, F, G and H . Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions . Because of its greater dependence on host functions and its low copy number, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B . subtilis . pKW1 should also be a useful shuttle vector for cloning of genes in B . subtilis in cases when high gene dosage might be a problem. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Dec, 267(2), 173 - 85 Different mechanisms of insensitivity to the staphylococcin-like peptide Pep 5; Sahl HG et al.; Three different mechanisms of insensitivity to the bactericidal membrane disrupting action of the staphylococcin-like peptide Pep 5 were found to occur among certain Gram-positive bacteria . i) The immunity of the producer of Pep 5 Staphylococcus epidermidis 5 was shown to be due to a Pep 5 antagonist, which was excreted along with Pep 5 during growth . The cells were killed by Pep 5 when incubated with high doses exceeding the concentration of the antagonist . ii) Bacillus subtilis W23 produced a protease which cleaved Pep 5 into fragments with greatly reduced bactericidal activity . Like with S . epidermidis 5, cells of B . subtilis were susceptible to the membrane damaging action of the peptide and growth in culture was only possible after inactivation of Pep 5 . iii) By prolonged incubation in the presence of Pep 5 resistant mutants of S . cohnii 22 and S . epidermidis 5 Pep 5- could be selected . Their cytoplasmic membrane was not sufficiently disrupted by the peptide to promote active killing, resulting in unhindered growth of these mutants in the presence of high doses (200 AU/ml) of Pep 5. J Appl Bacteriol, 1987 Dec, 63(6), 559 - 64 Effects of Freon-113 on the survival of bacteria; Sousa S et al.; An initial investigation was made of the bactericidal and sporicidal activity of the dry cleaning solvent Freon-113 (1,1,2-trichlorotrifluoroethane) under various conditions . Representative bacterial strains selected for this study were Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 6538) and spores of Bacillus globigii (Bacillus subtilis var . niger) . Various conditions were studied, including temperature, moisture, detergents and organic load . The results of this study indicate that there is the potential for survival of significant levels of micro-organisms in Freon-113 within the conditions evaluated . However, the bactericidal efficiency of this solvent increases significantly against vegetative forms at elevated temperature and, to a greater extent, upon the addition of detergents . Organic material present in the form of soiled fabric was observed to depress this efficacy whereas bacterial spores were virtually unaffected under all conditions evaluated. Mol Gen Genet, 1987 Dec, 210(2), 347 - 51 Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages; Errington J et al.; We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage phi 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes . The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome . This places an upper limit of about 5 kb on the size of insertions that can be accommodated in phi 105 transfection cloning vectors, such as phi 105J27 . Induction of prophages that exceed that upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants . A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis. Mol Gen Genet, 1987 Dec, 210(3), 578 - 80 Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions; Bashkirov VI et al.; The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied . It was found that nucleotide sequences of both parental plasmids could be involved in this process . The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined . The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome . The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process . Two different pathways of illegitimate recombination in B . subtilis are suggested. Mol Gen Genet, 1987 Dec, 210(3), 518 - 22 Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1; Temeyer KB et al.; The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin . BamHI digests of plasmid pUB110 (Kanr/Neor) and Bg/II digests of pTL12 (Tmpr, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis . Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids . Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb) . Plasmid pKBT1 was stably maintained in recE4 strains of B . subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy . At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment . The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B . subtilis chromosomal DNA . At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA . This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event. Mol Gen Genet, 1987 Dec, 210(3), 498 - 503 Localization of ribosomal protein L27 at the peptidyl transferase centre of the 50 S subunit, as determined by immuno-electron microscopy; Lotti M et al.; Protein L27 has been localized on the ribosomal surface by immuno-electron microscopy by using antibodies specific for Escherichia coli L27, and by reconstituting 50 S subunits from an E . coli mutant, which lacks protein L27, with the homologous protein from Bacillus subtilis and using antibodies specific for the B . subtilis protein . With both approaches, protein L27 has been located at the base of the central protuberance at the interface side of the 50 S particle and thus in proximity to the peptidyl transferase centre . The immuno-electron microscopic data also suggest that the interface region of the 50 S particle is not as flat as most of the proposed three-dimensional models suggest, but instead there is a significant depression. Mol Gen Genet, 1987 Dec, 210(3), 476 - 84 Initiation of plasmid pC194 replication and its control in Bacillus subtilis; Alonso JC et al.; By deletion analysis we have defined a 1.1 kb segment required for driving autonomous replication of the plasmid pC194 . The minimal replicon specifies a positive, RepH, and a negative, Inc8A, trans-acting product and their target sites . The RepH product has a Mr of 34.1 kDa, could be overproduced, and binds specifically to the pC194 origin region . By trans complementation studies we have shown that pC194 replication is indirectly controlled at the level of RepH synthesis by a negative product, IncA, that is transcribed within the repH transcription unit in the opposite direction ("antisense RNA") . The antisense RNA regulates the RepH synthesis by a mechanism that seems to involve RNA/RNA interaction in a manner that interferes with translation . In addition, an autoregulatory control might be operative. Mol Gen Genet, 1987 Dec, 210(3), 468 - 75 Construction and characterization of multicopy expression-vectors in Streptomyces spp; Horinouchi S et al.; A strong transcriptional signal previously cloned from the Streptomyces griseus genome in S . lividans was subcloned and its nucleotide sequence was determined . Upstream of the transcriptional start point which was determined by high-resolution S1 nuclease mapping, -35 (5'-TTGCCG-3') and -10 (5'-TAGCGT-3') sequences, separated by 18 nucleotides, were present . By replacing the tet promoter of pBR322 with the Streptomyces promoter, no expression of the tet gene was observed in Escherichia coli cells . The result suggests that notwithstanding a similarity to the E . coli -35 and -10 sequences, the Streptomyces promoter is not functional in E . coli . The strong promoter was inserted in multi-copy and wide host range plasmids pIJ702 and pKS11, resulting in the pSEV series of expression-vectors with several unique restriction endonuclease cleavage sites downstream of the promoter for cloning of foreign genes . The extremely heat-stable malate dehydrogenase of Thermus flavus, when its coding sequence with a ribosome-binding site was located downstream of the strong promoter in pSEV2, was produced in large quantities in S . lividans throughout growth . When an extracellular cellulase from Bacillus subtilis was expressed in a cellulase-negative S . lividans strain, virtually all of the cellulase activity was found in the culture supernatant. J Antibiot (Tokyo), 1987 Dec, 40(12), 1677 - 81 Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis . I . Production, taxonomy and antibacterial activity; Zimmerman SB et al.; Difficidin and oxydifficidin, two novel macrocyclic polyene lactone phosphate esters were discovered in fermentation broths of each of two strains of Bacillus subtilis: ATCC 39320 and ATCC 39374 . Difficidin and oxydifficidin each showed a broad spectrum of activity against aerobic and anaerobic bacteria . Many of the susceptible aerobes and anaerobes were human pathogens resistant to one or more antibiotics . Difficidin and oxydifficidin when administered intraperitoneally protected mice against an otherwise lethal bacteremia caused by Klebsiella pneumoniae (ED50 in mg/kg of 1.31 and 15.6 respectively) . Neither difficidin nor oxydifficidin were effective when administered via the subcutaneous route. Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8773 - 7 Bacillus subtilis sucrose-specific enzyme II of the phosphotransferase system: expression in Escherichia coli and homology to enzymes II from enteric bacteria; Fouet A et al.; Sucrose is transported into Bacillus subtilis cells by way of a phosphotransferase system, which consists of a specific enzyme II, a nonspecific enzyme I, and a histidine-containing phosphocarrier protein . Mutations in the sacP locus abolish the specific transport of sucrose . The B . subtilis sacP gene was cloned and expressed in Escherichia coli, and transformed cells could transport and phosphorylate sucrose . This indicates that the sacP gene product is enzyme II of the sucrose phosphotransferase system of B . subtilis . The nucleotide sequence of the sacP gene was determined and was found to overlap with the sacA gene at the tetranucleotide ATGA, which may allow a translational coupling between sacP and sacA . The two genes are therefore probably organized in an operon structure with the promoter located 5' to sacP gene . The deduced amino acid sequence gave a Mr of 48,945 for the sucrose-specific enzyme II polypeptide . The amino acid sequence was compared to that of three other known enteric bacterial enzymes II (beta-glucoside-specific enzyme II, mannitol-specific enzyme II, and glucose-specific enzyme II) . Homology was found with beta-glucoside enzyme II, and well conserved regions were identified through the comparison of the proteins. J Bacteriol, 1987 Dec, 169(12), 5867 - 9 Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis; Nicholson WL et al.; Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions . The effect of decoyinine on alpha-amylase synthesis in B . subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined . Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B . subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis . Decoyinine had no effect on alpha-amylase enzymatic activity . Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B . subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components. J Bacteriol, 1987 Dec, 169(12), 5838 - 40 Helix hand fidelity in Bacillus subtilis macrofibers after spheroplast regeneration; Briehl MM et al.; Left- and right-handed Bacillus subtilis macrofibers produced by strains FJ7 and C6D were converted to spheroplasts . Intact cells were regenerated and macrofibers were produced under conditions conducive for production of left- and right-handed structures . The resulting helix hand phenotypes always corresponded to those expected on the basis of the parental genotype. J Bacteriol, 1987 Dec, 169(12), 5771 - 5 Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis; Dowds BC et al.; The sensitivity of Bacillus subtilis to hydrogen peroxide (oxidative stress) was found to vary with the position of the culture in the growth cycle . The most dramatic change occurred at the stationary phase, when the cells became totally resistant to 10 mM H2O2, in contrast to the loss of 3 to 4 log units of viability when treated at the early log phase . Two of the eight proteins induced by a protective concentration of H2O2 (50 muM) were also induced (in the absence of oxidative stress) on entry into the late log phase of growth . The response of five isogenic spo0 mutants (spo0B, spo0E, spo0F, spo0H, and spo0J) to oxidative stress was identical to that of the wild-type parental strain . In an isogenic spo0A strain, mid-log-phase cells were 100-fold less sensitive to 10 mM H2O2 than was the wild type . Pretreatment with 50 microM H2O2 induced little further protection, suggesting that the response is constitutive in this strain . By comparison of proteins induced by 50 microM H2O2 in the wild-type, spo0A, spo0H, and spo0J strains, four proteins were identified that may be essential for protection against lethal concentrations of H2O2 . The presence of multiple copies of the spo0H gene in a spo0A background converted the stress phenotype of the spo0A mutant to that of the wild type but left the sporulation phenotype unaltered. J Bacteriol, 1987 Dec, 169(12), 5766 - 70 Oxidative stress and growth temperature in Bacillus subtilis; Murphy P et al.; Pretreatment of Bacillus subtilis with low concentrations of hydrogen peroxide protected the cells against the lethal effects of higher levels of oxidative stress . During the period of adaptation, eight proteins were induced, as detected by one-dimensional gel electrophoresis . Four of these proteins were the same size as four of the proteins induced by the temperature upshift . The range of proteins synthesized in response to an elevation in temperature depended both on the starting (lower) temperature and on the temperature to which the cells were shifted . Both catalase and superoxide dismutase were present at high levels in B . subtilis, but neither was induced by oxidative stress or temperature upshift . In fact, catalase activity was reduced after the temperature upshift. Microb Pathog, 1987 Dec, 3(6), 461 - 8 Expression and secretion of pertussis toxin subunit S1 in Bacillus subtilis; Runeberg-Nyman K et al.; Pertussis toxin (PT) is an important virulence determinant of Bordetella pertussis and one of the major protective antigens against whooping cough . The genes coding for PT have recently been cloned, but attempts to express them in Escherichia coli have been unsuccessful . We therefore explored the possibility of expressing these genes in Bacilius subtilis for which efficient vectors are available . The lack of endotoxin in the Gram-positive Bacillus might be an additional advantage for the production of a vaccine component . A DNA fragment coding for S1, one of the subunits of pertussis toxin, was inserted into an alpha-amylase secretion vector and the recombinant plasmid was introduced into B . subtilis . This resulted in high expression of S1, most of which was secreted and therefore found in the culture supernatant . This supernatant had ADP-ribosylating activity similar to that of PT . Western blot with antiserum to B . pertussis holotoxin showed several proteins ranging in size from 28 kDa to 20 kDa reacting in specific manner . About 10% of the protein recognized by the antiserum was of the size expected for native-size S1 . The total amount of S1 proteins (full size and truncated) in the culture supernatant was about 100 mg/l . S1 protein made in B . subtilis was partially purified using chromatography with P-cellulose and Blue Sepharose . This preparation was used to immunize rabbits; the immune serum thus obtained recognized subunit S1 of native pertussis toxin. J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3481 - 93 The gtaB marker in Bacillus subtilis 168 is associated with a deficiency in UDPglucose pyrophosphorylase; Pooley HM et al.; Fifty-six mutants of Bacillus subtilis 168 were selected for resistance to bacteriophages phi 29 or phi 25 . The mutations were all linked to previously described teichoic acid markers gtaA, gtaB or gtaC, for the first and last of which, the gene products have previously been identified . Each linkage group was shown to have two distinct phenotypes with respect to phage resistance and cell-wall galactosamine content . Recombination indexes of 0.35, 0.13 and 0.41 for groups A, B and C respectively were consistent with the presence of two average-sized genes in groups A and C . Correlation between genetic and phenotypic differences supported this conclusion and led to the designation of two new markers, gtaD and gtaE . Two- and three-factor transformation crosses suggested the order hisA-gtaB-gtaD-gtaA-tag-1 and gtaC-gtaE-argC . Assays for UDPglucose pyrophosphorylase and phosphoglucomutase activities in soluble extracts of representative mutants revealed that, in contrast to previous findings, the former activity was virtually undetectable in all nine group B mutants examined, suggesting that gtaB is the structural gene of this enzyme . Our results allow us to account for discrepancies with respect to previous reports . The thermosensitive mutation previously designated rodC1 was shown to be 90% cotransformable with tag-1 . In view of their extremely similar phenotypes the former mutation was renamed tag-3, and the likely order obtained was gtaA-tag-3-tag-1 . This suggests that many mutations associated with deformation of cell shape in B . subtilis are located in the region where teichoic acid genes map. J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3299 - 312 Genetical and molecular studies on gerM, a new developmental locus of Bacillus subtilis; Sammons RL et al.; A transposon Tn917 insertion between gerE and ilvB has identified a new developmental locus, gerM, in Bacillus subtilis . gerM96::Tn917 affects both sporulation and germination . DNA on either side of the transposon has been cloned and includes the previously cloned sdhC and gerE loci . gerE terminates 2.1 kb from the end of the transposon . The gerM96::Tn917 mutant is oligosporogenous, yielding approximately 1% of the number of wild-type heat resistant spores in liquid medium and 10% on solid medium . Six hours after the onset of sporulation alkaline phosphatase and glucose dehydrogenase levels were 90% and 7%, respectively, of those of the wild-type . At this time 50% of the mutant cells were still dividing . The occurrence of multiple polar septa and 'pygmy' cells suggested a block at stage II of sporulation . Following addition of germinants, mutant spores prepared on nutrient agar lost heat resistance normally but released slightly less dipicolinic acid than wild-type spores . They also showed only partial loss of optical density, associated with a phase-grey appearance and striations in the cortex suggesting partial degradation . Expression of the gerM gene was monitored by production of beta-galactosidase encoded by a promotorless lacZ gene fused to the gerM96::Tn917 insertion . It occurred 1.5-4 h after commencement of sporulation . Transcription was directed from a promoter on the gerE side of gerM and was unaffected by a mutation in the gerE gene. Genetics, 1987 Dec, 117(4), 603 - 17 Genetic analysis of Bacillus subtilis spo mutations generated by Tn917-mediated insertional mutagenesis; Sandman K et al.; Mutations that cause sporulation defects (spo mutations) often identify developmentally regulated transcription units or genes whose products are required for the expression of sporulation-specific regulons . We report here the isolation, genetic analysis and phenotypic characterization of spo mutations produced by insertional mutagenesis with transposon Tn917, a form of mutagenesis that facilitates genetic and physical manipulation of mutated genes in many ways . Twenty-four insertional spo mutations were studied in detail . On the basis of transformation-mediated and transduction-mediated linkage analysis and a range of phenotypic tests, these mutations were assigned to 20 distinct loci, at least 9 of which are different from the 40 previously described spo loci . The insertional mutations caused blocks at a variety of different stages of sporulation, and therefore probably identify genes active at different times during sporulation . In addition to increasing substantially the total of known spo loci, we anticipate that this collection will include representatives of many of the temporally regulated sets of genes that comprise the overall program of sporulation-specific gene activation in Bacillus subtilis . Given the kinds of manipulations that are possible with genes disrupted by Tn917 insertions, this should significantly facilitate efforts to understand the regulation of these gene sets. J Bacteriol, 1987 Dec, 169(12), 5848 - 51 Genetic mapping of katA, a locus that affects catalase 1 level in Bacillus subtilis; Loewen PC et al.; Several mutants of Bacillus subtilis deficient in catalase synthesis generated by nitrosoguanidine mutagenesis have been used to map a locus affecting catalase activity . Two- and three-factor bacteriophage PBS1 transductional crosses were used to locate the locus, named katA, between recH and thiA with 98% linkage to thiA at 70 degrees on the B . subtilis genome . The synthesis of catalase 1, found only in vegetative cells, was affected by katA. J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3495 - 504 Autostimulation of dihydrostreptomycin uptake in Bacillus subtilis; Emling F et al.; In Bacillus subtilis it was shown that the membrane potential (delta psi) has to reach a threshold value of -180 to -190 mV for efficient uptake of dihydrostreptomycin to occur . The magnitude of delta psi is raised above this threshold, and dihydrostreptomycin uptake greatly enhanced, not only by dihydrostreptomycin itself (autostimulation) and by other miscoding aminoglycoside antibiotics, but also by puromycin, bacitracin and N,N'-dicyclohexylcarbodiimide . Stimulation of uptake by dihydrostreptomycin or puromycin was dependent on a specific interference with ongoing protein synthesis . Thus, chloramphenicol prevented the stimulating effect of puromycin by lowering the magnitude of delta psi . Although normally severely antagonizing streptomycin accumulation, K+, as well as spermidine and putrescine, which are known to stabilize ribosomes, consequently enhanced autostimulation of dihydrostreptomycin uptake in a K+-retention mutant with impaired protein synthesis . It is suggested that miscoding aminoglycosides and puromycin both enhance dihydrostreptomycin uptake by increasing delta psi due to ion fluxes, which are themselves caused by a dramatic stimulation of intracellular proteolysis of faulty proteins. J Antibiot (Tokyo), 1987 Dec, 40(12), 1692 - 7 Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis . III . Mode of action of difficidin; Zweerink MM et al.; The mode of action of difficidin (DIF) was investigated . Upon addition of DIF to log phase cultures of Escherichia coli, growth ceased immediately and small round cells accumulated after 30 minutes incubation . No cell lysis was observed . DIF was rapidly bactericidal to both growing and stationary phase cultures, and inhibited protein synthesis more rapidly than RNA, DNA, or cell-wall synthesis in growing cells . Protein synthesis was also inhibited in a cell-free system . The frequency of natural mutation to resistance in E . coli was less than 1 in 10(10) cells. Eur J Biochem, 1987 Nov 2, 168(3), 695 - 701 Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558; Friden H et al.; Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase . The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced . In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants . Mutant cytochrome was analyzed both in B . subtilis and after amplification in Escherichia coli . It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation . Mutagenesis of His-113 excludes this residue as an axial heme ligand . A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B . subtilis, whereas the mutation had no effect on translation in E . coli . Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC . Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B . subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation. Proc Natl Acad Sci U S A, 1987 Nov, 84(21), 7398 - 402 Occurrence of unmodified adenine and uracil at the first position of anticodon in threonine tRNAs in Mycoplasma capricolum; Andachi Y et al.; Codon usage pattern in the threonine four-codon (ACN) box in Mycoplasma capricolum is strongly biased towards adenine and uracil for the third base of codons . Codons ending in uracil or adenine, especially ACU, predominate over ACC and ACG . This bacterium contains two isoacceptor threonine tRNAs having anticodon sequences AGU and UGU, both with unmodified first nucleotides . It would thus appear that ACN codons are translated in an unusual way; tRNA(Thr)(AGU) would translate the most abundantly used codon ACU exclusively, because adenine at the first anticodon position can, according to the wobble rule, pair only with uracil of the third codon position . The tRNA(Thr)(UGU) would mainly be responsible for translation of three other codons, ACA, ACG, and ACC . Anticodon UGU would also be used for reading codon ACU as a redundancy of tRNA(Thr)-(AGU), as deduced from the mitochondrial code where unmodified uracil at the first anticodon position can pair with adenine, cytosine, guanine, and uracil by four-way wobble . The tRNA(Thr)(AGU) has much higher sequence homology to tRNA(Thr)(UGU) from M . capricolum (88%), Bacillus subtilis (77%) and Escherichia coli (86%) than to tRNA(Thr)(GGU) from B . subtilis (66%) and E . coli (63%), suggesting that tRNA(Thr)-(AGU) has been derived from tRNA(Thr)(UGU), but not from tRNA(Thr)(GGU). Antibiot Med Biotekhnol, 1987 Nov, 32(11), 820 - 4 {Design of a hybrid gene coding for the leader sequence of Bacillus amyloliquefaciens alpha-amylase and for human proinsulin}; Dem'ianova NG et al.; The chemically synthesized structure gene of human proinsulin was cloned in E . coli on the secretory vector containing regulatory elements of the Bacillus amyloliquefaciens alpha-amylase gene . The proinsulin gene was inserted by the EcoRI site located immediately after the DNA area encoding the alpha-amylase signal peptide . The E . coli cells transformed by such a plasmid produced hybrid protein consisting of the alpha-amylase signal peptide, five amino acid residues after the gene mating and human proinsulin . For accurate mating of the alpha-amylase gene leader sequence and proinsulin gene directed mutagenesis was performed on the filiform phage M13 mp9 with synthetic oligonucleotide . The hybrid gene was transferred to the vector molecule capable of replicating in Bacillus subtilis . It was shown that in the cells of both E . coli and B . subtilis there is synthesized protein interacting by the radio-immunological data with antibodies to porcine insulin, a large portion of immunologically active protein being detected in the periplasmic space of E . coli cells and in the culture fluid of B . subtilis cells which was indicative of proinsulin secretion directed by the alpha-amilase regulatory elements. J Antibiot (Tokyo), 1987 Nov, 40(11), 1588 - 95 Action of iturin A, an antifungal antibiotic from Bacillus subtilis, on the yeast Saccharomyces cerevisiae: modifications of membrane permeability and lipid composition; Latoud C et al.; The action of iturin A on non-growing cells of Saccharomyces cerevisiae was tested . This antibiotic gave important modifications in the membrane permeability which permitted nucleotides, proteins, polysaccharides and lipids to escape from cells . The lipid content of cells was strongly disturbed; the level of phospholipids, essentially phosphatidylcholine, decreased while the level of fatty acids increased . A part of these fatty acids were extruded from yeast cells . The role of iturin A in these modifications was discussed. J Gen Microbiol, 1987 Nov, 133 ( Pt 11), 3271 - 7 Protein processing to form extracellular thermostable alpha-amylases from a gene fused in a Bacillus subtilis secretion vector; Sohma A et al.; A thermostable alpha-amylase gene (amyT631) from Bacillus stearothermophilus A631 was cloned into pBR322 and recloned into pUB110: the resulting plasmid was designated pTUB607 . To investigate the processing from preproenzyme to mature enzyme, amyT631 from pTUB607, after digestion with BAL31, was introduced into the B . subtilis alpha-amylase secretion vector pTUB285 . Three chimaeric plasmids, pTUB613, pTUB616, and pTUB617, were isolated . The fused alpha-amylases expressed from the three plasmids seemed to be synthesized as preproenzymes . From analysis of the NH2-terminal amino acid sequences of purified extracellular alpha-amylases, the precursors of the fused enzymes appeared to be cleaved at first between amino acids 31 and 32 from the translation initiator Met (positions -11 and -10 with respect to the beginning of the mature enzyme), and processed to mature extracellular enzymes in which the NH2-terminal amino acid sequences were the same as that of the parental pTUB607 alpha-amylase, in spite of the lengths of the prosequences and the amino acid composition near the secondary cleavage sites being different in each enzyme. Biochem Cell Biol, 1987 Nov, 65(11), 939 - 47 Purification and characterization of catalase-1 from Bacillus subtilis; Loewen PC et al.; The catalase activity produced in vegetative Bacillus subtilis, catalase-1, has been purified to homogeneity . The apparent native molecular weight was determined to be 395,000 . Only one subunit type with a molecular weight of 65,000 was present, suggesting a hexamer structure for the enzyme . In other respects, catalase-1 was a typical catalase . Protoheme IX was identified as the heme component on the basis of the spectra of the enzyme and of the isolated hemochromogen . The ratio of protoheme/subunit was 1 . The enzyme remained active over a broad pH range of 5-11 and was only slowly inactivated at 65 degrees C . It was inhibited by cyanide, azide, and various sulfhydryl compounds . The apparent Km for hydrogen peroxide was 40.1 mM . The amino acid composition was typical of other catalases in having relatively low amounts of tryptophan and cysteine. Mutat Res, 1987 Nov, 184(3), 187 - 96 Effect of ultraviolet radiation on the Bacillus subtilis phages SPO2, SPP1 and phi 29 and their DNAs; Freeman AG et al.; A comparative study of the effects of ultraviolet radiation on three Bacillus subtilis phages is presented . Phages phi 29, SPP1 and SPO2c12 or their DNAs were irradiated by UVC (254 nm) and quantum yields for inactivation were calculated . For each phage, the purified DNA was found to be more sensitive than the intact virus when assayed in a uvr+ host . The data imply that this is because transfecting DNA is repaired less efficiently than DNA of the intact phage; rather than because of differences in sensitivity to lesion production . Even though phi 29 has the smallest target size of the three phages, phi 29 and its DNA are the most sensitive . Phages SPO2 and SPP1 code for gene products which complement the repair system of the host . The transfecting DNA of phage SPP1 is extremely sensitive to UV damage when assayed in a uvr-host . This is attributed to the fact that in transfection SPP1 DNA must undergo recombination for productive infection to occur . The recombination process strongly interferes with the repair of damaged DNA. J Bacteriol, 1987 Nov, 169(11), 5333 - 5 Efficient utilization and operation of the gluconate-inducible system of the promoter of the Bacillus subtilis gnt operon in Escherichia coli; Miwa Y et al.; A DNA fragment containing the promoter of the Bacillus subtilis gluconate (gnt) operon and its first gene (gntR) was cloned into Escherichia coli . E . coli recognized this promoter efficiently and precisely . Moreover, the gluconate-inducible system of this operon operated even in E . coli. J Bacteriol, 1987 Nov, 169(11), 5301 - 3 Absence of penicillin-binding protein 4 from an apparently normal strain of Bacillus subtilis; Buchanan CE; The phenotype of a Bacillus subtilis 168 strain with no detectable penicillin-binding protein 4 was examined . Despite the fact that penicillin-binding protein 4 is one of the most penicillin-sensitive proteins in the species, its apparent loss had no obvious effect on the organism or its susceptibility to various beta-lactam antibiotics. J Bacteriol, 1987 Nov, 169(11), 5258 - 62 Crystal protein formed by Bacillus subtilis cells; Rubikas J et al.; Electron microscopic investigation of ultrathin sections of Bacillus subtilis Cgr4 cells revealed the presence of crystal-like inclusions which were formed of spheric homogeneous subunits . The frequency of cells with a crystal-like inclusion in the culture approached 1% . The appearance of the crystal protein in cells coincided in time with spore morphogenesis . However, the process of crystal protein formation and sporulation are two alternatives: the cells either form the crystal protein or continue spore morphogenesis . Fractionation of cells in the stationary growth phase on a Percoll density gradient showed that the cells containing the crystal protein accumulated in the fraction corresponding to a 1.14-g/ml Percoll density . The cells were disintegrated by sonication, and alkaline-extracted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . After sodium dodecyl sulfate-gel electrophoresis, the fraction enriched with crystal-containing cells showed practically a single band with a molecular weight of 47,000 that corresponded to the crystal-forming protein . The antigenic features and amino acid composition indicated certain similarities between the crystal-forming protein in B . subtilis Cgr4 cells and the spore coat protein. Mol Gen Genet, 1987 Nov, 210(1), 116 - 21 Recombination between short repeated sequences is more frequent in plasmids than in the chromosome of Bacillus subtilis; Janniere L et al.; We measured in Bacillus subtilis the recombination frequency between directly repeated 9 bp sequences flanking the transposon Tn5 . Four different repeats were analyzed . They recombined with frequencies between 10(-7) and 10(-6) when they were carried on plasmids, and 150-1500 times less frequently when they were carried on the chromosome . We propose that this difference is due to the generation of single stranded DNA during plasmid replication. EMBO J, 1987 Nov, 6(11), 3543 - 9 Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions; Balganesh TS et al.; Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC . They differ in their capacity to methylate additional sequences . These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T . Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes . These expressed Mtase activity at a level comparable to that of the parental genes . The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions . This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme {CCGG, CC(A/T)GG or GCNGC} are represented by the central non-conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region. J Bacteriol, 1987 Nov, 169(11), 5131 - 9 Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus; Projan SJ et al.; A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers . The complete sequence of pIM13 has been reported (M . Monod, C . Denoya, and D . Dubnau, J . Bacteriol . 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194 . The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S . aureus plasmid pSN2 and is probably involved in plasmid replication . The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S . aureus . pIM13 is unstable in S . aureus but still multimerizes in that host, while pE5 is unstable in B . subtilis and does not form multimers in either host . The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13 . It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B . subtilis . The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein . It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication . It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5. Nucleic Acids Res, 1987 Oct 26, 15(20), 8501 - 9 Sequence features of the replication terminus of the Bacillus subtilis chromosome; Carrigan CM et al.; The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtilis 168 chromosome has been determined . The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence . The arrest site occurs in an A + T-rich region between two open reading frames and very close to one of two imperfect inverted repeats (47-48 nucleotides each) which are separated by 59 nucleotides . The closeness of approach of the arrested clockwise fork to the first imperfect inverted repeat encountered in this region raises the possibility of a role for the inverted repeats in the mechanism of fork arrest. J Mol Biol, 1987 Oct 20, 197(4), 729 - 36 Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase; Swanberg SL et al.; The gene gyrA of Escherichia coli, which encodes the A subunit of DNA gyrase (topoisomerase II), has been cloned and a region of approximately 3300 base-pairs sequenced . An open reading frame of 2625 nucleotides coding for a protein of 97,000 Mr is located . The peptide weight of the subunit predicted from this open reading frame is in close agreement with previously published estimates of that of the A subunit . There is a "TATAAT" promoter motif located 44 bases upstream from the first "ATG" of the open reading frame . The amino acid sequence derived from the nucleotide sequence is about 50% homologous with that derived from the Bacillus subtilis gyrA gene sequence, with several regions showing greater than 90% homology. Nucleic Acids Res, 1987 Oct 12, 15(19), 7781 - 93 Purification in an active form of the phage phi 29 protein p4 that controls the viral late transcription; Barthelemy I et al.; The phage phi 29 protein p4, that controls viral late transcription, was highly purified from Escherichia coli cells harbouring a gene 4-containing plasmid . This protein, representing about 6% of the total cellular protein, was obtained in a highly purified form . The protein was characterized as p4 by amino acid analysis and NH2-terminal sequence determination . The purified protein was active in an in vitro transcription assay, allowing specific initiation of transcription at the phi 29 A3 late promoter in the presence of Bacillus subtilis sigma 43-RNA polymerase holoenzyme. Nucleic Acids Res, 1987 Oct 12, 15(19), 8023 - 40 Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases; Shields DC et al.; Codon usage data for 56 Bacillus subtilis genes show that synonymous codon usage in B . subtilis is less biased than in Escherichia coli, or in Saccharomyces cerevisiae . Nevertheless, certain genes with a high codon bias can be identified by correspondence analysis, and also by various indices of codon bias . These genes are very highly expressed, and a general trend (a decrease) in codon bias across genes seems to correspond to decreasing expression level . This, then, may be a general phenomenon in unicellular organisms . The unusually small effect of translational selection on the pattern of codon usage in lowly expressed genes in B . subtilis yields similar dinucleotide frequencies among different codon positions, and on complementary strands . These patterns could arise through selection on DNA structure, but more probably are largely determined by mutation . This prevalence of mutational bias could lead to difficulties in assessing whether open reading frames encode proteins. J Gen Microbiol, 1987 Oct, 133 ( Pt 10), 2937 - 44 H2, a temperate bacteriophage isolated from Bacillus amyloliquefaciens strain H; Zahler SA et al.; Bacillus amyloliquefaciens strain H is lysogenic for a large temperate phage we call H2 . H2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm . H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA . The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B . subtilis to prototrophy . The H2 genome is a linear DNA molecule about 129 kb in length . DNA extracted from phage particles grown in B . subtilis is not cut by the restriction endonucleases HaeIII, Fnu4HI, Bsp1286I, and BamHI; the latter enzyme is produced by B . amyloliquefaciens strain H . The prophage in lysogenic B . subtilis cells can be cut by these enzymes . We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both. Mol Gen Genet, 1987 Oct, 209(3), 467 - 74 Mutations of the Escherichia coli lacUV5 promoter resulting in increased expression in Bacillus subtilis; Henkin TM et al.; The Escherichia coli lacUV5 promoter is used inefficiently by the major vegetative form of Bacillus subtilis RNA polymerase, despite very close adherence in the -35 and -10 regions to consensus sequences for promoters recognized by this enzyme . To select derivatives of this promoter with increased activity in B . subtilis, the lacUV5 promoter was fused to a promoter-less chloramphenicol acetyltransferase gene and mutagenized by passage through an E . coli mutD5 mutator strain . Derivatives that conferred resistance to chloramphenicol in B . subtilis were isolated . Twenty-three independent isolates each contained single mutations in the 207 bp lac fragment . These mutations, which were in ten different positions, fell in two clusters . One set of mutations, located between positions -18 and -14, resulted in greater homology to a consensus sequence previously noted for this region in B . subtilis vegetative promoters . The remaining mutations were located near the transcription initiation site . The effects of these mutations and additional mutations constructed by oligonucleotide-directed mutagenesis on expression in B . subtilis and E . coli was determined by measurements of chloramphenicol acetyltransferase activities directed by these promoters . While most mutations had little effect on expression in E . coli, the increase in activity in B . subtilis was as much as 28-fold. Genetika, 1987 Oct, 23(10), 1900 - 2 {Determination of the position of the spo-genes in region 210 of the Bacillus subtilis chromosome}; Chikindas ML et al.; Genes controlling sporulation of Bacillus subtilis SHgW are localized in the region of 210 degrees of Bac . subtilis chromosome between lys genes and the rib operon . The method of constructing deletions of definite size was used in the deletion mapping. Genetika, 1987 Oct, 23(10), 1839 - 46 {Integration and expression of the human dihydrofolate reductase gene in the Bacillus subtilis chromosome}; Poluektova EU et al.; Integration of functionally active human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis chromosome was performed . The clones obtained contained 1 to 7 copies of hDHFR gene per chromosome equivalent and were resistant to trimethoprim . In cell lysates of such clones a protein with the molecular mass of hDHFR was detected . The hDHFR gene was stably maintained in all clones having this gene integrated into the bacterial chromosome, when grown under non-selective conditions. J Bacteriol, 1987 Oct, 169(10), 4743 - 9 Purification and characterization of the F1 ATPase from Bacillus subtilis and its uncoupler-resistant mutant derivatives; Hicks DB et al.; The F1 ATPase of Bacillus subtilis BD99 was extracted from everted membrane vesicles by low-ionic-strength treatment and purified by DEAE-cellulose chromatography, hydrophobic interaction chromatography, and anion-exchange high-performance liquid chromatography . The subunit structure of the enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of urea . In the absence of urea, the alpha and beta subunits comigrated and the ATPase was resolved into four bands . The mobility of the beta subunit, identified by immunoblotting with anti-beta from Escherichia coli F1, was altered dramatically by the presence of urea, causing it to migrate more slowly than the alpha subunit . The catalytic activity of the ATPase was strongly metal dependent; in the absence of effectors, the Ca2+-ATPase activity was 15- to 20-fold higher than the Mg2+ -ATPase activity . On the other hand, sulfite anion, methanol, and optimally, octylglucoside stimulated the Mg2+ -ATPase activity up to twice the level of Ca2+ -ATPase activity (specific activity, about 80 mumol of Pi per min per mg of protein) . The F1 ATPase was also isolated from mutants of B . subtilis that had been isolated and characterized in this laboratory by their ability to grow in the presence of protonophores . The specific activities of the ATPase preparations from the mutant and the wild type were very similar for both Mg2+- and Ca2+ -dependent activities . Kinetic parameters (Vmax and Km for Mg-ATP) for octylglucoside-stimulated Mg2+ -ATPase activity were similar in both preparations . Structural analysis by polyacrylamide gel electrophoresis and isoelectric focusing indicated that the five F1 subunits from ATPase preparations from the mutant and wild-type strains had identical apparent molecular weights and that no charge differences were detectable in the alpha and beta subunits in the two preparations . Thus, the increased ATPase activity that had been observed in the uncoupler-resistant mutants is probably not due to a mutation in the F1 moiety of the ATPase complex. J Bacteriol, 1987 Oct, 169(10), 4848 - 51 Cloning and expression in Escherichia coli of chromosomal mercury resistance genes from a Bacillus sp; Wang Y et al.; A 7.9-kilobase (kb) chromosomal fragment was cloned from a mercury-resistant Bacillus sp . In Escherichia coli, in the presence of a second plasmid carrying functional transport genes, resistance to HgCl2 and to phenylmercury acetate (PMA) was expressed . Shortening the cloned fragment to 3.8 kb abolished resistance to PMA but not to HgCl2 . In Bacillus subtilis, the 3.8-kb fragment produced mercuric reductase constitutively but did not produce resistance to HgCl2 or to PMA. J Bacteriol, 1987 Oct, 169(10), 4479 - 85 Incorporation of specific exogenous fatty acids into membrane lipids modulates protonophore resistance in Bacillus subtilis; Krulwich TA et al.; Attempts to manipulate the level of C16:1 fatty acids in membrane phospholipids were made by using Bacillus subtilis and its protonophore-resistant mutants to test the hypothesis that C16:1 fatty acid levels relate to the bioenergetic properties of the mutant strains . Growth of the three mutants in the presence of palmitoleic acid restored the level of C16:1 fatty acids in the membrane lipids to somewhat above those found in the wild type . The palmitoleic acid was preferentially incorporated into diphosphatidylglycerol (cardiolipin) and phosphatidylethanolamine and was associated with increased levels of these phospholipids . These membrane preparations showed no increase in the levels of free fatty acids . The increase in C16:1 fatty acids achieved by growth in the presence of palmitoleic acid was accompanied by secondary changes in membrane lipids as well as a pronounced diminution in the protonophore resistance of growth and ATP synthesis . Other membrane-associated properties that had been observed in these mutants, e.g., elevated ATPase levels, were not altered coordinately with protonophore resistance and C16:1 fatty acid levels . Growth of the wild type in the presence of palmitic acid caused a modest elevation of the C16:0 of the membrane lipids and a modest increase in the protonophore resistance of growth and ATP synthesis . Growth of the wild type at elevated temperatures, in the absence of fatty acid supplementation, also enhanced its resistance to protonophores . The results support the hypothesis that specific changes in membrane lipid composition underlie the bioenergetic changes associated with protonophore resistance. J Bacteriol, 1987 Oct, 169(10), 4469 - 78 Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis; Guffanti AA et al.; Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) . The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin . The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential . Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force . Studies of revertants indicated that the phenotype resulted from a single mutation . Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP . The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids . Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons. J Mol Biol, 1987 Sep 5, 197(1), 55 - 67 Transcriptional control in the EcoRI-F immunity region of Bacillus subtilis phage phi 105 . Identification and unusual structure of the operator; Van Kaer L et al.; We present the first evidence, in Bacillus subtilis, for gene regulation through the classical mechanism of repressor-operator interaction . The EcoRI-F immunity region (immF) of lysogenic phage phi 105 contains two promoters, PM and PR, in divergent orientations . PM initiates transcription of the phi 105 repressor (c phi 105) gene, whereas PR most probably signals the onset of the lytic pathway . Fusions between each of these promoters and the cat-86 gene were constructed, and in-vivo promoter activities were determined, in both the presence and absence of the functional c phi 105 product, using S1 nuclease analysis and chloramphenicol acetyl transferase assays . The results showed that transcription from PM is stimulated, whereas PR activity is negatively controlled by the repressor . This differential regulation appears to be mediated by recognition of a 14 base-pair (bp) sequence, 5' GACGGAAATACAAG 3', three identical copies of which are present as direct repeats . Two copies, OR1 and OR2, are located closely together in the non-transcribed region between PM and PR, but do not overlap with the -35 and -10 regions of these promoters . The third copy, OR3, is located some 250 bp downstream from PR, within the coding region (ORF3) of the proximal gene of the PR transcription unit . When a 231 bp restriction fragment containing only OR3 was inserted between a strong constitutive promoter (P138) and the cat-86 gene, the in-vivo expression of chloramphenicol resistance was considerably reduced in the presence, but not in the absence, of phi 105 repressor . This hybrid P138-OR-cat-86 construct was subsequently used to select in vivo for operator-constitutive (Oc) mutations . Of 25 Oc mutants analyzed, all showed base alterations or deletions affecting the 14 bp sequence . We show further that insertion of a chemically synthesized oligonucleotide, containing the 14 bp OR sequence, at a site more than 100 bp downstream from the constitutive P138 is sufficient for transcription to become negatively controlled by phi 105 repressor . In comparison with previously identified Gram-negative bacterial and phage operators, the most unusual aspect of the phi 105 OR sequence appears to be its complete lack of 2-fold rotational symmetry. J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2567 - 72 Early changes in bacterial envelopes after inhibition of peptidoglycan synthesis, as shown by the use of a fluorescent probe; Rogers HJ et al.; The probe 8-anilino-1-naphthalene sulphonate (ANS) showed increasing fluorescence with Bacillus subtilis metC lyt-2 cells taken from exponentially growing cultures treated with antibiotics that inhibit cell-wall peptidoglycan synthesis . This increase was due to the probe reaching hydrophobic cell constituents, probably membranes, and started within 30 min of the addition of the antibiotics . This corresponded to the time at which membrane function had been shown to be damaged . The increased fluorescence of the cells with ANS persisted after removal of the antibiotics. J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2381 - 91 The possible DNA-binding nature of the regulatory proteins, encoded by spoIID and gerE, involved in the sporulation of Bacillus subtilis; Holland SK et al.; The predicted polypeptide products of two genes, spoIID and gerE, which appear to be concerned in the regulation of spore formation in Bacillus subtilis have been compared by modelling methods with known DNA-binding proteins . The results indicate that both polypeptides may have DNA-binding properties and the conclusion is drawn that this may account for their regulatory action. J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2371 - 80 Regulation of stage II of sporulation in Bacillus subtilis; Clarke S et al.; A mutation, spo-87, in the spo0J locus of Bacillus subtilis allows appreciable transcription of spoIIA, spoIID and spoIIG and later operons, even though most of the cells are morphologically blocked at stage 0 and the incidence of heat-resistant spores is about 1 per 10(4) cells . This mutation therefore appears to disengage the genetic control of sporulation from the morphological changes to which it should be connected . spoIIA and spoIIG are transcribed independently of one another . However, the products of both operons are needed for the activation of spoIID which occurs later . This indicates a convergence of parallel pathways of operon expression . We have also shown that nonsense mutations in spoIIAC (which codes for a sigma factor of RNA polymerase) prevent transcription of spoIID; by contrast, missense mutations in the same gene allow transcription of spoIID. J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2359 - 70 Nucleotide sequence of the sporulation operon, spoIIIE, of Bacillus subtilis; Butler PD et al.; A fragment of Bacillus subtilis DNA 3490 bp long, capable of complementing spoIIIE mutations, was sequenced . The region of the fragment that encodes functions required for sporulation was delimited using integrational plasmids . Sequencing showed that this region contained an operon with two open reading frames together with associated ribosome-binding sites . The deduced translation products would be polypeptides of 518 and 252 amino acid residues . Several sequences resembling promoters recognized by RNA polymerase containing sigma 29 occur in the region preceding the larger open reading frame . Although no transcription-termination signal was identified downstream of the smaller coding region, analysis with integrational plasmids and determination of the size of spoIIIE messenger RNA suggest that the locus does not contain a third gene. J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2349 - 57 Studies of transcriptional regulation of the Bacillus subtilis developmental gene spoVE; Bugaichuk UD; The structural gene of the spoVE locus of Bacillus subtilis was replaced with the promoterless lacZ gene of Escherichia coli . The spoVE::lacZ gene fusion was transferred to the B . subtilis chromosome and beta-galactosidase activity was measured under sporulation conditions . Expression of the hybrid gene could be detected as early as 40 min after the induction of sporulation . Transcription of the spoVE::lacZ gene was dependent on the products of two stage 0 loci, spo0H and spo0K . Mutations in the spoIIA and spoIIG loci did not impede expression of spoVE and, therefore, neither of the sigma factors coded for by these loci seems to be necessary for its transcription . Consequently, the spoVE locus does not seem to be part of the dependent sequence of operons involved in the developmental change, although its protein product is clearly needed for the completion of spore formation. Zh Mikrobiol Epidemiol Immunobiol, 1987 Sep, (9), 12 - 8 {Patterns in the immunoenzyme analysis of bacterial cells}; Karulin AIu et al.; Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora . Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis) . The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined . Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates . The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed . Four EIA methods for measurement of contaminant microflora have been developed and optimized . These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes. Appl Environ Microbiol, 1987 Sep, 53(9), 2133 - 7 Mechanism of microwave sterilization in the dry state; Jeng DK et al.; With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp . niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven . The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical . Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects . Nonthermal effects were not significant in a dry microwave sterilization process . Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C . The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively . The Z value was estimated to be 18 degrees C. Mol Gen Genet, 1987 Sep, 209(2), 333 - 4 Effect of precisely identified mutations in the spoIIAC gene of Bacillus subtilis on the toxicity of the sigma-like gene product to Escherichia coli; Yudkin MD et al.; Yudkin (1986) has shown that the spoIIAC gene of Bacillus subtilis cannot be cloned in Escherichia coli in such an orientation that it is expressed . This toxicity of the gene product has been attributed to its close homology with the sigma subunit of the E . coli RNA polymerase . The effect of six individual mutations in spoIIAC has now been studied . All six mutant genes could be cloned in E . coli in an orientation that does not allow expression . When in the orientation that permits expression, one mutant gene could not be cloned, and a second substantially hampered growth; both mutations lie in the region that is believed to encode the DNA-binding domain of the protein . By contrast, two missense mutations in the region of the gene thought to encode the domain that binds to the core RNA polymerase rendered the protein harmless in E . coli, as did two nonsense mutations. J Bacteriol, 1987 Sep, 169(9), 4235 - 41 Drug-free induction of a chloramphenicol acetyltransferase gene in Bacillus subtilis by stalling ribosomes in a regulatory leader; Duvall EJ et al.; The plasmid gene cat-86 is induced by chloramphenicol in Bacillus subtilis, resulting in the synthesis of the gene product chloramphenicol acetyltransferase . Induction is due to a posttranscriptional regulatory mechanism in which the inducer, chloramphenicol, activates translation of cat-86 mRNA . We have suggested that chloramphenicol allows ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the coding sequence . In the present report we show that cat-86 expression can be activated by stalling ribosomes in the act of translating a regulatory leader peptide . Stalling was brought about by starving host cells for specific leader amino acids . Ribosomal stalling, which led to cat-86 expression, occurred upon starvation for the amino acid specified by the leader codon located immediately 5' to the RNA stem-loop structure and was independent of whether that codon specified lysine or tyrosine . These observations support a model for chloramphenicol induction of cat-86 in which the antibiotic stalls ribosome transit in the regulatory leader . Stalling of ribosomes in the leader can therefore lead to destabilization of the RNA stem-loop structure. J Bacteriol, 1987 Sep, 169(9), 4135 - 40 Membrane protein binding to the origin region of Bacillus subtilis; Laffan J et al.; Binding of membrane proteins extracted from Bacillus subtilis to an 11.6-kilobase region containing the origin of replication was examined by Western blotting (protein blotting) procedures . Two adjacent origin probes in the double-stranded form (spanning a length of 4 kilobases) were found to bind very strongly to a 63-kilodalton (kDa) protein in that they resisted dissociation after a high-concentration salt wash . This region encompasses both a site implicated in initiation in vivo and a gene coding for a DNA gyrase subunit (gyrA) . In contrast, flanking origin and nonorigin double-stranded probes were dissociated after washing with a high salt concentration . Another protein of 67 kDa bound less intensely to the putative initiation site but not to the gyrA region . All of the origin and nonorigin probes in the double- or single-stranded form were found to bind nonspecifically to a subset of 10 to 12 proteins of 50 to 60 separated by gel electrophoresis after a low-concentration salt wash . They ranged in size from 14 to over 100 kDa (including 63 kDa) . However, in contrast to the double-stranded forms, most of the single-stranded probes resisted dissociation from the protein subset after a high-concentration salt wash. J Bacteriol, 1987 Sep, 169(9), 4068 - 75 Characterization of nutrition-induced helix hand inversion of Bacillus subtilis macrofibers; Wolfe AJ et al.; The kinetics of Bacillus subtilis macrofiber helix hand inversion was examined . Inversion was induced by transfer of structures produced in one medium to another medium . When cultured at 20 degrees C in either medium, the doubling time was approximately 100 min . To establish a baseline, the macrofiber twist state produced in one medium was measured over the same time course during which other macrofibers underwent inversion after transfer to a second medium . The baseline was used to identify the time of inversion initiation: the point at which curves representing changes of twist as a function of time after transfer to the new medium intersected the baseline . Right- and left-handed macrofibers of different twists were produced by growth in mixtures of TB and S1 media . These were used to determine the influence of initial twist on the time course of inversion initiation . In the right to left inversion, a positive correlation was found between initial twist and the time of inversion initiation . The left to right inversion differed, however, in that a constant time was required for inversion initiation regardless of the starting left-handed twist . When a nutritional pulse was administered by transferring fibers from TB to S1 to TB medium, the time to initiation of inversion was found to decrease with incubation of increasing duration in S1 medium . A similar pulse protocol was used in conjunction with inhibitors to examine the protein and peptidoglycan synthesis requirements for the establishment of nutrition-induced memory that leads to initiation of inversion . Nutritionally induced right to left inversion but not left to right inversion required protein synthesis . The addition of trypsin to left-handed macrofibers apparently required, as described previously for the temperature-regulated twist system (D . Favre, D . Karamata, and N . H . Mendelson, J . Bacteriol . 164:1141-1145, 1985), for the production of left-handed twist states in the nutrition system. Eur J Biochem, 1987 Sep 1, 167(2), 233 - 8 Purification and characterization of the spoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli; Ikeuchi T et al.; The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation . To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter . When expression of the spoOA gene was induced in Escherichia coli cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein . The SpoOA protein was purified to homogeneity from these cells . We found that the NH2-terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA-cat protein) from B . subtilis, and that the NH2-terminal methionine of the SpoOA protein from E . coli was formylated presumably because of insufficient amounts of the deformylating enzyme . The T signal {Ganoza, M . C., Marliere, P., Kofoid, E . C . and Louis, B . G . (1985) Proc . Natl Acad . Sci . USA 82, 4587-4591}, in addition to the Shine-Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E . coli as well as in B . subtilis . We also found that the purified SpoOA protein had a DNA-binding activity . It was preferentially bound to the 175-bp BclI fragment of phi 105 DNA, and was released in the presence of 0.3 M KCl. J Bacteriol, 1987 Sep, 169(9), 4190 - 5 Developmental expression of three proteins from the first gene of the RNA polymerase sigma 43 operon of Bacillus subtilis; Wang LF et al.; The first gene of the Bacillus subtilis RNA polymerase sigma 43 operon, P23, has a protein-coding capacity of 23,000 daltons . Sequence analysis revealed three potential translational initiation sites within the same reading frame, which could encode proteins of 23,000 (P23), 19,000 (P19), and 9,000 (P9) daltons, respectively . An internal promoter (P3), which is expressed only during the sporulation stage, is located between the second and the third translational start sites . By protein fusion to the Escherichia coli beta-galactosidase gene, we showed that all three translational initiation sites of the P23 gene are used in vivo in both E . coli and B . subtilis, and regulation for differential expression of the three proteins during the development of B . subtilis is coupled to the transcriptional promoter switching mechanism . The physiological function of these multiple gene products is unknown and is currently under investigation. J Bacteriol, 1987 Sep, 169(9), 4141 - 6 Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid; Watabe K et al.; The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane . We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2 . In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature . In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked . We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1 . The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1 . In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature . On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature . These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways. J Bacteriol, 1987 Sep, 169(9), 3952 - 62 Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis; Chang S et al.; The Bacillus plasmid pLS11 partitions faithfully during cell division . Using a partition-deficient plasmid vector, we randomly cloned DNA fragments of plasmid pLS11 and identified the locus that regulates plasmid partition (par) by cis complementation in Bacillus subtilis . The cloned par gene conferred upon the vector plasmid a high degree of segregational stability . The par locus was mapped to a 167-base-pair segment on pLS11, and its nucleotide sequence was determined . The cloned par fragment regulated the partition of several different Bacillus replicons, and it only functioned in cis; it did not contain the replication function nor elevate the plasmid copy number in B . subtilis . The expression of par was orientation specific with respect to the replication origin on the same plasmid . We propose that the pLS11-derived par functions as a single-stranded site that interacts with other components involved in plasmid partition during cell division. J Bacteriol, 1987 Sep, 169(9), 3873 - 8 A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis; Kobayashi T et al.; A second regulatory locus (blaR1) required for the induction of beta-lactamase synthesis in Bacillus licheniformis 749 was cloned and sequenced . The gene was located on a 5.2-kilobase-pair SphI DNA fragment which also contained the beta-lactamase (blaP) and repressor (blaI) genes . Bacillus subtilis BD224 carrying these three genes synthesized beta-lactamase on exposure to cephalosporin C, whereas Escherichia coli HB101 carrying the genes did not show any detectable induction of the enzyme . An open reading frame of 1,803 bases was identified as the blaR1 gene by subcloning and DNA sequencing . The gene started 2 bases downstream of the termination codon of bla1 and was preceded by a putative Shine-Dalgarno sequence (AAGGA) with a spacing of 5 bases . The deduced blaR1 product (601 amino acids) had a molecular weight of 68,425 . Five transmembrane regions were predicted from the hydrophobicity profile . The region around Phe-Ala-Pro-Ala-Ser-Thr-Tyr-Lys (amino acids 398 to 405), which appeared to be located outside the membrane, was homologous to the binding regions of penicillin-binding proteins, including the beta-lactamases . The segment of 22 amino acids from 400 to 421 showed more than 70% homology to the penicillin-binding region of PBP 2 of E . coli . The blaR1 gene encodes a potential penicillin receptor which is required for the induction of beta-lactamase in B . licheniformis 749. J Bacteriol, 1987 Sep, 169(9), 3867 - 72 Cloning and nucleotide sequence of the penicillinase antirepressor gene penJ of Bacillus licheniformis; Imanaka T et al.; The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid . The penicillinase gene, penP, its repressor gene, penI, and penJ were encoded on the cloned 5.2-kilobase HindIII fragment of the recombinant plasmid pTTE71 . The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388) . A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site . Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the penI promoter . Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B . licheniformis by chromosomal recombination . The transformant B . licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible . Only when these three genes (penP, penI, and PenJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B . licheniformis . It was concluded that penJ is involved in the penicillinase induction . The regulation of penP expression by penI and penJ is discussed. Antimicrob Agents Chemother, 1987 Sep, 31(9), 1348 - 54 Accumulation of enoxacin by Escherichia coli and Bacillus subtilis; Bedard J et al.; Several methods were used to determine enoxacin uptake in Escherichia coli strains because washing of cells removed all or most cell-associated enoxacin whereas no washing was associated with large amounts of cell-bound enoxacin . Washing after up to 40 to 45 min of exposure to enoxacin followed by suspension in drug-free medium prevented a significant effect of enoxacin on cell growth . Cell uptakes obtained with different methods showed no difference in the shape of the timed uptake curves but did show significant quantitative differences . These results are consistent with cell-associated enoxacin comprising a freely exchangeable pool of drug . Lineweaver-Burk plots of uptake were consistent with uptake of enoxacin by simple diffusion . No saturability and no competition with ciprofloxacin were observed . Low temperature (4 degrees C) was associated with decreased uptake . Arsenate, carbonyl cyanide m-chlorophenylhydrazone, sodium fluoride, sodium azide, and 2,4-dinitrophenol had no effect on uptake . We conclude that the mechanism of transport of enoxacin into cells is by simple diffusion . Mutants of E . coli with deficiency of outer membrane proteins F and C and an enoxacin-resistant mutant selected by serial passage with increasing enoxacin concentrations demonstrated that F porins play a significant role in enoxacin uptake and influence susceptibility to enoxacin . Uptake was shown to be similar in a strain of Bacillus subtilis. Mol Gen Genet, 1987 Sep, 209(2), 335 - 42 The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg; Haima P et al.; Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied . In a restriction-deficient but modification-proficient mutant of B . subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E . coli (10(4) clones per microgram target DNA) . Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb) . In the restriction-proficient B . subtilis strain, the class of large inserts was underrepresented . Transformation of B . subtilis with E . coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways . First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host . The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb) . Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid . In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0-16 kb), and no structural instability was observed . It is concluded that for shotgun cloning in B . subtilis, the use of restriction-deficient strains is highly preferable . Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction . It is postulated that AsuII sites are additional target sites for BsuM restriction. Nucleic Acids Res, 1987 Aug 25, 15(16), 6349 - 67 Generation of linear multigenome-length plasmid molecules in Bacillus subtilis; Viret JF et al.; Linear multigenome-length double and single stranded plasmid DNA was identified in a Bacillus subtilis ATP-dependent DNAase mutant strain (addA5) bearing plasmids pC194 or pBD95ts . Plasmid pBC30, a seg mutant of pC194, as well as some pUB110 derivatives with rearrangements external to the minimal replicon, produce high amounts of such a concatemeric DNA, even in Rec+ cells . The synthesis of this type of plasmid DNA occurs in the absence of an active plasmid-encoded Rep protein and is markedly affected in polA5 and recE4 genetic backgrounds . To account for these observations, we propose that the AddAB complex serves to prevent a sigma-type replication of plasmid DNA. Biochim Biophys Acta, 1987 Aug 25, 909(3), 213 - 21 Molecular studies on thiaminase I; Abe M et al.; The thiaminase I gene of Bacillus thiaminolyticus was cloned on a 1.6 kb DNA fragment (enzyme molecular weight 42,000), and was expressed in both Escherichia coli and Bacillus subtilis . When a selection drug was absent, the plasmid was maintained stably for approx . 100 generations in wild-type E . coli . Instability of the thiaminase gene was demonstrated in the thiamin pyrophosphate-requiring mutant of E . coli from which the plasmid was deleted rapidly . Wild-type E . coli accumulated the enzyme in its periplasm . A method for the detection of thiaminase I enzyme in SDS-polyacrylamide gel was developed . Thiaminase I of B . thiaminolyticus was found to exist in two sizes, 44 and 42 kDa, among different strains . Moreover, thiaminase of 42 kDa became approximately 41 kDa after a long-term culture, most likely because of the action of proteinases . Thiaminase expressed in E . coli from a thiaminase-positive recombinant plasmid was 42 kDa, and showed the same mobility on SDS-polyacrylamide gele electrophoresis as the enzyme isolated from the young culture of the parent strain of B . thiaminolyticus used for cloning . This value was, therefore, considered to represent intact thiaminase that had escaped from the attack of bacilli proteinases. J Mol Biol, 1987 Aug 5, 196(3), 721 - 7 Breakdown and quantitation of the forked termination of replication intermediate of Bacillus subtilis; Hanley PJ et al.; Using a procedure that minimizes shear forces, the BamHI-derived forked termination of replication intermediate of Bacillus subtilis, called band I DNA, can be extracted with little or no accompanying band II DNA . It has been shown that band II DNA is a product of band I breakdown . Nuclease P1-mediated breakdown of the forked band I DNA proceeds in two steps . The first causes the release of one of the arms as band II DNA; in the second step, the remaining arm is cleaved away to yield the free stem . It is concluded that band I represents the primary termination of replication intermediate . A quantitative assessment of the level of band I in DNA from cells of the merodiploid strain, GSY1127, growing at different rates has been made . For cells grown in a minimal medium, at least, the experimentally measured level of band I is of the order (approx . 60%) of that predicted for a complete block to movement of the clockwise fork at the replication terminus, terC. Eur J Biochem, 1987 Aug 3, 166(3), 589 - 95 Spectral and potentiometric analysis of cytochromes from Bacillus subtilis; de Vrij W et al.; Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain . At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography . They include two terminal oxidases with CO-binding properties and cyanide sensitivity . One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions . In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration . The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm . Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV . Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found . This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels . A protein with a molecular mass of about 30 kDa is responsible for this activity . A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase . Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected. Eur J Biochem, 1987 Aug 3, 166(3), 581 - 7 Kinetic characterization of cytochrome c oxidase from Bacillus subtilis; de Vrij W et al.; Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins . The kinetic properties of the enzyme are influenced by ionic strength . The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant . An increase of Vmax is observed when the enzyme is incorporated in artificial membranes . Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase . The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax . These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme . The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH . The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state. Genetika, 1987 Aug, 23(8), 1414 - 20 {The role of metabolic and regulatory factors in the mutagenic activity of purine derivatives in microorganisms}; Mikhailova NP et al.; The results concerning analysis of the mutagenic activity of analogues of nitrogen bases are given for Saccharomyces cerevisiae, Streptomyces antibioticus, Bacillus subtilis . The mutagenic activity of purine derivatives depends on their metabolic transformations in cell and on the activity of enzyme systems, involved in regulation of purine biosynthesis . The criterion of selection of purine analogues with genetic activity is proposed for yeasts, based on retroinhibitory ability of analogues of nitrogen bases. Microbiol Sci, 1987 Aug, 4(8), 238 - 44 Dependent sequences of gene expression controlling spore formation in Bacillus subtilis; Mandelstam J et al.; Sporulation in Bacillus subtilis is a simple form of differentiation that involves the formation of a two-cell organism; mother cell and prospective spore . It is regulated by at least 50 operons, some mono- and some polycistronic . These are expressed in a dependent sequence which branches at an early stage so that there are independent lines of expression in both cells; further ramifications then occur in both cell compartments . Three DNA-binding proteins and two sigma factors have so far been identified as probable regulators of the dependent sequence . Most of the operons of the sequence are expressed in the first four hours and the last two stages occur mainly by self-assembly of proteins. J Gen Microbiol, 1987 Aug, 133 ( Pt 8), 2079 - 88 Analysis of the inhibition of sporulation of Bacillus subtilis caused by increasing the number of copies of the spo0F gene; Chapman JW et al.; The Bacillus subtilis spo0F gene was cloned on a 6.3 kbp Bg/II fragment . The effect on sporulation of amplification of the spo0F region was examined . Sporulation was inhibited to less than 5% of that of the parental strain when as few as four copies of the spo0F region were present . Subclones, constructed in autonomous or integrative vectors, were used to demonstrate that the region responsible for the copy-number-dependent asporogeny corresponded closely with the spo0F structural gene . A possible mechanism for this effect is discussed. Mol Gen Genet, 1987 Aug, 209(1), 8 - 14 Molecular cloning of Bacillus subtilis genes involved in DNA metabolism; Perego M et al.; Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101 . A lambda recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a lambda Charon 4A library . A restriction map of the cloned DNA fragments was constructed . The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well. J Appl Bacteriol, 1987 Aug, 63(2), 183 - 8 The sporicidal activity and inactivation of chlorhexidine gluconate in aqueous and alcoholic solution; Gorman SP et al.; The sporicidal activity of chlorhexidine gluconate in aqueous and alcoholic solution against spores of Bacillus subtilis was examined over a broad temperature range . Activity was not observed at 20 degrees C even with concentrations as high as 10% chlorhexidine . Temperatures of 37 degrees-70 degrees C in combination with such high concentrations were required for reductions in spore viability . No viable spores were recoverable after 4 h contact at 55 degrees C with 10% aqueous chlorhexidine and none after 3 h contact with the alcoholic solution . Because of the high concentrations necessary for activity and the possibility of sporostasis occurring from inefficient chlorhexidine inactivation, existing inactivation systems were examined and modified to obtain satisfactory results . The spores of other Bacillus species examined (B . cereus, B . megaterium and B . stearothermophilus) proved to be considerably less resistant than those of B . subtilis . Presence of organic matter had little effect on the activity. J Bacteriol, 1987 Aug, 169(8), 3633 - 7 Different small, acid-soluble proteins of the alpha/beta type have interchangeable roles in the heat and UV radiation resistance of Bacillus subtilis spores; Mason JM et al.; Spores of Bacillus subtilis strains which carry deletion mutations in one gene (sspA) or two genes (sspA and sspB) which code for major alpha/beta-type small, acid-soluble spore proteins (SASP) are known to be much more sensitive to heat and UV radiation than wild-type spores . This heat- and UV-sensitive phenotype was cured completely or in part by introduction into these mutant strains of one or more copies of the sspA or sspB genes themselves; multiple copies of the B . subtilis sspD gene, which codes for a minor alpha/beta-type SASP; or multiple copies of the SASP-C gene, which codes for a major alpha/beta-type SASP of Bacillus megaterium . These findings suggest that alpha/beta-type SASP play interchangeable roles in the heat and UV radiation resistance of bacterial spores. J Bacteriol, 1987 Aug, 169(8), 3601 - 7 Multiple catalases in Bacillus subtilis; Loewen PC et al.; Vegetative cells of Bacillus subtilis in logarithmic growth phase produced one catalase, labeled catalase 1, with a nondenatured molecular weight of 205,000 . As growth progressed, other activity bands with slower electrophoretic mobilities on polyacrylamide gels appeared, including a series of bands with a common nondenatured molecular weight of 261,000, collectively labeled catalase 2, and a minor band, with a molecular weight of 387,000, labeled catalase 3 . Purified spores contained only catalase 2, and it was not produced in spo0A- or spo0F-containing mutants . Strains deficient in catalase 1 or catalase 2 or both were selected after mutagenesis . Sensitivities of the two main catalases to NaCN, NaN3, hydroxylamine, and temperature were similar, but the apparent Kms for H2O2 differed, being 36.6 and 64.4 mM, respectively, for catalase 1 and catalase 2 . The levels of catalase 1 increased 15-fold during growth into stationary phase and could be increased 30-fold by the addition of H2O2 to the medium . Catalase 2, which was not affected by H2O2, appeared only after the cells had reached stationary phase, and the maximum levels were only half of the basal level of catalase 1. J Bacteriol, 1987 Aug, 169(8), 3508 - 14 Secretion and processing of staphylococcal nuclease by Bacillus subtilis; Miller JR et al.; We have studied the secretion and processing of Staphylococcus aureus nuclease in Bacillus subtilis . We show that the initial species of nuclease found in the cell supernatants during short-term radioactive labeling (pulse-chase) had a molecular weight of approximately 18,800 and comigrated in a sodium dodecyl sulfate-polyacrylamide gel with staphylococcal nuclease B . This nuclease B form was processed to the mature nuclease A extracellularly by a phenylmethylsulfonyl fluoride-sensitive protease . The nuclease B-processing site is a consensus signal peptidase site, and the processing of nuclease B was coupled to secretion as judged by pulse-chase experiments . The nuclease A was shown by microsequencing of the N terminus to be 2 amino acid residues shorter than the nuclease A described for S . aureus Foggi . The nuclease B form was still the first species found in the culture supernatant after removal of the N-terminal 26 amino acids of the native 60-amino-acid signal peptide . However, removal of the N-terminal 72 amino acids abolishes secretion of any nuclease form and leads to the intracellular accumulation of nuclease. J Bacteriol, 1987 Aug, 169(8), 3464 - 9 Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis; Igo M et al.; sigma B (sigma 37) is a secondary species of RNA polymerase sigma factor found in the gram-positive bacterium Bacillus subtilis . To study the function of sigma B genetically, we sought mutations that block the expression of a gene (ctc) known to be transcribed by sigma B-containing RNA polymerase in vitro . One such mutation, called crl, was found to map in or near the structural gene (sigB) for sigma B . To determine directly whether mutations in sigB would prevent transcription of ctc, we replaced sigB in the B . subtilis chromosome with insertion and deletion mutations that disrupted the sigma B coding sequence . Like crl, these in vitro-constructed mutations blocked expression of ctc, but had little or no effect on viability, sporulation, expression of the sporulation gene spoVG, or production of sporulation-associated alkaline protease . Using fusions of ctc to the reporter genes xylE and lacZ, we also identified mutations that enhanced ctc expression . One such mutation, called socB, was found to be located in an open reading frame immediately downstream of sigB. Proc Natl Acad Sci U S A, 1987 Aug, 84(15), 5207 - 10 A common origin for enzymes involved in the terminal step of the threonine and tryptophan biosynthetic pathways; Parsot C; Comparison of the amino acid sequence of Bacillus subtilis threonine synthase with the National Biomedical Research Foundation protein sequence library revealed a statistically significant extent of similarity between the sequence of the tryptophan synthase beta chain from various organisms and that of threonine synthase . This homology in the primary structure of threonine synthase and tryptophan synthase beta chain, which catalyze the last step in the threonine and the tryptophan biosynthetic pathways, respectively, correlates well with some of their catalytic properties and indicates that they have evolved from a common ancestor . The evolutionary relationship between these enzymes supports the hypothesis that primitive enzymes possessed a broad substrate specificity and were active in several metabolic pathways. Contact Dermatitis, 1987 Aug, 17(2), 73 - 9 Piroxicam-induced photosensitivity; Figueiredo A et al.; During the last 3 years, 9 patients with a photosensitive eruption related to piroxicam therapy were seen . In all but one, it occurred within 4 days of first exposure to the drug . 7 patients required systemic corticosteroids, and 2 hospitalization . Clinical, histological and provocation studies were not conclusive in classifying the eruption as photoallergic or phototoxic . Experimental studies including photohaemolysis, Bacillus subtilis culture and nuclear magnetic resonance showed: (i) in irradiated piroxicam solutions, there was more haemolysis; (ii) in irradiated Petri dishes, piroxicam solutions showed greater inhibition of growth of B . subtilis; (iii) Piroxicam's NMR spectrum is not modified after irradiation . The results provide evidence of piroxicam phototoxic potential. J Gen Microbiol, 1987 Aug, 133 ( Pt 8), 2237 - 46 The major acid-soluble proteins of Bacillus subtilis spores: partial amino acid sequence and forespore location of their mRNAs; Blom H et al.; In Bacillus subtilis the alpha, beta, gamma and delta components comprise 80-90% of the total acid-soluble spore proteins (ASSPs) . Sequence analysis demonstrates that alpha and beta share 32 of their first 36 amino acids and are closely related to the A and C ASSPs of Bacillus megaterium spores, confirming the results of analysis of their cloned genes . Despite the difference in apparent size of gamma and delta, they have identical N-terminal sequences (37 residues) . Unless gamma and delta derive from very recently duplicated genes, it appears that gamma is derived from delta, either in vivo or during isolation . Although the sequenced regions of gamma and delta have no homology to alpha and beta, outside of the previously recognized pentapeptide recognition sequence for the spore endopeptidase, they share 10 and 15 residue peptides flanking this sequence with ASSP B of B . megaterium, but in reverse order . At least two groups of ASSPs have, therefore, been conserved between B . subtilis and B . megaterium: the multigene AC alpha beta family and the B gamma (delta) group . Sequence conservation in each group implies selection for functions in addition to storage . Both the alpha and beta components of B . subtilis ASSPs and their mRNAs are located in the forespore compartment of cells at t5.5 of sporulation, the time of most rapid ASSP synthesis . The sizes of these transcripts (250-350 bp) and their ability to direct the in vitro synthesis of ASSPs of mature size, indicate that genes for these ASSPs are monocistronic, consistent with dispersed map location . Synthesis of ASSPs is, therefore, coordinately controlled by selective transcription in the forespore. J Bacteriol, 1987 Aug, 169(8), 3857 - 60 Site and substrate specificity of the ermC 23S rRNA methyltransferase; Denoya CD et al.; The purified ermC methyltransferase described here incorporates two methyl groups per Bacillus subtilis 23S rRNA molecule in vitro . The Km for S-adenosyl-L-methionine was 12 microM, and for B . subtilis 23S rRNA the Km was 375 nM . In vivo methylation specified by several related resistance determinants prevented in vitro methylation by the ermC enzyme, suggesting that methylation specified by all of these determinants occurs at homologous sites . Since methyl groups were incorporated in protein-free 23S rRNA molecules, the structure of rRNA alone must contain sufficient information to specify the methylation site. J Mol Biol, 1987 Jul 20, 196(2), 347 - 54 3'-terminal polyadenylate sequences of Escherichia coli tryptophan synthetase alpha-subunit messenger RNA; Karnik P et al.; Our earlier studies have shown that the mRNA from many bacterial species, including Escherichia coli and Bacillus subtilis, is extensively polyadenylated, but with shorter poly(A) segments than those associated with eukaryotic mRNA . In this paper, we show that about 40% of the mRNA for the tryptophan synthetase alpha-subunit (TrpA) of E . coli carries a 3'-terminal polyadenylate sequence of 15 to 20 residues . This conclusion was supported by several independent lines of evidence . About 40% of trpA mRNA bound to oligo(dT)-cellulose at high ionic strength and was eluted with water . Treatment with RNase H in the presence of oligo(dT)12-18 destroyed the ability of trpA mRNA to bind to oligo(dT)-cellulose, presumably through the degradation of the poly(A) tract . trpA mRNA could be used as template for complementary DNA synthesis with reverse transcriptase in a reaction that was absolutely dependent on oligo(dT)12-18 as primer . The identity of the cDNA product as a complement to trpA mRNA was established by specific hybridization . In addition, it was possible to synthesize polyadenylated trpA mRNA in toluene-permeabilized cells of E . coli transformed with a recombinant plasmid carrying the trpA gene . In view of the fact that the trpA gene and its 3'-untranslated region contain no continuous deoxyadenylate sequences larger than five nucleotides, one can conclude that the polyadenylate moiety is added post-transcriptionally. Biochem Biophys Res Commun, 1987 Jul 15, 146(1), 73 - 9 Modulation of Bacillus subtilis alpha-amylase promoter activity by the presence of a palindromic sequence in front of the gene; Takano J et al.; Upstream of the promoter of the Bacillus subtilis alpha-amylase gene (amyE) derived from an alpha-amylase hyper-producing strain, there is an inverted repeat sequence (palindromic sequence), which has a free energy of 21.2 kcal/mol due to the formation of stable stem-loop structure . The role of the palindromic sequence for the expression of amyE was studied using a plasmid encoding the amyE'-'bla (E . coli beta-lactamase) fused gene in an alpha-amylase-deficient B . subtilis mutant as the host . By the presence of the palindromic sequence, the transcription activity of the