Bacillus subtilis

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 amyE promoter was enhanced approximately 6 fold by starch (3%) in the medium and was less repressed by glucose.

J Mol Biol, 1987 Jul 5, 196(1), 39 - 48
Plasmid replication stimulates DNA recombination in Bacillus subtilis; Noirot P et al.; The effects of plasmid replication on the frequency of homologous recombination have been investigated . For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed . The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B . subtilis chromosomal sequences . As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated . Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate . It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid . These results show that plasmid replication stimulates DNA recombination.

J Mol Biol, 1987 Jul 5, 196(1), 1 - 10
Genes encoding spore coat polypeptides from Bacillus subtilis; Donovan W et al.; Endospores of the Gram-positive bacterium Bacillus subtilis are encased in a tough protein shell, known as the coat, that consists of a dozen or more different polypeptides . We have cloned structural genes designated cotA, cotB, cotC and cotD that encode spore coat proteins of Mr 65,000, 59,000, 12,000 and 11,000, respectively . These genes were cloned by using as hybridization probes synthetic oligonucleotides that were designed on the basis of partial NH2-terminal sequence determinations of the purified coat proteins . To determine the location of the cot genes on the chromosome and to study their function genetically, we tagged each gene by insertion of a chloramphenicol-resistance determinant (cat) within its coding sequence . We then replaced each wild-type cot gene in the chromosome with the corresponding, insertionally inactivated gene . Genetic mapping experiments showed that cotA, cotB, cotC and cotD were located at 52 degrees, 290 degrees, 168 degrees and 200 degrees, respectively, on the B . subtilis chromosome . None of the cot::cat insertion mutants were Spo-, but spores of the cotD mutant were found to germinate somewhat more slowly than did wild-type spores, and the cotA mutant was found to be blocked in the appearance of the brown pigment characteristic of colonies of wild-type sporulating cells . Physical and genetic experiments established that cotA was identical to a previously identified gene called pig, known to be responsible for sporulation-associated pigment production . Spores from all four insertion mutants exhibited the wild-type pattern of coat polypeptides, except for the absence in each instance of the corresponding product of the cot gene that had been insertionally inactivated.

J Assoc Off Anal Chem, 1987 Jul-Aug, 70(4), 691 - 7
Thin-layer chromatographic determination of erythromycin and other macrolide antibiotics in livestock products; Petz M et al.; A method is described for determination of 4 macrolide antibiotics in livestock products . Erythromycin, tylosin, oleandomycin, and spiramycin were extracted from animal tissues, milk, and egg with acetonitrile at pH 8.5 . Cleanup was done by adding sodium chloride and dichloromethane, evaporating the organic layer, and subsequent acid/base partitioning . After the antibiotics were separated by thin-layer chromatography (TLC), they were reacted with xanthydrol and could be detected as purple spots down to 0.02 mg/kg without interference by other commonly used therapeutic drugs (23 were tested) . Anisaldehyde-sulfuric acid, cerium sulfate-molybdic acid, phosphomolybdic acid, and Dragendorff's reagent proved to be less sensitive as visualizing agents . For quantitation, TLC plates were scanned at 525 nm . Recoveries were between 71 and 96% for erythromycin and tylosin in liver, muscle, and egg at the 0.1-0.5 mg/kg level and 51% for erythromycin in milk at the 0.02 mg/kg level (coefficient of variation = 10-18%) . Bioautography with Bacillus subtilis was used to confirm results, in addition to TLC analysis of derivatized antibiotics and liquid chromatography with electrochemical detection . Various derivatization procedures for erythromycin were investigated for improved ultra-violet or fluorescence detection in liquid chromatography.

J Gen Microbiol, 1987 Jul, 133 ( Pt 7), 1775 - 82
Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli; Tachibana K et al.; The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion . On induction of the phoA promoter, the B . subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E . coli . The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E . coli signal peptidase at the end of the phoA signal peptide.

J Gen Microbiol, 1987 Jul, 133 ( Pt 7), 1733 - 42
Mutations affecting penicillin-binding proteins 2a, 2b and 3 in Bacillus subtilis alter cell shape and peptidoglycan metabolism; Shohayeb M et al.; Bacillus subtilis mutants with altered penicillin-binding proteins (PBPs), or altered expression of PBPs, were isolated by screening for changes in susceptibility to beta-lactam antibiotics . Mutations affecting only PBPs 2a, 2b and 3 were isolated . Cell shape and peptidoglycan metabolism were examined in representative mutants . Cells of a PBP 2a mutant (UB8521) were usually twisted whereas PBP 2b (UB8524) and 3 (UB8525) mutants produced helices, particularly after growth at 41 degrees C . The PBP 2a mutant (UB8521) had a higher peptidoglycan synthetic activity than its parent strain whereas the opposite applied to the PBP 2b mutant UB8524 . The PBP 3 mutant (UB8525) had a similar peptidoglycan synthetic activity to that of the parent strain when grown at 37 degrees C, but 40% higher activity after growth at 41 degrees C . The PBP 2a mutant (UB8521) exhibited the same wall thickening activity as the parent, but the PBP 2b and 3 mutants (UB8524 and UB8525) were partially defective in this respect . The changes in the susceptibility of PBP 2a, 2b and 3 mutants to beta-lactam antibiotics imply that these PBPs are killing targets, consistent with the fact that these PBPs are also important for shape determination and peptidoglycan synthesis.

Prikl Biokhim Mikrobiol, 1987 Jul-Aug, 23(4), 542 - 6
{Sorption immobilization of alpha-amylase from Bacillus subtilis using meshed carboxyl polyelectrolytes}; Glazova NV et al.; Immobilization of alpha-amylase from Bacillus subtilis by adsorption on carboxyl polyelectrolytes, copolymers of methacrylic acid and ethylene glycol dimethacrylate, was being investigated depending on the structure of the polymeric matrix . It is demonstrated that cation exchange resins can be successfully used for reversible sorption and immobilization of alpha-amylase . It was found that the more heterogeneous the structure of the polymeric carrier, the higher is reversibility of the enzyme adsorption . The isothermic process of Bac . subtilis alpha-amylase adsorption on copolymers of different structures was studied as well . The equilibrium of the sorption system was proved to be true.

Radiobiologiia, 1987 Jul-Aug, 27(4), 449 - 54
{{Analysis of synergistic effects during thermoradiation exposure of bacteriophage T4 and Bacillus subtilis spores}; Komarov VP et al.; Experimental data are interpreted in terms of a half-empiric model of synergism obtained for bacteriophage T4 and Bacillus subtilis spores exposed to ionizing radiation of different dose rates at elevated temperatures . The model permits to optimize the ratio of both factors for effective sterilization.

Arch Biochem Biophys, 1987 Jul, 256(1), 325 - 34
Glyphosate sensitivity of 5-enol-pyruvylshikimate-3-phosphate synthase from Bacillus subtilis depends upon state of activation induced by monovalent cations; Fischer RS et al.; The 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase from Bacillus subtilis was activated by monovalent cations, catalytic activity being negligible in the absence of monovalent cations . The order of cation effectiveness (NH4+ greater than K+ greater than Rb+ greater than Na+ = Cs+ = Li+) indicated that the extent of activation was directly related to the unhydrated cation radius . Ammonium salts, at physiological concentrations, were dramatically more effective than other cations . Activation by ammonium was instantaneous, was not influenced by the counter ion, and gave a hyperbolic saturation curve . Hill plots did not show detectable cooperativity in the binding of ammonium . Double-reciprocal plots indicated that ammonium increases the maximal velocity and decreases the apparent Michaelis constants of EPSP synthase with respect to both phosphoenol pyruvate (PEP) and shikimate 3-phosphate (S3P) . A direct relationship between sensitivity to inhibition by glyphosate and the activation state of EPSP synthase was demonstrated . Hill plots indicated a single value for glyphosate binding throughout the range of ammonium activation . Double-reciprocal plots of substrate saturation data obtained with ammonium-activated enzyme in the presence of glyphosate showed glyphosate to behave as a competitive inhibitor with respect to PEP and as a mixed-type inhibitor relative to S3P . The increased glyphosate sensitivity of ammonium-activated EPSP synthase is attributed to a lowering of the inhibitor constant of glyphosate with respect to PEP . Erroneous underestimates of sensitivities of some bacterial EPSP synthases to inhibition by glyphosate may result from failure to recognize cation requirements of EPSP synthases.

J Bacteriol, 1987 Jul, 169(7), 3372 - 4
Chromosomal locations of three Bacillus subtilis din genes; Gillespie K et al.; Previously isolated DNA damage-inducible (din) genes of Bacillus subtilis have been mapped on the bacterial chromosome by bacteriophage PBS1-mediated transduction . The din genes have been localized to three positions on the B . subtilis map . dinA cotransduction with the hisA locus was 80%, while dinC cotransduction with this marker was about 56% . dinB is unlinked to hisA, but its cotransduction with the dal-1 and purB loci was 84 and 22%, respectively.

J Bacteriol, 1987 Jul, 169(7), 3362 - 4
Pseudosecretion of Escherichia coli chloramphenicol acetyltransferase by Bacillus subtilis; Le Grice SF et al.; Bacillus subtilis harboring the vector 25RBSII secrets an Escherichia coli-derived chloramphenicol acetyltransferase into culture supernatants . The secreted enzyme lacks 18 amino acids; these are removed externally rather than during secretion.

J Bacteriol, 1987 Jul, 169(7), 3321 - 8
Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis; Smith H et al.; To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis . The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence . By shotgun cloning of restriction fragments from B . subtilis chromosomal DNA, a great variety of different export-coding regions were selected . These regions were functional both in B . subtilis and in E . coli . In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products . The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.

J Bacteriol, 1987 Jul, 169(7), 3110 - 7
Expression of competence genes in Bacillus subtilis; Albano M et al.; A set of competence (com) mutants of Bacillus subtilis was constructed by using Tn917lacZ as a mutagen . In about half of the mutants, the promoterless lacZ element on the transposon was placed under control of putative com promoters . Expression of the mutant com genes was studied by using the beta-galactosidase tag . Two of the mutant genes (those represented by com-124 and com-138) were expressed early in the growth cycle in all of the media tested and were not dependent on the spo0A or spo0H product for expression . The remaining mutants, which represented a minimum of four additional genes, expressed beta-galactosidase in stationary phase during the period in which competence developed . We conclude that expression of com genes is probably regulated transcriptionally and in a growth stage-specific manner . Expression of these genes was also dependent on growth in competence medium and, like competence development, required the presence of glucose and was dependent on the spo0H products . The dependence on the spo0A gene product was partially bypassed by the abrB703 mutation . These effects were qualitatively equivalent to those on competence development . The latter was dependent on spo0A and spo0H, and the spo0A dependency was partially suppressed by abrB703 . Several of the mutants were still capable of resolution into light and heavy buoyant density cell fractions when grown in competence medium . All of these expressed beta-galactosidase to a greater extent in the light fraction, showing that expression of these com genes was cell type specific . Development of competence was not markedly affected by mutations in spo0B, spo0E, spo0F, spo0J, or sigB, the structural gene of sigma 37.

J Bacteriol, 1987 Jul, 169(7), 3068 - 75
Relationship between aconitase gene expression and sporulation in Bacillus subtilis; Dingman DW et al.; The citB of Bacillus subtilis codes for aconitase (D . W . Dingman and A . L . Sonenshein, J . Bacteriol . 169:3060-3065) . By direct measurements of citB mRNA levels and by measurements of beta-galactosidase activity in a strain carrying a citB-lacZ fusion, we have examined the expression of citB during growth and sporulation . When cells were grown in nutrient broth sporulation medium, citB mRNA appeared in mid- to late-exponential phase and disappeared by the second hour of sporulation . This timing corresponded closely to the kinetics of appearance of aconitase enzyme activity . Decoyinine, a compound that induces sporulation in a defined medium, caused a rapid simultaneous increase in aconitase activity and citB transcription . After decoyinine addition, the rate of increase in aconitase activity in a 2-ketoglutarate dehydrogenase (citK) mutant and in a citrate synthase (citA) mutant was significantly less than in an isogenic wild-type strain . This is apparently due to a failure to deplete 2-ketoglutarate and accumulate citrate . These metabolites might act as negative and positive effectors of citB expression, respectively . Mutations known to block sporulation at an early stage (spo0H and spo0B) had no appreciable effect on citB expression or aconitase activity . These results suggest that appearance of aconitase is stimulated by conditions that induce sporulation but is independent of certain gene products thought to act at an early stage of sporulation.

J Bacteriol, 1987 Jul, 169(7), 3062 - 7
Purification of aconitase from Bacillus subtilis and correlation of its N-terminal amino acid sequence with the sequence of the citB gene; Dingman DW et al.; The DNA sequence for the promoter region of the Bacillus subtilis citB gene has been determined . Presumed "-10" and "-35" regions of the promoter have been identified, and transcriptional and translational start points of citB have been located . To correlate the DNA sequence of citB with the amino acid sequence of its presumed product, aconitase, it was necessary to devise a scheme for purification of this labile enzyme . This procedure relies on the ability to restore enzyme activity at each stage of purification by incubation in a reducing buffer containing a source of ferrous ions . B . subtilis aconitase appears to be a monomer with a molecular weight of approximately 120,000 . The amino-terminal amino acids of aconitase fit the sequence predicted by analysis of the citB gene . Thus, citB codes for aconitase.

J Bacteriol, 1987 Jul, 169(7), 3044 - 50
prtR enhances the mRNA level of the Bacillus subtilis extracellular proteases; Tanaka T et al.; Studies were performed on the prtR gene which enhances the production of the Bacillus subtilis extracellular proteases and levansucrase, but not the alpha-amylase, RNase, and alkaline phosphatase . To investigate the mode of action of prtR, the Escherichia coli bla gene was placed under the control of two promoters . One was the promoter of the alkaline protease gene (aprE), and the other was the promoter of B . subtilis dihydrofolate reductase gene (dfrA) . Expression of the bla gene was enhanced by prtR only when the apr promoter was used . From these results, it was concluded that the apr promoter or its vicinity was the target of prtR and that prtR does not affect the process after transcription . The mRNA levels of aprE and nprE (the neutral protease gene) were significantly increased by prtR, but the half-life of the aprE mRNA was not affected . These results show that the prtR gene product enhances protease production by increasing the rate of transcription initiation.

J Bacteriol, 1987 Jul, 169(7), 2977 - 83
Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs; Saxild HH et al.; Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs . Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake . By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides . Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity . Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source . Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine . Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity . The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems . The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B . subtilis linkage map at 243, 55, and 198 degrees, respectively.

J Bacteriol, 1987 Jul, 169(7), 2917 - 25
Secretion of human serum albumin from Bacillus subtilis; Saunders CW et al.; We have fused the structural gene (hsa) for human serum albumin (HSA) to the expression elements and signal sequence coding region of each of two genes from Bacillus amyloliquefaciens P, an alpha-amylase gene (amyBamP) and a neutral protease gene (nprBamP) . Bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of HSA . Results from pulse-labeling studies indicated that the bacterially produced HSA was secreted from cells which had been converted to protoplasts . Results from similar studies with intact cells suggested that the signal sequence was removed from the hybrid protein, providing further evidence that B . subtilis can translocate this foreign protein across the cell membrane . Signal sequence removal was efficient when the level of HSA synthesis was low . However, in strains which synthesized HSA at a high level, signal sequence removal was less efficient.

Proc Natl Acad Sci U S A, 1987 Jul, 84(13), 4524 - 8
The gluconate operon gnt of Bacillus subtilis encodes its own transcriptional negative regulator; Fujita Y et al.; The gluconate (gnt) operon of Bacillus subtilis consists of four gnt genes; the second and third genes code for gluconate kinase (gluconokinase, EC 2.7.1.12) and gluconate permease, respectively . A fragment carrying the promoter of this operon (gnt promoter) and the first gene (gntR) was subcloned into a promoter probe vector (pPL603B) . Repression of the expression of cat-86 gene, encoded in the vector portion of a constructed plasmid (pgnt21), that is under the control of the gnt promoter was removed by gluconate . The results of deletion analysis and of insertional inactivation of the gntR gene cloned in pgnt21 suggested that the product of the gntR gene, actually synthesized as a 29-kDa protein in vivo, is involved in repression of the gnt promoter . A 4-base-pair insertional mutation within the gntR gene constructed in vitro was introduced into the B . subtilis chromosomal gnt operon by use of linkage of the 4 base pairs to gntK10 in transformation . The introduced mutation gntR1 caused the constitutive expression of the gluconate kinase and gluconate permease genes . S1 nuclease analysis indicated that the mRNA of this operon is synthesized in the gntR1 strain and amounts of mRNA are not changed very much by gluconate, which acts as an inducer in the wild-type gene . These results strongly indicate that the gntR gene codes for a transcriptional negative regulator for the gnt operon.

J Bacteriol, 1987 Jul, 169(7), 3232 - 6
Identification of the promoter of the Bacillus subtilis sdh operon; Melin L et al.; The Bacillus subtilis sdhCAB operon contains the structural genes for the three subunits of the membrane bound succinate dehydrogenase complex . An sdh-specific transcript of about 3,450 nucleotides was detected in vegetative bacteria . S1 nuclease mapping experiments showed that the sdh operon is transcribed from a sigma-43 promoter; the transcript starts at a guanosine residue 90 base pairs upstream from the first gene of the operon, sdhC . No sdh transcript was found in B . subtilis carrying the sdh-115 mutation, which decreases expression of the sdh operon by more than 99% . The sdh-115 mutation is a G-to-A transition in the -35 region of the sigma-43 promoter . The sdh operon is sensitive to glucose repression . When the sdh promoter region was used to drive transcription of the cat-86 gene this gene also became glucose repressed.

J Bacteriol, 1987 Jul, 169(7), 3104 - 9
Isolation and characterization of Tn917lac-generated competence mutants of Bacillus subtilis; Hahn J et al.; We isolated 28 mutants of Bacillus subtilis deficient in the development of competence by using the transposon Tn917lacZ as a mutagen . The mutant strains were poorly transformable with plasmid and chromosomal DNAs but were normally transducible and exhibited wild-type resistance to DNA-damaging agents . The mutations were genetically mapped, and the mutants were characterized with respect to their abilities to bind and take up radiolabeled DNA . All were defective in uptake, and some failed to bind significant amounts of DNA . The abilities of the mutant strains to resolve into two buoyant density classes on Renografin gradients were studied . Most resolved normally, but several banded in Renografin only at the buoyant density of noncompetent cells . The genetic mapping studies and the other analyses suggested that the mutations define a minimum of seven distinct com genes.

J Bacteriol, 1987 Jul, 169(7), 2913 - 6
Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis; Seki T et al.; Two DNA fragments which complement the alkaline phosphatase-negative mutation phoP of Bacillus subtilis were cloned from a B . subtilis chromosome with the prophage vector phi CM (a derivative of phi 105) . One of the fragments contained the regulatory gene phoR in addition to phoP . Nucleotide sequence analysis of the phoP region revealed that the phoP gene product consists of 241-amino-acid residues and that the sequence of these amino acids is extensively homologous with the sequence of the phoB gene product . This protein is the positive regulator for the phosphate regulon in Escherichia coli . It therefore appears that phoP is a regulatory gene for alkaline phosphatase synthesis in B . subtilis.

Z Naturforsch {C}, 1987 Jul-Aug, 42(7-8), 941 - 7
{Synthesis of heat shock proteins following amino acid or oxygen limitation in Bacillus subtilis relA+ and relA strains}; Hecker M et al.; Some of the presumable heat shock proteins will be produced in Bacillus subtilis in response to different environmental conditions, e.g . heat shock, amino acid limitation or oxygen limitation . During amino acid limitation or during oxygen limitation the relA+ strain is able of synthesizing this set of proteins but the relA strain is not . We suggest that the accelerated rate of the synthesis of some heat shock proteins depends on the induction of the stringent response because the (p)ppGpp production does not occur in the relA strain during amino acid or oxygen limitation . On the other hand the relA strain can produce heat shock proteins under heat stress . Therefore different mechanisms must be responsible for the expression of this set of genes during heat and other stress stimuli . It can be supposed that in B . subtilis the (p)ppGpp-dependent stringent control is a central defense reaction against different adverse environmental conditions and furthermore, that the synthesis of "stress" proteins as an essential component of the stringent response is part of a general adaptation mechanism under non-growing conditions.

Mol Microbiol, 1987 Jul, 1(1), 125 - 32
Isolation and sequence of the spo0E gene: its role in initiation of sporulation in Bacillus subtilis; Perego M et al.; The pleiotropic stage 0 sporulation locus spo0E was isolated and sequenced . The spo0E gene was found to code for a protein of 9791 molecular weight . Two spo0E mutations were identified by sequence analysis and were found to give rise to nonsense codons within the gene . The results indicated that it is the lack of the spo0E gene product that is responsible for the sporulation-defective phenotype . The DNA fragment containing the spo0E locus was inhibitory to sporulation when present on a multicopy plasmid . Since DNA fragments containing only the upstream region of the gene were also inhibitory, this effect was not due to over-production of the spo0E gene product . Coupling the transcription of the spo0E gene to beta-galactosidase in an integrative plasmid vector revealed that active transcription of this gene begins at the end of exponential growth and continues through the early part of sporulation . Studies of the regulation of this gene have allowed the generation of a hypothesis to explain the interactions of those five stage 0 genes involved in the activation of sporulation-specific transcription.

Plasmid, 1987 Jul, 18(1), 89 - 92
A protoplast transformation system for Listeria sp; Vicente MF et al.; We describe protoplast transformation of members of the genus Listeria with plasmid DNA . Shuttle vectors able to replicate in both Escherichia coli and Listeria have been constructed by fusing the E . coli plasmid pUC8 with the Bacillus subtilis plasmids pBD9 and pBD10.

Plasmid, 1987 Jul, 18(1), 8 - 15
Stability function in the Bacillus subtilis plasmid pTA 1060; Bron S et al.; Plasmid pBB2 (11.3 kb) was constructed by genetically labeling the cryptic Bacillus subtilis plasmid pTA 1060 with the pC194-derived CmR and the pUB110-derived KmR markers . In nonselective media pBB2 was segregationally almost completely stable (loss rates less than or equal to 0.02% per cell generation) . In contrast, pBB3, obtained by deleting from pBB2 a region consisting of two ClaI fragments (1.45 and 0.20 kb, respectively), was unstable (loss rates greater than or equal to 0.5% per cell generation) . This indicates that a genetic element required for stability is located on one or both of these fragments . In pBB3 cop, a mutant with a two- to threefold increased copy number, the rate of plasmid loss was reduced compared to that of pBB3 . The insertion of a 4.2-kb Escherichia coli DNA fragment reduced the stability of pBB2 only slightly, suggesting that this vector may be useful for the cloning of relatively large fragments.

J Bacteriol, 1987 Jul, 169(7), 3329 - 39
Organization and regulation of an operon that encodes a sporulation-essential sigma factor in Bacillus subtilis; Kenney TJ et al.; Deletion of sigE, the structural gene for the sporulation-induced RNA polymerase sigma factor, sigma E, prevented endospore formation by Bacillus subtilis . The effects of integration of plasmids into the sigE region of the chromosome and the use of complementation analyses demonstrated that sigE is part of an operon that includes a promoter-proximal gene, spoIIGA, that is essential for sporulation . Gene fusions to the promoter of this operon, spoIIG, demonstrated that transcription from this promoter is induced at the beginning of sporulation and is dependent on several spoO genes.

J Biol Chem, 1987 Jun 25, 262(18), 8787 - 98
Nucleotide sequence of the overlapping genes for the subunits of Bacillus subtilis aspartokinase II and their control regions; Chen NY et al.; The nucleotide sequence of a 2.9-kilobase Bacillus subtilis DNA fragment containing the entire coding region of aspartokinase II and adjacent chromosomal regions (Bondaryk, R . P., and Paulus, H . (1985a) J . Biol . Chem . 260, 585-591) has been determined . The results confirmed the earlier prediction that the two subunits of aspartokinase II, alpha and beta, are encoded by in-phase overlapping genes . The nucleotide sequence showed strong ribosome binding sites before the translation initiation codons of the alpha and beta subunits . Deletion of most of the coding region unique to the alpha subunit had no effect on the synthesis of the smaller beta subunit, demonstrating that the beta subunit is indeed the product of independent translation . The site of transcription initiation of the aspartokinase gene was found to be more than 300 nucleotides upstream from the translation start of the alpha subunit . The intervening region contained a short reading frame capable of encoding a 24-residue lysine-rich polypeptide, which overlaps a region of extensive dyad symmetry culminating in a rho-independent transcription terminator . This region may be an attenuator control element that regulates the expression of the aspartokinase gene in response to the availability of lysine, the end product of the pathway . The coding sequence of the aspartokinase II subunits was immediately followed by a rho-independent transcription terminator . This termination site has an unusual symmetry, which allows it also to serve as transcription terminator for a gene that converges on the aspartokinase II gene from the opposite direction, an interesting example of genetic economy . The deduced amino acid sequence of B . subtilis aspartokinase II was compared with the sequences of the three aspartokinases from Escherichia coli (Cassan, M., Parsot, C., Cohen, G . N., and Patte, J . C . (1986) J . Biol . Chem . 261, 1052-1057) . Significant sequence similarities suggest a close evolutionary relationship between the four enzymes.

Biochem Biophys Res Commun, 1987 Jun 15, 145(2), 712 - 8
Differences between Saccharomyces cerevisiae and Bacillus subtilis in secretion of human lysozyme; Yoshimura K et al.; Saccharomyces cerevisiae secreted human lysozyme in the medium as an active form when the signal peptides of chicken lysozyme and a chicken lysozyme-Aspergillus awamori glucoamylase hybrid were used, whereas it did not synthesize any human lysozyme protein by using the signal peptide of A . awamori glucoamylase . The secreted lysozyme was easily purified and crystallized . On the other hand, Bacillus subtilis secreted an inactive human lysozyme, which seemed to have incorrect disulfide bonds, with the signal peptide of amylase and its mutants . The free energy changes for the membrane translocation of the signal peptides are related to the secretion of human lysozyme in S . cerevisiae, but not in B . subtilis . These results indicate that differences exist between S . cerevisiae and B . subtilis in the secretion of human lysozyme.

J Biol Chem, 1987 Jun 15, 262(17), 8274 - 87
Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis; Ebbole DJ et al.; An approximately 16-kilobase pair region of the Bacillus subtilis chromosome at 55 degrees containing genes for de novo purine nucleotide synthesis (Piggot, P . J., and Hoch, J . A . (1985) Microbiol . Rev . 49, 158-179) was cloned . The nucleotide sequence of over 13 kilobase pairs indicates that this region contains a cluster of 12 genes, 11 of which encode enzymes that catalyze the 10 reactions for de novo purine nucleotide synthesis from 5-phosphoribosyl 1-pyrophosphate to IMP . The genes were identified by complementation of Escherichia coli pur mutants and by sequence comparisons with homologous enzymes . The cluster is likely an operon and is organized into three groups of overlapping genes followed by the last gene: purEKB-purC(orf)QLF-purMNH(J)-purD . Sequence comparisons provide evidence for homology of monofunctional purine nucleotide biosynthetic enzymes from B . subtilis with the corresponding multifunctional enzymes from yeast and Drosophila . Sequence alignment of the phosphoribosylaminoimidazole carboxylase heterodimer from B . subtilis with the monomeric enzyme from Methanobrevibacter smithii indicates an evolutionary relationship between these two enzymes . S1 nuclease analysis was used to map the mRNA 5' and 3' ends and to estimate levels of mRNA . These experiments indicate that synthesis of purine nucleotides is regulated independently by adenine and guanine nucleotides . Adenine nucleotides regulate transcription initiation . Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide 5' untranslated mRNA leader region . Groups of overlapping genes, regulated at least in part by transcription termination-antitermination is likely to be a common theme for genetic organization and regulation of biosynthetic genes in this Gram-positive organism.

Biochem Biophys Res Commun, 1987 Jun 15, 145(2), 861 - 7
Identification of the product of dnaB gene in Bacillus subtilis; Watabe K et al.; In order to detect the product of dnaB gene in B . subtilis, a gene which is involved in the initiation of DNA replication and the formation of the DNA-membrane complex, we synthesized an origopeptide of 15 amino acids which corresponds to a region near the carboxyl-terminal of the gene product, and raised antibody against the synthetic peptide . We have also employed a filter binding assay to measure the predicted DNA binding activity of the product of the dnaB gene, using the plasmid pUB110 . The binding activity was detected after fractionation of cell lysates of B . subtilis on sucrose-density gradients . When the active fraction was prepared from a mutant which was temperature-sensitive for the dnaB gene, the DNA binding activity in the fraction showed significant thermolability . Furthermore, the binding activity was inhibited by the purified antibody raised against the synthetic peptide . These results suggest that the product of the dnaB gene does indeed have DNA binding activity, and that the filter binding assay and the antibody can be used for the detection and characterization of the gene product.

J Biol Chem, 1987 Jun 5, 262(16), 7859 - 64
Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli; Ikemura H et al.; Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin . When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space . When the pre-sequence was replaced with the E . coli OmpA signal peptide, active subtilisin E was also produced . When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein . When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced . These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin . It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.

J Pharm Sci, 1987 Jun, 76(6), 466 - 70
Inimical effects of compaction speed on microorganisms in powder systems with dissimilar compaction mechanisms; Fassihi AR et al.; Tablets were prepared from powders that were consolidated by plastic deformation and fragmentation mechanisms . Cells of Staphylococcus aureus and spores of Bacillus subtilis were incorporated in the tablets by compaction at various pressures and compression speeds . The extent of inactivation of organisms was dependent on the compression behavior of the powders, on the speed of compaction, and on the degree of densification of compacts . Scanning electron micrographs of fractured compacts revealed that particle rearrangement, fragmentation, and deformation significantly influence the "survivor" . The "pressure-survivor plots" showed a linear relationship with greater inactivation in brittle material, while the extent in inactivation was reduced, and followed a nonlinear pattern in those powders exhibiting plastic deformation.

Mol Gen Genet, 1987 Jun, 208(1-2), 353 - 6
A new mutator strain of Bacillus subtilis; Viret JF et al.; Bacillus subtilis strain SB1207, widely used in our laboratory, was found to be highly temperature-sensitive and to exhibit a strong SOS-independent mutator phenotype at elevated temperatures . Both chromosomal and plasmid-borne genes were affected by the mutator . Lethality and mutator phenotype could not be attributed to a replication shut off or to thymine starvation . Due to the high frequency of base misincorporation, the mutator phenotype probably results from an editing defect rather than from a post-replication defect (mismatch repair).

Mol Gen Genet, 1987 Jun, 208(1-2), 349 - 52
Plasmid maintenance in Bacillus subtilis recombination-deficient mutants; Alonso JC et al.; An isogenic set of 11 recombination-deficient mutant strains of Bacillus subtilis has been constructed . Whereas plasmid pUB110 is stably maintained in such Rec- cells, the high copy number plasmid pC194 is unstable . Instability in Rec- strains could be mostly attributed to the deleterious effect of the presence of the plasmid on the Rec- cells' growth capability . In part, instability of pC194 derivatives could also be correlated with the presence of an unusually high amount of multimeric DNA molecules.

Mol Gen Genet, 1987 Jun, 208(1-2), 254 - 62
Genetic mapping by means of protoplast fusion in Bacillus subtilis; Akamatsu T et al.; A new mapping method involving protoplast fusion in Bacillus subtilis is described . Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, "x", to be mapped were fused with polyethylene glycol, and purA+ purB+ fusants were selected . After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers . This method was fully applicable to PBS1-resistant strains (e.g., lyt strains) . The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152) . The properties of lyt-15 are also discussed.

Mol Gen Genet, 1987 Jun, 208(1-2), 177 - 84
Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase; Martin I et al.; The structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli . The cloned gene was mapped by PBS1 transduction near the sacL locus on the B . subtilis chromosome, between leuA and aroD . Expression of the enzyme was demonstrated both in B . subtilis and in E . coli . The presence of sacC allowed E . coli to grow on sucrose as the sole carbon source . The complete nucleotide sequence of sacC was determined . It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli . The apparent molecular weight of purified levanase is 73 kDa . The sacC gene product was characterized in an in vitro system and in a minicell-producing strain of E . coli, confirming the existence of a precursor form of levanase of about 75 kDa . Comparison of the predicted aminoacid sequence of levanase with those of the two other known beta-D-fructofuranosidases of B . subtilis indicated a homology with sucrase, but not with levansucrase . A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.

Mol Gen Genet, 1987 Jun, 208(1-2), 37 - 44
Chromosomal initiation in Bacillus subtilis may involve two closely linked origins; Levine A et al.; When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45 degrees C, a synchronous round of DNA replication immediately ensues . Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication . Such treatments are necessary to achieve even modest synchrony in germinating spores . Our results showed that the first fragment to be replicated was a 4 kb BamHI-SalI restriction fragment, BS6 . In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS6 . Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB . The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores . We also discuss the possibility that B . subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.

Mol Gen Genet, 1987 Jun, 208(1-2), 114 - 20
Cloning and preliminary characterization of the sacS locus from Bacillus subtilis which controls the regulation of the exoenzyme levansucrase; Aymerich S et al.; The regulation of sacB, the gene encoding Bacillus subtilis levansucrase is altered by mutations located in several loci unlinked to sacB . Amongst these, the sacS locus seems to play an important role in the induction of sacB by sucrose . We have cloned sacS and found evidence suggesting that it contains two genes . The product of the first gene might repress the expression of the second; the second gene encodes a positive regulator of levansucrase synthesis, since its deletion abolishes this synthesis . There is a palindromic sequence resembling Q-independent terminators between the sacB promoter and the structural gene . Mutations affecting this palindrome make sacB constitutive . This suggests that the putative terminator is involved in the induction of sacB by sucrose . We discuss the possibility that the sacS-encoded positive regulator is a sucrose-dependent antiterminator which modulates transcription termination between the sacB promoter and the structural gene.

J Bacteriol, 1987 Jun, 169(6), 2579 - 90
Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes; Schnetz K et al.; Wild-type Escherichia coli cells are unable to grow on beta-glucosides . Spontaneous mutants arise, however, which are able to utilize certain aromatic beta-glucosides such as salicin or arbutin as carbon sources, revealing the presence of a cryptic operon called bgl . Mutations activating the operon map within (or close to) the promoter region of the operon and are due to the transposition of an IS1 or IS5 insertion element into this region . This operon was reported to consist of three genes coding for a phospho-beta-glucosidase, a specific transport protein (enzyme IIBgl), and a positively regulating protein . We have defined the extent and location of three structural genes, bglC, bglS, and bglB, and have determined their DNA sequence . The amino acid sequences deduced from the open reading frames together with deletion and subcloning analyses suggest that the first gene, bglC, codes for the regulatory protein, the second, bglS, codes for the transport protein, and the third, bglB, for phospho-beta-glucosidase . A fourth gene may exist which codes for a product of unknown function . We discuss structural features of the DNA sequence which may bear on the regulation of the operon . Homologies to sequences preceding the gene for an excreted levansucrase of Bacillus subtilis, which are known to be involved in the regulation of this gene, and to sequences preceding the gene for an excreted beta-endoglucanase of B . subtilis, for which data pertaining to regulation are not yet available, suggest a close evolutionary relationship among the regulatory components of all three systems.

Can J Microbiol, 1987 Jun, 33(6), 566 - 8
Wall turnover deficiency of Bacillus subtilis Ni15 is due to a decrease in teichoic acid; Vitkovic L; Bacillus subtilis Ni15 is deficient in cell wall turnover . The deficiency is removed if the medium contains 0.2 M NaCl, which does not affect growth . The levels of amidase and glucosaminidase, the most likely enzymes involved in turnover, were, in stationary phase Ni15 cells, similar to those in late-exponential phase cells of a standard strain . The Ni15 enzymes were not salt sensitive . However, the Ni15 walls contained 4.7-fold less phosphorus than the walls of the standard strain . Since the phosphorus content of B . subtilis walls reflects the level of teichoic acid, it is proposed that the turnover deficiency of this strain is due to a decrease in wall teichoic acid.

Can J Microbiol, 1987 Jun, 33(6), 563 - 5
Bacillus subtilis Lyt+ and Lyt- strains secrete peptidoglycan hydrolases; Vitkovic L; The phenotypes characteristic of the pleiotropic lytic-deficient Bacillus subtilis mutants have been attributed to reductions in N-acetyl-muramoyl-L-alanine amidase (EC 3.5.1.28) and endo-beta-N-acetyl-glucosaminidase (EC 3.2.1.30) . It is reported here that these peptidoglycan hydrolases are secreted . The FJ3 (lyt-1), FJ6 (lyt-2), and Ni15 (lyt-15) mutants secreted the enzymes in amounts comparable to a Lyt+ strain . Thus, the Lyt- mutants appear not be as deficient in the enzymes' synthesis as their cell-bound activities indicated . Based on the levels of cell-bound and extracellular activities measured during growth, it is suggested that the Lyt- phenotype may be due to a deficiency of the enzymes' acceptor(s) in cell walls.

J Bacteriol, 1987 Jun, 169(6), 2819 - 27
DNA replication by a DNA-membrane complex extracted from Bacillus subtilis: site of initiation in vitro and initiation potential of subcomplexes; Laffan J et al.; A DNA-membrane complex extracted from Bacillus subtilis was studied further as a model system for initiation of bacterial DNA replication in vitro . Of three subcomplexes purified from the crude complex by a combination of CsCl and sucrose gradient centrifugation, the synthetic capability of only one was inhibited significantly by streptovaricin, a known inhibitor of RNA primer formation . A selective enrichment in the level of this subcomplex was obtained by manipulating a thymine-requiring mutant . The synthetic capabilities of an enriched and nonenriched DNA-membrane complex were compared in the presence and absence of streptovaricin . Although the rate and extent of DNA synthesis per unit of protein were approximately the same in the absence of the antibiotic, there was a much greater inhibition of synthesis shown by the enriched complex in the presence of streptovaricin . Although the amount of DNA present in the putative initiation subcomplex was less than 0.3 to 0.4% of the total DNA present in the crude complex, such DNA, except for a few quantitative differences, was still representative of genomic DNA . Newly synthesized DNA hybridized to specific origin- and non-origin-derived restriction fragments of the B . subtilis genome . However, when an elongation inhibitor (ddCTP) was added, hybridization of such DNA to almost all of the nonorigin fragments disappeared or was reduced drastically, whereas origin region hybridization patterns remained strong . The highest level of hybridization in the origin region occurred with a BamHI (B7) restriction fragment of 5.6 kilobases that has been implicated by others as one site initiation in vivo (N . Ogasawara, M . Seiki, and H . Yoshikawa, Nature (London) 281:702-704, 1979; S . J . Seror-Laurent and G . Henckes, Proc . Natl . Acad . Sci . USA 82:3586-3590, 1985).

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Jun, 265(1-2), 136 - 45
Curing of plasmid pE194 with novobiocin and coumermycin A1 in Bacillus subtilis and Staphylococcus aureus; Gado I et al.; Plasmid pE194 determining macrolide-lincosamid-streptogramin B resistance could be eliminated with novobiocin and coumermycin A1 in Bacillus subtilis and Staphylococcus aureus . In both organisms the curing effect depended on the temperature . In the case of Staphylococcus aureus it increased very much at high temperatures, while in the case of Bacillus subtilis only a moderate increase could be observed . The curing activity of coumermycin was substantially weaker than that of novobiocin in both bacteria . This difference decreased at higher temperatures only in Staphylococcus aureus . In Bacillus subtilis the curing with novobiocin could be antagonized with coumermycin in accordance with the growth experiments reported previously.

J Mol Biol, 1987 May 20, 195(2), 299 - 310
The normal replication terminus of the Bacillus subtilis chromosome, terC, is dispensable for vegetative growth and sporulation; Iismaa TP et al.; The Bacillus subtilis strains CU1693, CU1694 and CU1695 were shown by hybridization analysis to carry large deletions of the terminus region that originated within discrete fragments of the SP beta prophage genome . The absence of terC in CU1693 was demonstrated definitively by the identification of a novel junction fragment comprising SP beta DNA and DNA that lies on the other side of terC in the parent strain . This represented the deletion of approximately 230 kb of CU1693 DNA, with the removal of approximately 150 kb to the left of terC and approximately 80 kb to the right of terC . The lack of hybridization of CU1694 and CU1695 DNA to cloned DNA carrying the terC sequence and to cloned DNAs flanking terC suggested that terC is absent from the chromosome of each of these strains also, and that the deletions in CU1694 and CU1695 extend beyond the segment of the terminus region that has been mapped and cloned . The normal growth rate and morphology of CU1693, CU1694 and CU1695 relative to the parent strain when grown in complex medium indicated dispensability of terC for vegetative growth and division . B . subtilis SU153 was constructed using a specific deletion-insertion vector that was designed to effect the deletion of 11.2kb of DNA spanning terC, with the removal of approximately 9.7kb to the left of terC and approximately 1.kb to the right of terC . This manipulation did not introduce any readily detectable auxotrophic requirement . Physiological characterization of SU153 confirmed the dispensability of terC for vegetative growth and cell division, and also established the lack of requirement of terC for the specialized cell division that is associated with formation of the bacterial endospore.

Nucleic Acids Res, 1987 May 11, 15(9), 3689 - 702
Bacillus subtilis phage SPR codes for a DNA methyltransferase with triple sequence specificity; Gunthert U et al.; SPR, a temperate Bacillus subtilis phage, codes for a DNA methyltransferase that can methylate the sequences GGCC (or GGCC) and CCGG at the cytosines indicated . We show here that it can also methylate the sequence CC(A/T)GG and protect it from cleavage with EcoRII and ApyI . This methylation can be seen in vivo as well as in vitro with purified SPR methyltransferase . SPR19 and SPR83 are two mutant phages, defective in GGCC or CCGG methylation, respectively . These mutants have not lost their ability to methylate CC(A/T)GG sites . Mutation SPR26 has lost the ability to methylate all three sites . Thus the SPR methyltransferase codes for three genetically distinguishable methylation abilities.

Science, 1987 May 8, 236(4802), 690 - 4
A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA; Guo PX et al.; A small RNA of Bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral DNA packaging in vitro . This requirement for RNA in the encapsidation of viral DNA provides a new dimension of complexity to the attendant protein-DNA interactions . The RNA is a constituent of the viral precursor shell of the DNA-packaging machine but is not a component of the mature virion . Studies of the sequential interactions involving this RNA molecule are likely to provide new insight into the structural and possible catalytic roles of small RNA molecules . The phi 29 assembly in extracts and phi 29 DNA packaging in the defined in vitro system were strongly inhibited by treatment with the ribonucleases A or T1 . However, phage assembly occurred normally in the presence of ribonuclease A that had been treated with a ribonuclease inhibitor . An RNA of approximately 120 nucleotides co-purified with the phi 29 precursor protein shell (prohead), and this particle was the target of ribonuclease action . Removal of RNA from the prohead by ribonuclease rendered it inactive for DNA packaging . By RNA-DNA hybridization analysis, the RNA was shown to originate from a viral DNA segment very near the left end of the genome, the end packaged first during in vitro assembly.

J Biochem (Tokyo), 1987 May, 101(5), 1191 - 8
A thermostable Gm-methylase recognizes the tertiary structure of tRNA; Matsumoto T et al.; The efficiency of methylation of tRNA by a thermostable tRNA(guanosine-2')-methyltransferase (Gm-methylase) was examined at various temperatures using several species of tRNA isolated from Escherichia coli, yeast and Bacillus subtilis, each possessing different thermal properties . The optimal temperature for the methylation reaction was ca . 20 degrees C lower than the melting temperature of the tRNA in each case . Arrhenius plots of the methylation reactions with various tRNAs gave straight lines below the optimal temperatures in all cases, with similar activation energies of between 10 and 14 kcal/mol . Above the optimal temperatures, the methyl acceptor activity decreased as the incubation temperature was raised to 80 degrees C, at which point the methylase was still active . A correlation was observed between the remaining methyl acceptor activity and the hyperchromicity of tRNA . These results suggest that Gm-methylase recognizes the tertiary structure of tRNA, and it is not the substrate tRNA but the enzyme which is activated by heat.

J Gen Microbiol, 1987 May, 133 ( Pt 5), 1337 - 42
Characterization of a Ca2+ uniporter from Bacillus subtilis by partial purification and reconstitution into phospholipid vesicles; Kusaka I et al.; Ca2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin . Uptake was dependent on the magnitude of the membrane potential . This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+ . The system showed saturation kinetics with an apparent Km for Ca2+ of about 250 microM . Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed . A fraction at pH 5.28-5.33 contained the activity . The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+) . In addition, uptake was not influenced by a pH gradient imposed on the vesicles . The apparent Km for Ca2+ in the reconstituted system was about 260 microM . The specific activity was increased about 50-fold by purification with isoelectric focusing.

J Gen Microbiol, 1987 May, 133 ( Pt 5), 1159 - 70
Thermosensitive Bacillus subtilis mutants which lyse at the non-permissive temperature; Brandt C et al.; A collection of 655 thermosensitive mutants of Bacillus subtilis 168, obtained by indirect selection, was screened for those lysing at the non-permissive temperature . Thirty-three mutations thus identified were distributed by transformation into eight linkage groups designated lssA to lssH . The distribution was non-random . With the exception of group A, all groups were small, suggesting that mutations identified in each of them may map in one gene only . Linkage groups identified here were mapped in four different regions of the B . subtilis chromosome and their positions relative to reference markers were the following: (i) aroI-lssA-dal-purB; (ii) metC-lssB-lssC-furA-pyrE-cysC-lssD; (iii) lssF-gtaA-lssG-hisA-lssH-cysB; and (iv) cysA-lssE-dnaC-purA . Kinetics of N-acetyl-D-{1-14C}glucosamine incorporation revealed that groups A, B, C, D and F are deficient in peptidoglycan synthesis at the restrictive temperature . In group G, anomalies at the cell wall level were suggested by incorporation and growth curves . It appears that in almost all known cases, thermosensitive lysis mutations in B . subtilis either affect genes involved in peptidoglycan synthesis or lead, more or less directly, to induction of prophages.

Prikl Biokhim Mikrobiol, 1987 May-Jun, 23(3), 327 - 30
{Purification of alkaline proteinase from Bacillus subtilis 72 using ion-exchange chromatography}; Motina LI et al.; Alkaline proteinase was isolated from the culture fluid filtrate obtained as a result of cultivation of Bacillus subtilis st . 72 using ion-exchange chromatography on a modified silochrome . Sorption of the enzyme at pH 6.0-6.3 and desorption at pH 7.5-7.8 followed by ultrafiltration through the membrane UAM-150 (USSR) resulted in a 5-fold increase of the enzyme specific activity calculated in respect of protein and an activity yield of 84%.

Prikl Biokhim Mikrobiol, 1987 May-Jun, 23(3), 291 - 8
{Isolation of a highly active preparation of beta-D-galactosidase}; Vikha IV et al.; Methods for isolation and purification of beta-galactosidase from Bacillus subtilis, st . IBP-101 are described . The bacterial cells were disrupted by different procedures such as freezing and thawing with subsequent autolysis at 37 degrees C, disrupting in a French press DKM-3 or in ultrasonic disintegrators UZDN-1 (USSR) and Soniprep-150 . It is shown that the specific activity and yield of the enzyme depends to a great extent on the disrupting procedure used . The best results were obtained in case of sonication . The preparation was purified by precipitation with ammonium sulphate (25-75% saturated) and chromatography on DEAE-cellulose and DEAE-Sephadex . The purified enzyme had a specific activity of 3155 units per mg protein . The molecular weight of the homogeneous according to gel polyacrylamide electrophoresis preparation was 215,000, as estimated by gel filtration, and 105,000, as estimated by SDS gel electrophoresis . The enzyme retains the activity in the presence of Na+, Mn2+ or Mg2+ ions or the thiolic reagents, dithiothreitol or 2-mercaptoethanol . The pH optimum of the enzyme activity is 6.3 and it is stable in water solutions at pH from 6 to 9 and can be lyophilized . The given preparation of beta-galactosidase has a high affinity for synthetic substrates such as o- and p-nitrophenyl-beta-D-galactopyranosides and 4-methylumbelliferyl-beta-D-galactopyranoside.

Mol Gen Genet, 1987 May, 207(2-3), 335 - 41
Expression of the Bacillus subtilis polC gene in Escherichia coli; Ott RW et al.; We have cloned a 14 kb DNA segment containing the coding sequence (polC) for DNA polymerase III (PolIII) from the Bacillus subtilis chromosome . The plasmid carrying the sequence, pRO10, directs the synthesis of the 160 kDa PolIII protein and three additional polypeptides in Escherichia coli maxicells from strain CSR603 . A set of deletion derivatives of pRO10 was constructed in vitro . The location of the PolIII coding sequence and the direction of transcription through the polC gene were determined by analysis of the truncated polypeptides observed in extracts of CSR603 transformants . Two HindIII segments subcloned from pRO10 were found to contain promoter sequences which function in E . coli and in vegetative phase B . subtilis cells . The location of the promoter sequence was determined with respect to the polC gene . The B . subtilis polC gene did not complement the temperature-sensitive defect of an E . coli PolIII mutant (dnaE486) . The presence of the complete B . subtilis polC gene on a multicopy plasmid inhibited the growth of E . coli cells.

Antibiot Med Biotekhnol, 1987 May, 32(5), 331 - 3
{Lysosubtilin G10x--a new antimicrobial agent for treatment and prophylaxis . Physico-chemical properties}; Biziuliavichius GA et al.; Lysosubtilin G10x, a lytic enzyme from Bacillus subtilis SK-52 was studied with respect to its physico-chemical properties such as effect of optimal conditions and stability . The maximum activity of the enzyme was observed in phosphate buffer at a concentration of the buffer mixture equal to 0.004 M, pH 7.2 and the temperature of 37-50 degrees C . Aqueous solutions of lysosubtilin G10x were stable at pH 5-9 . The lytic activity of the enzyme aqueous solutions rapidly lowered at a temperature above 50 degrees C . The ions of some metals, especially those of mercury, copper and ferrous iron inhibited the enzyme . Lysis activation was recorded in the presence of surface active substances such as sodium dodecylsuphate, tritone X-100 and certain twins . Comparison of the findings with the results of our previous studies on the lytic properties of enzymes from other strains of B . subtilis showed that irrespective of the strain lysosubtilins were sensitive to changes in the medium ionic strength, they were mostly active in neutral solutions of low ionic strength, their activity was inhibited by heavy metal ions and they were stable within wide ranges of pH . This should be taken into account in practical use of lysosubtilin G10x.

J Bacteriol, 1987 May, 169(5), 2287 - 90
Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene; Gonzy-Treboul G et al.; The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis . A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes . In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I . Therefore, ptsH and ptsI are adjacent in B . subtilis, as in E . coli . In E . coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon . The 4.1-kilobase fragment from B . subtilis was shown to contain a gene that enables an E . coli crr mutant to use glucose . This gene, unlike the E . coli crr gene, was located to the left of ptsH.

J Bacteriol, 1987 May, 169(5), 2202 - 6
Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster; Lerner CG et al.; A 10.5-kilobase PstI endonuclease fragment encoding the entire Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster was cloned in Escherichia coli by transformation of a carB strain to uracil-independent growth . The cloned fragment also complemented E . coli pyrB, pyrC, pyrD, pyrE, and pyrF mutants . From the ability of subclones to complement E . coli pyr mutants, the gene order was deduced to be pyrBCADFE . The B . subtilis pyrB gene was shown to be expressed in E . coli, but synthesis of the enzyme was not repressible by the addition of uracil to the growth medium . The approximate molecular weights of the polypeptides encoded by B . subtilis pyrA, pyrB, pyrC, pyrD, pyrE, and pyrF were found to be 110,000, 36,000, 46,000, 34,000, 25,000, and 27,000, respectively.

J Bacteriol, 1987 May, 169(5), 2017 - 25
Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG; Robson LM et al.; The DNA sequence of the Bacillus subtilis DLG endo-beta-1,4-glucanase gene was determined, and the in vivo site of transcription initiation was located . Immediately upstream from the transcription start site were sequences closely resembling those recognized by B . subtilis sigma 43-RNA polymerase . Two possible ribosome-binding sites were observed downstream from the transcription start site . These were followed by a long open reading frame capable of encoding a protein of ca . 55,000 daltons . A signal sequence, typical of those present in gram-positive organisms, was observed at the amino terminus of the open reading frame . Purification of the mature exocellular beta-1,4-glucanase and subsequent amino-terminal protein sequencing defined the site of signal sequence processing to be between two alanine residues following the hydrophobic portion of the signal sequence . The probability of additional carboxy-terminal processing of the beta-1,4-glucanase precursor is discussed . S1 nuclease protection studies showed that the amount of beta-1,4-glucanase mRNA in cells increased significantly as the culture entered the stationary phase . In addition, glucose was found to dramatically stimulate the amount of beta-1,4-glucanase mRNA in vivo . Finally, the specific activities of purified B . subtilis DLG endo-beta-1,4-glucanase and Trichoderma reesei QM9414 endo-beta-1,4-glucanase (EC 3.2.1.4) were compared by using the noncrystalline cellulosic substrate trinitrophenyl-carboxymethyl cellulose.

J Bacteriol, 1987 May, 169(5), 1985 - 92
Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis; Hackett RH et al.; The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined . The amino acid sequence of SASP-gamma is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs . The SASP-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 340 nucleotides long . The results of hybridization of an sspE gene probe to Southern blots of B . subtilis DNA suggested that there is only a single gene coding for the SASP-gamma type of protein in B . subtilis . This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B . subtilis chromosome, with concomitant loss of the wild-type gene . This sspE deletion strain sporulated well, but lacked the SASP-gamma type of protein.

J Bacteriol, 1987 May, 169(5), 1929 - 37
Nucleotide sequence of the streptothricin acetyltransferase gene from Streptomyces lavendulae and its expression in heterologous hosts; Horinouchi S et al.; The nucleotide sequence of the streptothricin acetyltransferase (STAT) gene from streptothricin-producing Streptomyces lavendulae predicts a 189-amino-acid protein of molecular weight 20,000, which is consistent with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme . The amino acid composition and the NH2-terminal sequence determined by using the purified protein are in good agreement with those predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein . High-resolution S1 nuclease protection mapping suggests that transcription initiates at or near the adenine residue which is the first position of the translational initiation triplet (AUG) of STAT . Another open reading frame located just upstream of the STAT gene was detected and contains a region bearing a strong resemblance to DNA-binding domains which are conserved in known DNA-binding proteins . By addition of promoter signals and a synthetic ribosome-binding (Shine-Dalgarno) sequence at an appropriate position upstream of the STAT translational start codon, the STAT gene confers streptothricin resistance on Escherichia coli and Bacillus subtilis . The STAT coding sequence with both the promoter of a B . subtilis cellulase gene and a synthetic Shine-Dalgarno sequence was functionally expressed in Streptomyces lividans, which suggests that the addition of an artificial leader upstream of the translational initiation codon (AUG) does not significantly influence the translation of STAT.

J Bacteriol, 1987 May, 169(5), 1807 - 11
Genetic analysis of RNA polymerase-promoter interaction during sporulation in bacillus subtilis; Ray C et al.; The discovery of secondary sigma factors in Bacillus subtilis that enable RNA polymerase to transcribe cloned sporulation genes in vitro has led to the proposal that the appearance of new sigma factors during sporulation directs RNA polymerase to the different temporal classes of sporulation genes . One sigma factor, which appears 2 h after the initiation of sporulation, is sigma E (formerly sigma 29) . Mutations that inactivate the structural gene for sigma E prevent transcription from promoter G4 . To determine whether sigma E-RNA polymerase interacts with the G4 promoter in vivo, we examined the effects of six single-base-pair substitutions in the G4 promoter on its utilization in vivo and in vitro by sigma E-RNA polymerase . The mutations in the G4 promoter affected utilization of the promoter in vivo in the same way that they affected its utilization in vitro by purified sigma E-RNA polymerase; therefore, we conclude that this polymerase interacts directly with the G4 promoter in vivo . The effects of these mutations also support the model in which sigma E-RNA polymerase utilizes promoters by interacting with two distinct sets of nucleotides located 10 and 35 base pairs upstream from the start point of transcription.

J Bacteriol, 1987 May, 169(5), 2215 - 22
Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis; Marahiel MA et al.; Tyrocidine is a cyclic decapeptide antibiotic which is produced and secreted by stationary-phase cells of the sporeforming bacterium Bacillus brevis . We identified the promoter for the B . brevis structural gene (tycA) for tyrocidine synthetase I, the enzyme catalyzing the first step in tyrocidine biosynthesis, and studied its regulation in cells of B . brevis and Bacillus subtilis . Transcription from the tycA promoter was induced at the end of the exponential phase of the growth cycle in B . brevis cells growing in sporulation medium . To study the regulation of tycA in B . subtilis, we constructed a derivative of the B . subtilis bacteriophage SP beta containing a transcriptional fusion of the tycA promoter to the lacZ gene of Escherichia coli and introduced the tycA-lacZ operon fusion by means of specialized transduction into sporulation mutants known to be blocked in sporulation-associated antibiotic production . Our principal finding was that tycA-directed lacZ expression was impaired in the stage-0 mutants with mutations spo0A, spo0B, and spo0E but not in spo0C, spo0F, spo0H, or spo+ bacteria . The dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation, which caused tycA-lacZ to be transcribed constitutively at all stages of growth . A simple model is proposed for the mechanism of tycA induction based on the Spo0A-dependent inactivation of Ab-B protein, which is proposed to be a negative regulator of tycA transcription.

Isr J Med Sci, 1987 May, 23(5), 342 - 6
Organization of the regions flanking the rRNA cistrons of Mycoplasma strain PG50; Rasmussen OF et al.; Four clones harboring fragments of the rRNA cistrons have been isolated from a genomic library of Mycoplasma PG50 DNA . By heteroduplex and displacement loop analysis, supplemented with sequence data, we demonstrate that the four clones together cover the two rRNA cistrons of Mycoplasma PG50 as well as the flanking regions . Parts of the cloned fragments have been analyzed in detail, and the following conclusions have been reached . Genes for tRNALys and tRNALeu are located about 450 bp upstream of the 16S rRNA gene of rrnA . An open reading frame of at least 215 codons is located just upstream of and in the same direction as rrnB . Analysis of the 3' flanking region of rrnA reveals that no tRNA genes succeed this operon . Also here an open reading frame can be identified, which is at least 83 codons long and has a putative promoter and a possible ribosome binding site . Its direction is the same as that of rrnA . None of the open reading frames has any function assigned yet . The last 880 bp of the 23S and the 5S genes of rrnA are compared with the corresponding Bacillus subtilis sequence and sequences of other Mycoplasma species, respectively.

J Bacteriol, 1987 May, 169(5), 2223 - 30
Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis; Zuber P et al.; Transcription of the Bacillus subtilis gene spoVG is induced at the onset of sporulation and is dependent on the products of the stage-0 regulatory genes spo0A, spo0B, and spo0H . We show here that the dependence of spoVG transcription on Spo0A and Spo0B (but not Spo0H) can be bypassed by a mutation at abrB, a previously identified locus at which mutations that suppress some of the phenotypes of spo0A are often located, or by a cis-acting mutation within the spoVG promoter . To explain the epistatis of abrB to spo0A and spo0B mutations, we propose that AbrB acts, directly or indirectly, to block transcription of spoVG and that Spo0A and Spo0B cause inactivation of the abrB gene product(s) . Spo0A-Spo0B-dependent inactivation of AbrB could be a general explanation for the pleiotropic effects of spo0A and spo0B mutations on B . subtilis gene expression.

Eur J Biochem, 1987 Apr 15, 164(2), 317 - 20
Nucleotide sequence of a cellulase gene of Bacillus subtilis; Nakamura A et al.; The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme . The mature protein appeared to be extended by a signal sequence of 36 amino acids . The putative AUG initiation codon was preceded by a sigma 43-type promoter of B . subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites . Partial homology of amino acid sequences was found between B . subtilis cellulase and an alkalophilic Bacillus cellulase.

J Biol Chem, 1987 Apr 5, 262(10), 4610 - 5
Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus; Porumb H et al.; Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL . Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate . The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH . The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps . Cibacron blue interfered with renaturation, presumably by competition with NADH . Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity . By immobilization on Sepharose 4B, alanine dehydrogenase from B . cereus retained 66% of the specific activity of the soluble enzyme . After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits . The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment . The formation of "hybrids" between subunits of enzyme from B . cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.

J Antibiot (Tokyo), 1987 Apr, 40(4), 437 - 42
Isolation and characterization of new iturins: iturin D and iturin E; Besson F et al.; Two new antibiotics, iturin D and iturin E, were isolated from a strain of Bacillus subtilis producing iturin A . These compounds belong to the iturin group, the acid hydrolysates contained alpha-amino acids Asp3, Glu1, Pro1, Ser1, Tyr1, and a mixture of n-C14, iso-C15, anteiso-C15, iso-C16 and n-C16 beta-amino acids . They differ from iturin A by the presence of a free carboxyl group in iturin D and a carboxymethyl group in iturin E.

Appl Environ Microbiol, 1987 Apr, 53(4), 860 - 3
Separation of organic dust from microorganism suspensions by partitioning in aqueous polymer two-phase systems; Strom G et al.; Conidia of Penicillium brevi-compactum and Aspergillus fumigatus, sporangiospores of Rhizopus rhizopodiformis, spores of Streptomyces griseus, and bacterial cells of Bacillus subtilis were partitioned in two-phase systems consisting of dextran, polyethylene glycol, substituted positively charged sulfonylpolyethylene glycol, and water . At a pH of 2.8 in the system, the microorganisms showed 60 to 90% affinity for the upper, polyethylene glycol-rich phase, except for cells of B . subtilis, which were entirely located in the lower, dextran-rich phase . This partition behavior was used to separate microorganisms in aqueous suspensions of peat, wood fuel chip, and straw samples from organic dust impurities prior to total count by acridine orange staining and epifluorescence microscopy . Only one extraction of the interphase and lower phase was needed to separate approximately 98% of the conidia of Penicillium chrysogenum from a suspension containing peat dust.

Mol Gen Genet, 1987 Apr, 207(1), 114 - 9
Promoter switching during development and the termination site of the sigma 43 operon of Bacillus subtilis; Wang LF et al.; Sequencing data indicated that the RNA polymerase sigma 43 operon of Bacillus subtilis consisted of three genes, P23 (function unknown), dnaE (DNA primase), and rpoD (sigma 43) (Wang and Doi 1986a) . S1 nuclease mapping experiments with RNA from various stages of growth demonstrated the presence of two overlapping sigma 43 promoters that controlled the expression of the operon during growth and a sigma 37 promoter that regulated the expression of the operon during the sporulation phase . This promoter switching mechanism ensured that this important operon would be expressed during different nutritional states of the cell and also illustrated a function for the minor RNA polymerase sigma 37 holoenzyme in the expression of genes which are normally expressed during the logarithmic phase of growth . The location of the transcription termination signal confirmed that the sigma 43 operon consists of three genes.

J Gen Microbiol, 1987 Apr, 133 ( Pt 4), 945 - 52
Recombinant derivatives of Bacillus subtilis phage Z containing the DNA methyltransferase genes of related methylation-proficient phages; Terschuren PA et al.; The DNA methyltransferase (Mtase) genes of temperate Bacillus subtilis phages SPR, phi 3T, SP beta and rho 11 can be transferred by transfection and recombination to the genome of the related non-modifying phage Z . Integration of the Mtase genes occurs in phage Z DNA at a unique location which is homologous with the flanking regions of the Mtase genes of the related phages . In lysogenic cells carrying recombinant phages, expression of the Mtase genes is repressed, irrespective of whether the Mtase genes were derived from phage donors which were homo- or heteroimmune to phage Z.

Mol Gen Mikrobiol Virusol, 1987 Apr, (4), 8 - 14
{Structural-functional characteristics of Bacillus mesentericus promoters cloned in Bacillus subtilis cells}; Bashkirov VI et al.; Bacillus mesentericus DNA fragments carrying promoters were cloned in Bacillus subtilis . The nucleotide sequences of four promoters, the sites of transcription initiation and the levels of promoter-mediated expression of pPL603 CAT gene at different stages of cell growth were determined . It was identified, that the two promoters (P435 and P442) are the typical sigma 43-RNA polymerase promoters . Promoter P462 possessed the unusual nucleotide sequence and induced the expression of indicator CAT gene at the middle of the stationary phase of cell growth . The next P428 promoter was found to be a complex promoter composed of three overlapping ones: P428-1, P428-2 and P428-3 . P428-3 is the sigma 43-specific promoter which revealed the high degree of homology with Bacillus subtilis spoOB gene promoter . We suggest the promoters P428-1 and P428-2 to be sigma 43- and sigma 37-RNA polymerase specific, respectively.

Mol Gen Mikrobiol Virusol, 1987 Apr, (4), 22 - 6
{The bounds of the riboflavin operon in Bacillus subtilis}; Chikindas ML et al.; All the structural genes of riboflavin biosynthesis are shown to be located on the 2.8 MD DNA fragment, using the collection of plasmids, carrying the Bacillus subtilis riboflavin operon fragments and Bacillus subtilis strains, containing various deletions of rib-operon for analysis . The proximal Bgl II site is shown to be located between promoter P1 and the first structural gene ribG . The distal Hind III site of fragment C is the left bound of the rib-operon.

Mol Gen Genet, 1987 Apr, 207(1), 73 - 81
Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168; Karamata D et al.; A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168 . Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient . Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids . Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient . All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA . In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes . Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined . Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated . We interpret these results as indicating an essential role for anionic wall polymers in the growth of B . subtilis.

EMBO J, 1987 Apr, 6(4), 1137 - 42
Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages; Behrens B et al.; B . subtilis phage rho 11s codes for a multispecific DNA methyltransferase (Mtase) which methylates cytosine within the sequences GGCC and GAGCTC . The Mtase gene of rho 11s was isolated and sequenced . It has 1509 bp, corresponding to 503 amino acids (aa) . The enzyme's Mr of 57.2 kd predicted from the nucleotide sequence was verified by direct Mr determinations of the Mtase . A comparison of the aa sequence of the rho 11s Mtase with those of related phages SPR and phi 3%, which differ in their methylation potential, revealed generalities in the building plan of such enzymes . At least 70% of the aa of each enzyme are contained in two regions of 243 and 109 aa at the N and C termini respectively, which are highly conserved among the three enzymes . In each enzyme, variable sequences separate the conserved regions . Variability is generated through the single or multiple use of related and unrelated sequence motifs . We propose that the recognition of those DNA target sequences, which are unique for each of the three enzymes, is determined by these variable regions . Evolutionary relationships between the three enzymes are discussed.

Proc Natl Acad Sci U S A, 1987 Apr, 84(7), 1784 - 8
Sporulation-specific sigma factor sigma 29 of Bacillus subtilis is synthesized from a precursor protein, P31; LaBell TL et al.; Evidence is presented that a sporulation-essential sigma factor of Bacillus subtilis, sigma 29, is synthesized as an inactive precursor (P31) and that its activation occurs by a developmentally regulated cleavage of 29 amino acids from the P31 amino terminus . A pulse-chase experiment demonstrated that sigma 29 was derived from a preexisting protein, with appearance of radioactively labeled sigma 29 paralleling the disappearance of labeled P31 . The disappearance of pulse-labeled P31 did not occur when the experiment was done with a B . subtilis strain carrying a mutation in a locus (spoIIE) required for sigma 29, but not P31, synthesis . Microsequencing of sigma 29 protein revealed that its amino terminus originates at amino acid 30 of the P31 amino acid sequence . In order to test whether a proteolytic event alone could activate P31 to a protein with sigma 29-like properties, a fusion protein (P31*) containing most of P31 was overproduced in Escherichia coli and converted in vitro into a protein with the electrophoretic mobility of sigma 29 by limited treatment with Staphylococcus aureus V8 protease . Protease-treated P31*, but not untreated P31*, was capable of directing B . subtilis core RNA polymerase to specifically initiate RNA synthesis at a sigma 29-recognized promoter in vitro.

J Bacteriol, 1987 Apr, 169(4), 1763 - 6
Nucleotide sequence and expression of the beta-lactamase gene from Staphylococcus aureus plasmid pI258 in Escherichia coli, Bacillus subtilis, and Staphylococcus aureus; Wang PZ et al.; The structural gene for beta-lactamase in the Staphylococcus aureus plasmid pI258 was cloned into a Staphylococcus aureus-Bacillus subtilis-Escherichia coli shuttle vector, pWN101, and the nucleotide sequence of the gene was determined . pWN101 was structurally stable and the beta-lactamase gene was expressed efficiently from its native promoter and ribosome-binding site in all three hosts.

Genetika, 1987 Apr, 23(4), 616 - 21
{Insertion mutagenesis in Bacillus subtilis (Marmur)}; Stoilova-Disheva MM et al.; A method for obtaining mutations in Bacillus subtilis using integration into the chromosome of the plasmid pHV60 ligated to chromosomal DNA fragments was developed . Auxotrophic mutants acquire, in addition, chloramphenicol-resistance, due to insertion of appropriate plasmid determinants . Chromosomal localization was established and the properties of several mutants were studied.

J Bacteriol, 1987 Apr, 169(4), 1480 - 4
Nucleotide sequence of the outB locus of Bacillus subtilis and regulation of its expression; Albertini AM et al.; The outB gene is one of the genes involved in the process of spore outgrowth in Bacillus subtilis . The gene has been cloned in bacteriophage lambda and subcloned in plasmids . We have determined the sequence of 2,553 base pairs around the outB locus . The locus was found to code for a protein of about 30,000 daltons . Analysis of the in vivo transcripts from this region by RNase protection experiments revealed the presence of two start sites for transcription . Two potential promoters for these transcripts can be tentatively assigned from the sequence data . The amount of one transcript is highest during outgrowth and vegetative growth and absent during the stationary phase . The second transcript is present at a low level throughout the cell cycle.

FEBS Lett, 1987 Mar 23, 213(2), 385 - 90
Processing of Bacillus subtilis succinate dehydrogenase and cytochrome b-558 polypeptides . Lack of covalently bound flavin in the Bacillus enzyme expressed in Escherichia coli; Hederstedt L et al.; The DNA sequence of the Bacillus subtilis sdh operon coding for the two succinate dehydrogenase subunits and cytochrome b-558 (the membrane anchor protein) has recently been established . We have now determined the extent of N-terminal processing of each polypeptide by radiosequence analysis . At the same time, direct evidence for the correctness of the predicted reading frames has been obtained . The cytochrome showed a ragged N-terminus, with forms lacking one residue, and is inserted across the membrane without an N-terminal leader-peptide . Covalently bound flavin was not detectable in B . subtilis succinate dehydrogenase expressed in Escherichia coli despite normal N-terminal processing of the apoprotein . This provides an explanation to why the succinate dehydrogenase synthesized in E . coli is not functional and demonstrates that host-specific factors regulate the coenzyme attachment.

Eur J Biochem, 1987 Mar 2, 163(2), 379 - 87
Secretion of Bacillus subtilis levansucrase: a possible two-step mechanism; Petit-Glatron MF et al.; The rate of exocellular levansucrase synthesis in an overproducing (sacUh) strain of Bacillus subtilis was shown to be directly proportional to the amount of two different transient forms of this enzyme located within the membrane fraction of the cells . The apparent Mr of the larger membrane form was 53,000, and that of the smaller form 50,000; the half-life time of each form was estimated in vivo to be 4-6 s and 32-42 s, respectively . Ethanol treatment of the cells lead to the accumulation of the 53,000-Mr form which may represent 1.5% of total membrane proteins . This latter form, partially purified, was transformed in vitro into the 50,000-Mr form by the action of the Escherichia coli leader peptidase . These enzyme forms were quite different from the exocellular levansucrase since they showed a weak affinity for hydroxyapatite and needed complexed iron to display enzyme activity . Assuming the membrane forms were precursors of exocellular levansucrase, we propose a two-step mechanism for the secretion process of levansucrase . The number of exoprotein synthesis/secretion sites in a B . subtilis cell is estimated to 2.5 X 10(4).

J Assoc Off Anal Chem, 1987 Mar-Apr, 70(2), 197 - 200
Thin layer chromatographic/bioautographic method for identification of antibiotic residues in animal tissues; Neidert E et al.; An analytical method for the identification of the residues from 14 commonly used antibiotics is presented . The technique is based on selective tissue extraction followed by thin layer chromatography (TLC)/bioautography . Antibiotic residues are extracted from the tissues with methanol and methanol-HCl (98 + 2) . The methanol extract is further extracted with chloroform to isolate groups of antibiotics . The extracts are spotted onto TLC plates and developed in suitable solvent systems . Developed plates are placed on set medium seeded with Bacillus subtilis and a bioautograph is produced . The locations of zones of inhibition are used to identify antibiotic residues . Recoveries of antibiotics were quantitative, while the effect of naturally inhibiting components of the matrix was minimized . The sensitivity of the method can be adjusted through minor modifications, which allows its use in routine regulatory analysis.

J Antibiot (Tokyo), 1987 Mar, 40(3), 251 - 7
7-Hydro-8-methylpteroylglutamylglutamic acid, a new anti-folate from an actinomycete . Fermentation, isolation, structure and biological activity; Murata M et al.; A new anti-folate, 7-hydro-8-methylpteroylglutamylglutamic acid, was isolated from the culture broth of a soil actinomycete . The antibiotic inhibited the growth of Enterococcus faecium which requires folic acid . The inhibitory action was reversed by thymidine or excess amounts of folate-related compounds such as pteroic acid, folic acid, dihydrofolic acid and leucovorin . It inhibited thymidylate synthase from E . faecium, Bacillus subtilis and Ehrlich ascites carcinoma cells.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 767 - 72
Influence of the culture medium on the production of iturin A by Bacillus subtilis; Besson F et al.; The production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium . Increasing phosphate concentrations did not modify the antibiotic yield . Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production . The nature of the nitrogen source was an important factor in the production of antibiotic . Among the amino acids which are components of iturin A, L-asparagine was the best substrate for the biosynthesis of iturin A; L-glutamine and L-serine were rather poor substrates while L-proline and D-tyrosine gave no antibiotic . Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 493 - 502
Cloning in Bacillus subtilis by transfection with bacteriophage vector phi 105J27: isolation and preliminary characterization of transducing phages for 23 sporulation loci; Errington J et al.; Bacteriophage cloning vector phi 105J27, the construction of which is described in an accompanying paper, has been used for shotgun cloning of sporulation genes in Bacillus subtilis . Various genomic libraries have been constructed and screened for the presence of recombinant phages capable of transducing strains containing sporulation (spo) mutations to Spo+ . Of a total of 30 spo loci tested, transducing phages have been isolated for 23, more than half of the known spo loci . Included are nine loci (spo0D, spo0J, spoIIIA, spoIIIE, spoIIIF, spoIVF, spoVB, spoVH and spoVJ) that do not appear to have been cloned previously . Preliminary genetic characterization of some of the new clones by a rapid screening procedure has enabled the status of various sporulation loci to be clarified.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 483 - 92
Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis; Jones D et al.; A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B . subtilis by direct transfection of protoplasts . The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105 . This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments . Two of the new vectors phi 105J27 and phi 105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible . This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA . The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B . subtilis sporulation genes.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 475 - 81
Structure and function in a Bacillus subtilis sporulation-specific sigma factor: molecular nature of mutations in spoIIAC; Yudkin MD; The spoIIAC gene was cloned from chromosomal DNA of seven spoIIAC mutants of Bacillus subtilis, and the complete sequence of the gene was determined for each mutant . Three of the mutants proved to have chain-terminating mutations (one of which, previously shown to be suppressible by sup-3, was identified as amber); these led in every case to complete failure either to manufacture spores or to synthesize two enzymes normally associated with stage II of sporulation . The four remaining mutations were missense, and these corresponded to a phenotype in which a few spores are formed and about half the wild-type quantities of the two enzymes are made . Of the four missense mutations, two were near the promoter-distal end of the gene, in a region believed to correspond to the DNA-binding domain of the sigma factor that spoIIAC encodes . The remaining two mutations were in the region of the gene that is thought to correspond to the domain of the protein that interacts with core RNA polymerase.

Plasmid, 1987 Mar, 17(2), 167 - 70
Structural plasmid instability in recombination- and repair-deficient strains of Bacillus subtilis; Peijnenburg AA et al.; Plasmid pGP1, containing a fusion between the penicillinase gene of Bacillus licheniformis and the beta-galactosidase gene of Escherichia coli, was constructed . This plasmid enabled a study of structural plasmid instability in Bacillus subtilis wild-type cells and a variety of B . subtilis strains, defective in recombination- and DNA-repair functions . Large differences with respect to the level of stability of this plasmid were observed in the various genetic backgrounds.

Pharmazie, 1987 Mar, 42(3), 167 - 8
{Germ reduction by microwaves--microwave specific effects}; Groning R et al.; A commercial 2450-MHz microwave oven has been used to inactivate microorganisms in pharmaceutical solutions and auxiliary materials . The present investigation is concerned with the possible existence of a specific microwave effect, potentiating the thermal effect of microwave irradiation . Bacillus subtilis suspensions in polyethylenglycol 300, distilled water and peanut oil were used . It is shown that microwave heating is significantly more lethal to Bacillus subtilis microorganisms than equivalent conventional heating.

Genetika, 1987 Mar, 23(3), 405 - 13
{Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors}; Nezametdinova VZ et al.; We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination . Plasmids consist of pBR322 replicon, different fragments of Bac . subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid . Frequency of transformation was 10(-4) per bacterial cell . Foreign DNA (genes for tryptophan metabolism of Bac . mesentericus) was introduced into the chromosome of Bac . subtilis with the help of these plasmids.

J Bacteriol, 1987 Mar, 169(3), 973 - 80
Lipoteichoic acid from Bacillus subtilis subsp . niger WM: isolation and effects on cell wall autolysis and turnover; Meyer PD et al.; Lipoteichoic acid (LTA) was extracted by means of hot aqueous phenol from Bacillus subtilis subsp . niger WM cells grown under various conditions in chemostat culture . The extracts were partially purified by nuclease treatment and gel permeation chromatography . Chemical analyses revealed a composition consistent with a polyglycerol phosphate polymer . The influence on autolysis of the LTAs thus obtained was studied with both whole cells and autolysin-containing native walls of B . subtilis subsp . niger WM . Lysis rates of phosphate-limited cells could be reduced to about 40% of the control rate by the addition of LTA, whereas lysis of cells grown under phosphate-sufficient conditions was affected to a much lesser extent . The lysis of native walls prepared from variously grown cells proved to be fairly insensitive to the addition of LTA . The effect of LTA on wall turnover was studied by following the release of radioactively labeled wall material during exponential growth . The most obvious effect of LTA was a lowered first-order rate of release of labeled wall material; calculations according to the model for cell wall turnover in Bacillus spp . formulated by De Boer et al . (W . R . De Boer, F . J . Kruyssen, and J . T . M . Wouters, J . Bacteriol . 145:50-60, 1981) revealed changes in wall geometry and not in turnover rate in the presence of LTA.

J Bacteriol, 1987 Mar, 169(3), 1212 - 6
In vitro expression of a Tn9-derived chloramphenicol acetyltransferase gene fusion by using a Bacillus subtilis system; Zaghloul TI et al.; A coupled in vitro protein-synthesizing system has been developed with components derived totally from Bacillus subtilis . The system synthesized specific gene products from various exogenous DNA templates, including B . subtilis phage phi 29, plasmid pUB110, and a heterologous B . subtilis-Escherichia coli gene fusion containing the transposon Tn9-derived chloramphenicol acetyltransferase (cat) gene . The gene fusion product was able to show CAT activity, bind specifically to a Sephacryl-chloramphenicol column, and react immunologically against anti-CAT antiserum . The fidelity of this in vitro system was demonstrated by the synthesis of gene products identical to that made in vivo . We suggest that this system may be used to study the regulation of gene expression in vitro.

Can J Microbiol, 1987 Mar, 33(3), 249 - 55
Genome homology and divergence in the SP beta-related bacteriophages of Bacillus subtilis; Spancake GA et al.; Chromosomal organization in related temperate Bacillus subtilis bacteriophages SP beta, phi 3T, rho 11, Z, and E was compared . DNA-DNA hybridization studies done in conjunction with available restriction fragment maps of SP beta, phi 3T, and rho 11 demonstrated that DNA homology between these three phages extended over most of their respective genomes, although each contained unique chromosomal segments, phi 3T, rho 11, Z, and E, but not SP beta, possessed apparently homologous structural genes (thyP) for thymidylate synthetase . DNA from all thyP-containing phages transformed thymine auxotrophs of B . subtilis SP beta lysogens to prototrophy . This transformation commonly involved incorporation of the thyP gene into SP beta prophage within a region corresponding to the middle of the viral chromosome . Chimeric plasmids containing the thyP gene from phi 3T or cloned fragments of SP beta DNA were used in DNA-DNA hybridization studies to locate the thymidylate synthetase gene near the center of the phi 3T chromosome, and to demonstrate that the organization of this region resembled the analogous portion of the SP beta genome . Profiles of virion structural proteins from the five phages were also very similar, further suggesting functional homology between these viruses . However, despite these evidences of relatedness, populations of fragments generated by digesting SP beta, phi 3T, rho 11, Z, and E DNA with restriction enzymes were quite dissimilar.

J Bacteriol, 1987 Mar, 169(3), 995 - 1002
Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli; Deneer HG et al.; rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT) . The level of expression of CAT in E . coli showed growth rate dependence when the CAT gene was linked to either E . coli or B . subtilis tandem promoters . The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E . coli tandem promoters, only the upstream promoter was growth rate regulated.

J Bacteriol, 1987 Mar, 169(3), 967 - 72
The mRNA for an inducible chloramphenicol acetyltransferase gene is cleaved into discrete fragments in Bacillus subtilis; Ambulos NP Jr et al.; cat-86 is a promoter-deficient plasmid gene that encodes chloramphenicol acetyltransferase (CAT) . Insertion of a promoter at a site 144 base pairs 5' to the cat-86 coding sequence activates transcription of the gene and allows cat-86 to specify chloramphenicol-inducible CAT activity in Bacillus subtilis . Induction of cat-86 by chloramphenicol has been shown to result from a regulatory event that activates translation of cat-86 mRNA that is present in cells before the addition of inducer (E . J . Duvall and P . S . Lovett, Proc . Natl . Acad . Sci . USA 83:3939-3943, 1986) . In the present study we show an unusual property of cat-86 mRNA . Full-length cat-86 transcripts, consisting of 920 nucleotides (nt), are cleaved in B . subtilis to yield two predominant fragmentation products: an 810-nt species that lacks sequences present at the 5' end of the 920-nt species and a 720-nt species that lacks sequences present at the 3' end of the 920-nt species . A third fragmentation product consisting of 620 nt may result from the cleavage of a single 920-nt transcript at both the 5' and 3' ends . The sequences which are missing from the 720- and 620-nt species suggest that these transcripts cannot be translated into functional CAT . The 810-nt species lacks sequences from the 5' regulatory region, and it is not yet certain whether or not translation of this species can be induced by chloramphenicol . The ratio of 920-nt molecules/720-nt molecules in rifampin-treated cells is increased when the cells are grown in chloramphenicol . Therefore, induction may partially stabilize full-length cat-86 transcripts against inactivation by a novel processing-like system.

J Bacteriol, 1987 Mar, 169(3), 1260 - 6
Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions; Piggot PJ et al.; The control of expression of the Bacillus subtilis spoIIA locus was analyzed by titrating gene expression against gene copy number . A plasmid integrated into the B . subtilis chromosome and carrying the spoIIA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance . DNA from the clones was digested with BglII, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number . Most clones had fairly homogeneous copy numbers . Gene expression was monitored by beta-galactosidase activity . The results indicate that spoIIA was under positive control by a moiety present at about five copies per chromosome . Spore formation was not affected by amplification, so spoIIA-lacZ reiteration did not sequester a molecule required elsewhere for sporulation.

J Bacteriol, 1987 Mar, 169(3), 1239 - 45
Cloning and characterization of the 5' region of the cell wall protein gene operon in Bacillus brevis 47; Yamagata H et al.; Bacillus brevis 47 secretes vast amounts of proteins derived from both middle wall protein (MWP) and outer wall protein into the medium . The 5' region of the cell wall protein gene operon was cloned into Bacillus subtilis and subsequently into B . brevis 47 . On the basis of the nucleotide sequence analysis, an open reading frame coding for MWP was identified on the cloned DNA fragment . Two potential translation initiation sites for the MWP gene are located tandemly in the same reading frame . Each of the sites contains a sequence highly homologous to the 3' end of B . brevis rRNA and an initiation codon . The translational fusion of the 5' region of the MWP gene with the Bacillus licheniformis alpha-amylase gene resulted in the efficient expression of the alpha-amylase gene in B . brevis 47 . Of the two potential translation initiation sites, the one located upstream could be eliminated without affecting the expression of the MWP-alpha-amylase fusion gene, suggesting that MWP is synthesized in a precursor form with a signal peptide of 23 amino acid residues . S1 nuclease mapping of the cell wall protein gene transcripts suggested the possibility of the existence of several promoters in the 5' region within 300 base pairs from the translation initiation sites; one promoter was definitely localized within this part of the 5' region, and it was capable of expressing a heterologous gene fusion at a high level . The roles of the apparent structural complexity of the 5' region of the cell wall protein gene operon are discussed in connection with the efficient gene expression.

J Bacteriol, 1987 Mar, 169(3), 1205 - 11
Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis; Weinrauch Y et al.; Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2H and 15N, in the presence of 32Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication . Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients . The distribution of total and donor plasmid DNA was examined, using specific hybridization probes . The synthesis of new DNA, associated with the integration of donor moiety, was also monitored . Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA . This recombinant DNA represented 1.4% of total plasmid DNA . The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker . Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA . These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism . The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases.

J Biol Chem, 1987 Feb 25, 262(6), 2811 - 6
Evidence for an intermediate methyl-acceptor for chemotaxis in Bacillus subtilis; Thoelke MS et al.; Bacillus subtilis responds to chemotactic attractants by demethylating certain membrane-bound proteins, termed methyl-accepting chemotaxis proteins (MCPs) and by augmenting the evolution of methanol . We propose that the methanol comes from a methylated intermediate rather than directly from the MCPs themselves . First, repellent blocks attractant-induced smooth swimming and methanol formation, but not MCP demethylation . Second, prior treatment of cells with much attractant to reduce radiolabeling of MCPs and increase that of the putative intermediate caused increased, rather than decreased, production of methanol upon addition and then removal of the repellent . Third, such cells also produced much, rather than little, methanol upon addition of less attractant than during the pretreatment . We speculate that unmethylated intermediate causes tumbling; attractant causes its methylation and hence absence of tumbling (smooth swimming) . Its demethylation during the period of smooth swimming affords adaptation.

J Biol Chem, 1987 Feb 5, 262(4), 1766 - 71
Demonstration of erythromycin-dependent stalling of ribosomes on the ermC leader transcript; Narayanan CS et al.; ermC encodes a methylase that modifies 23 S rRNA, conferring resistance to macrolide-lincosamide-streptogramin B antibiotics . The expression of this gene is induced by erythromycin using a translational mechanism . We have employed the inherent RNase activity of a Bacillus subtilis S-30 extract as a probe for studying the interaction of ribosomes with ermC mRNA in the presence of antibiotics . 5' end-labeled ermC runoff transcript is a substrate for this RNase activity, while the ribosome-bound region of the RNA appears to be protected . Erythromycin- and oleandomycin-dependent protection of fragments of length 79-81 was observed during the translation of end-labeled ermC transcript . This occurs only using unmethylated (erythromycin sensitive) ribosomes . Various other antibiotics including clindomycin, tylosin, and lincomycin do not show this specific protection . These effects parallel the in vivo specificity of ermC induction . The effect of erythromycin can be abolished by using oligonucleotides complementary to regions of the ermC transcript upstream from nucleotide 71 and not by using an oligonucleotide complementary to a region of ermC downstream from that position . These results are interpretable in terms of the translational attenuation model and demonstrate that erythromycin-bound ribosomes initiate translation of the leader peptide, stall upstream from nucleotide 80 on the ermC mRNA, and thus make the ribosome-binding site for methylase message available for ribosome interaction.

J Biol Chem, 1987 Feb 5, 262(4), 1756 - 65
An in vitro study of the translational attenuation model of ermC regulation; Narayanan CS et al.; We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation . The ermC gene product is known to methylate rRNA, rendering ribosomes unable to bind this antibiotic . We have shown that the induction of ermC methylase in vitro is post-transcriptional and specific for the macrolides erythromycin and oleandomycin . Erythromycin has no significant effect on the stability of the ermC transcript in vitro, and hence the post-transcriptional induction of methylase under these conditions occurs by stimulation of translation . The induction effect requires ribosomes able to bind erythromycin . By adding small proportions of unmethylated to a methylated extract in the presence of erythromycin, methylase synthesis could be induced . Conversely, when small amounts of methylated extracts were mixed with unmethylated extracts, methylase synthesis could be maintained at elevated levels in the presence of a high concentration of erythromycin . These effects were specific for the inducible ermC, were not observed with a constitutive variant, and could be explained satisfactorily by the translational attenuation model . The roles of three segments of the ermC leader in regulation were explored by probing with appropriate complementary synthetic oligodeoxynucleotides . The induction effect of erythromycin was mimicked by using an oligonucleotide that could free the ribosome binding site for methylase.

Mutat Res, 1987 Feb, 190(2), 83 - 7
Evidence that N-methyl-N'-nitro-N-nitrosoguanidine induces adaptive response in Bacillus thuringiensis; Boutibonnes P et al.; Bacillus thuringiensis is shown to have an inducible error-free repair system for alkylation damage as found in Escherichia coli and Bacillus subtilis . Growth of cells in the presence of low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces an adaptive response which is characterized by an increase in resistance to killing and mutagenesis by challenge with higher concentrations of MNNG . In addition, we have noted with interest that adaptive low doses seem to produce lesions at a rate sufficient to induce an increase of mutation frequency, and inhibition of cell division . The possibility of an interaction between SOS and adaptive responses with these low doses of MNNG is discussed.

Jpn J Antibiot, 1987 Feb, 40(2), 340 - 8
{Clinical studies on cefuroxime axetil in acute mastitis}; Nakamura T et al.; Cefuroxime (CXM), with its relatively broad antibacterial spectrum, good stability to beta-lactamases and high safety, has an established place in antibacterial therapy . However, like many other cephalosporins, its clinical application is rather limited due to the need for parenteral delivery . Recent work has led to the development of cefuroxime axetil (CXM-AX, SN407) which is the 1-acetoxyethyl ester of CXM is well absorbed from the gastrointestinal tract and promptly cleaved to cefuroxime thereafter . CXM-AX, in a dose of 500 mg, was given after meal by oral administration 3 times daily for 5-8 days to 10 lactating outpatients with acute mastitis . Ages of the patients ranged from 26 to 44 years, and body weights ranged from 48.0 to 59.0 kg . Clinical response was assessed as excellent in 2 cases, good in 8 cases and fair or poor in none . No adverse effects were observed . Seven strains of organisms were isolated from 7 cases . They included 5 strains of Staphylococcus aureus and 2 strains of Staphylococcus epidermidis . The MICs of CXM were under 3.13 micrograms/ml with inoculume size of 10(8) and 10(6) cells/ml . In 1 case, CXM concentrations in puruloid milk and in healthy milk were measured by bioassay with Bacillus subtilis ATCC 6633 as the test organism . The CXM concentrations in healthy milk ranged from 0.09 to 0.59 microgram/ml (mean: 0.32 +/- 0.25 microgram/ml) at 30 to 90 minutes after oral administration of 500 mg CXM-AX . The CXM concentrations in puruloid milk ranged from 0.57 to 1.05 micrograms/ml (mean: 0.74 +/- 0.27 microgram/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Microbiol, 1987 Feb, 147(1), 68 - 72
Buoyant density fluctuations during the cell cycle of Bacillus subtilis; Hart A et al.; A simple rapid method for preparing synchronous cultures of Bacillus subtilis has been used to investigate changes in density during the cell cycle . Asynchronous cells separated on a stepped Percoll density gradient had a mean cell density of 1.117 g ml-1 +/- 0.004 . Samples from a synchronous culture exhibited variation (ca . 1.5%) in mean cell density which was greatest at the onset of cell division . An asynchronous control culture showed little variation in density . These results are discussed in relation to previous work on Escherichia coli.

Mol Gen Mikrobiol Virusol, 1987 Feb, (2), 20 - 4
{Study of the 210-degree region of the Bacillus subtilis chromosome using recombinant plasmids}; Chikindas ML et al.; The 210 degrees region of Bacillus subtilis DNA containing the rib operon and genes for the first (dapA) and last (lysA) steps of lysine biosynthesis was cloned . PstI fragments of B . subtilis m.m . 4.7 MD DNA containing the lys and the proximal part of rib operon were isolated from different B . subtilis strains (SB25 and SHgW) and shown to have the same restriction and genetic maps . The restriction mapping of EcoRI fragment of B . subtilis m.m . 6.3 MD DNA containing the rib operon has been carried out.

Antibiot Med Biotekhnol, 1987 Feb, 32(2), 97 - 101
{Assessment by the material and energy balance method of the directed biosynthesis of riboxin by altering the external culture conditions}; Toitman AI et al.; The effect of various agitation conditions on growth of the cell population of Bacillus subtilis and production of riboxin was studied . The physiological state of the culture was estimated by the relations for the respiration coefficient CO2/O2, the yield of the constructive metabolism products (X + P) by oxygen Yx + p/O2, the ratio of consumed glucose to ammonium nitrogen S/NH4+, the coefficient of the cell biosynthetic activity Yp/x and the parameter of the energetic efficiency of the substrate usage (eta) . It was shown that the maximum biosynthetic activity was mainly defined by the culture state in the trophophase . The growth of the cell population in this case should proceed under conditions of conjugated aerobic metabolism . It was found that directed biosynthesis of riboxin could be provided by changing the cultivation conditions and estimation of the culture physiological state by the parameters of the material and energy balance.

Genetika, 1987 Feb, 23(2), 261 - 7
{Integration of the plasmid carrying the genes for tryptophan metabolism of Bacillus mesentericus into the chromosome of Bacillus subtilis}; Pavlova SI et al.; Integration of several tryptophan operon genes from Bacillus mesentericus into the chromosome of Bac . subtilis was carried out . The genetic material exchange between these two species is usually impossible in the process of transformation, because of poor homology . The integration only took place after the genes of Bac . mesentericus were introduced in Bac . subtilis cells on a hybrid plasmid . Plasmid markers also integrated into the chromosome, together with heterologous DNA . The integration occurred in the region of Bac . subtilis tryptophan operon, which is likely to be due to retained homology between corresponding DNAs . The integration acts occurred during prolonged maintenance of chimeric plasmid in autonomously replicating form . We suggest to designate this phenomenon "retarded homologous recombination".

J Bacteriol, 1987 Feb, 169(2), 587 - 92
Multiple species of Bacillus subtilis DNA alkyltransferase involved in the adaptive response to simple alkylating agents; Morohoshi F et al.; Three molecular species of methyl-accepting proteins exist in Bacillus subtilis cells, which collect methyl groups from methylated DNA . A 20-kilodalton (kDa) protein was constitutively present in the cells of the ada+ (proficient in adaptive response) strain as well as in those of six ada (deficient in adaptive response) mutant strains and was assigned to the O6-methylguanine:DNA methyltransferase . Another species of O6-methylguanine:DNA methyltransferase, which had a molecular size of 22 kDa, emerged after adaptive treatment of the ada+ but not any of the ada mutant cells . A 27-kDa methyl-accepting protein, which preferred methylated poly(dT) to methylated calf thymus DNA as a substrate, was assigned to the methylphosphotriester:DNA methyltransferase . It was produced, after adaptive treatment, in the cells of ada+, ada-3, ada-4, and ada-6 strains but not in the cells of ada-1, ada-2, or ada-5 strains . These results support and extend our proposition that ada mutants can be classified into two groups; one (the ada-4 group) is defective only in the inducible synthesis of O6-methylguanine:DNA methyltransferase (22-kDa protein), and the other (the ada-1 group) is deficient in the adaptive response in toto . The finding that inducible and constitutive methyltransferases reside in different molecular species of methyl-accepting proteins is intriguing compared with the regulatory mechanisms of the adaptive response to simple alkylating agents in other organisms.

J Bacteriol, 1987 Feb, 169(2), 519 - 25
Regulation of Bacillus subtilis macrofiber twist development by ions: effects of magnesium and ammonium; Mendelson NH et al.; The steady-state twist of Bacillus subtilis macrofibers produced by growth in complex medium was found to vary as a function of the magnesium and ammonium concentrations . Four categories of macrofiber-producing strains that differed in their response to temperature regulation of twist were studied . Macrofibers were cultured in the complex medium TB used in previous experiments and in two derivative media, T (consisting of Bacto Tryptose), in which most strains produced left-handed structures, and Be (consisting of Bacto Beef Extract), in which right-handed macrofibers arose . In nearly all cases, increasing concentrations of magnesium led to the production of macrofibers with greater right-handed twist . Some strains unable to form right-handed structures as a function of temperature could be made to do so by the addition of magnesium . Inversion from right- to left-handedness in strain FJ7 induced by temperature shift-up was blocked by the addition of magnesium . The presence of magnesium during a high-temperature pulse did not block the establishment of "memory," although it delayed the initiation of the transient inversion following return to low temperature . The twist state of macrofibers grown without a magnesium supplement was not instantaneously affected by the addition of magnesium . Such fibers were, however, protected from lysozyme attack and associated relaxation motions . Lysozyme degradation of purified cell walls (both intact and lacking teichoic acid) was also blocked by the addition of magnesium . Ammonium ions influenced macrofiber twist development towards the left-hand end of the twist spectrum . Macrofiber twist produced in mixtures of magnesium and ammonium was strain and medium dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1987 Feb, 53(2), 379 - 84
Protease-deficient Bacillus subtilis host strains for production of Staphylococcal protein A; Fahnestock SR et al.; We constructed strains of Bacillus subtilis which produced very low levels of extracellular proteases . These strains carried insertion or deletion mutations in the subtilisin structural gene (apr) which were constructed in vitro by using the cloned gene . The methods used to construct the mutations involved the use of a plasmid vector which allowed the selection of chromosomal integrates and their subsequent excision by homologous recombination to effect replacement of the chromosomal apr gene by a derivative carrying an inactivating insert with a selectable marker (a cat gene conferring chloramphenicol resistance) . The strains produced no subtilisin, no detectable extracellular metalloprotease activity, and residual extracellular serine protease levels as low as 0.5% of that of the standard strain from which they were derived . The strains proved to be superior host strains for the production of staphylococcal protein A, accumulating higher levels of intact protein than do previously available B . subtilis strains.

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 653 - 7
Nucleotide sequence of Bacillus subtilis dnaB: a gene essential for DNA replication initiation and membrane attachment; Hoshino T et al.; The complete nucleotide sequence of the Bacillus subtilis dnaB gene and its flanking regions was determined . The dnaB gene is essential for both replication initiation and membrane attachment of the origin region of the chromosome and plasmid pUB110 . It has been known that there are two different classes (dnaBI and dnaBII) in the dnaB mutants; dnaBI is essential for both chromosome and pUB110 replication, whereas dnaBII is necessary only for chromosome replication . The nucleotide sequence revealed that dnaBI and dnaBII are two functional domains in the single dnaB gene . The mutation sites of two mutants, belonging to dnaBI and dnaBII, respectively, were also determined as substitutions of amino acids . The putative DnaB protein deduced from nucleotide sequence consists of 472 amino acids (55 kDa) with no cysteine residue . A 55-kDa polypeptide produced in an in vitro transcription-translation system was labeled with {35S}methionine but not with {35S}cysteine . The DnaB protein has a highly hydrophobic sequence of 20 amino acids in its N-terminal region, a possible DNA binding site, and two possible ATP binding sites . The dnaBI domain is between the DNA binding site and one of the ATP binding sites; the dnaBII domain is close to the other ATP binding site . Comparison of the amino acid sequence between the "dnaB protein" and those of other dna genes of Escherichia coli showed no homology, suggesting that the dnaB gene of B . subtilis may be analogous to a hitherto undiscovered gene in E . coli.

J Bacteriol, 1987 Feb, 169(2), 864 - 73
Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex; Phillips MK et al.; The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined . This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB . The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B . subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli . Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E . coli in size, composition, and amino acid sequence . The sequence homologies further indicated that the B . subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E . coli but are less closely related than are the corresponding pairs of E . coli subunits . The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.

J Bacteriol, 1987 Feb, 169(2), 771 - 8
Gene encoding the 37,000-dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus, and cryptic function; Duncan ML et al.; We began an analysis of rpoF, the gene encoding the cryptic, 37,000-dalton minor sigma factor (sigma-37) of Bacillus subtilis RNA polymerase . Using antibody raised against sigma-37 holoenzyme to probe a lambda gt11 expression vector library, we isolated a 901-base-pair EcoRI fragment that expressed the COOH-terminal half of sigma-37 fused to lacZ . We used this fragment as a hybridization probe to isolate the entire rpoF gene and additional flanking sequences . Identity of the cloned gene was confirmed by the size and immunological reaction of its product expressed in Escherichia coli and, after DNA sequencing, by the homology of its predicted product (264 residues; 30,143 daltons) with other sigma factors . The DNA sequence also suggested that rpoF may lie in a gene cluster . Upstream of rpoF was an open reading frame that would encode a protein of 17,992 daltons; this frame overlapped the rpoF-coding sequence by 41 base pairs . Immediately following rpoF was a reading frame that would encode a protein of at least 20,000 daltons; expression of this region may be translationally coupled to that of rpoF . By plasmid integration and PBS1 transduction, we found the chromosomal locus of rpoF linked to ddl and dal at 40 degrees on the B . subtilis map and near no known lesions affecting growth regulation or development . Further, an rpoF null mutation resulting from gene disruption had no effect on cell growth or sporulation in rich medium, suggesting that sigma-37 may partly control a regulon not directly involved in the sporulation process.

J Bacteriol, 1987 Feb, 169(2), 612 - 8
Sequence of the Ampullariella sp . strain 3876 gene coding for xylose isomerase; Saari GC et al.; The nucleotide sequence of the gene coding for xylose isomerase from Ampullariella sp . strain 3876, a gram-positive bacterium, has been determined . A clone of a fragment of strain 3876 DNA coding for a xylose isomerase activity was identified by its ability to complement a xylose isomerase-defective Escherichia coli strain . One such complementation positive fragment, 2,922 nucleotides in length, was sequenced in its entirety . There are two open reading frames 1,182 and 1,242 nucleotides in length, on opposite strands of this fragment, each of which could code for a protein the expected size of xylose isomerase . The 1,182-nucleotide open reading frame was identified as the coding sequence for the protein from the sequence analysis of the amino-terminal region and selected internal peptides . The gene initiates with GTG and has a high guanine and cytosine content (70%) and an exceptionally strong preference (97%) for guanine or cytosine in the third position of the codons . The gene codes for a 43,210-dalton polypeptide composed of 393 amino acids . The xylose isomerase from Ampullariella sp . strain 3876 is similar in size to other bacterial xylose isomerases and has limited amino acid sequence homology to the available sequences from E . coli, Bacillus subtilis, and Streptomyces violaceus-ruber . In all cases yet studied, the bacterial gene for xylulose kinase is downstream from the gene for xylose isomerase . We present evidence suggesting that in Ampullariella sp . strain 3876 these genes are similarly arranged.

J Bacteriol, 1987 Feb, 169(2), 461 - 9
Effects of plasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subtilis; Zuber P et al.; Transcription of the sporulation gene spoVG of Bacillus subtilis is induced at the onset of spore formation and depends on the products of the regulatory genes spoOA, spoOB, and spoOH . We describe two effects of propagating the promoter region of spoVG on a multicopy plasmid replicon in B . subtilis cells . One effect is that transcription from the plasmid-borne spoVG promoter is altered with respect to the time of its induction and the dependence on spoO gene products . An example of this effect is that plasmid propagation was observed to relieve substantially the inhibitory effect of a mutation in spoOH, the spoO gene upon which spoVG promoter activity is most strongly dependent . We present results which suggest that propagation on a plasmid replicon causes an alteration in the conformation of spoVG promoter DNA which somehow compensates for the defective spoOH gene product . Plasmid propagation did not, however, entirely eliminate the requirement for the spoOH gene product; little or no spoVG-directed RNA synthesis was observed in cells bearing a putative spoOH deletion mutation, a finding which indicates that SpoOH protein plays an indispensable role in spoVG promoter utilization . Another effect of propagating the promoter region of spoVG on a multicopy plasmid is to inhibit sporulation . S1 nuclease mapping experiments suggest that amplification of spoVG on a multicopy plasmid causes the titration of a transcription factor or minor form of RNA polymerase holoenzyme required for utilization of one of the two overlapping promoters which comprise the spoVG transcription initiation region.

Biol Chem Hoppe Seyler, 1987 Feb, 368(2), 93 - 9
A new inhibitor of elastase from the sea anemone (Anemonia sulcata); Kolkenbrock H et al.; A proteinase inhibitor for elastases was isolated from extracts of the sea anemone Anemonia sulcata and purified to apparent homogeneity . The procedure comprises ethanolic extraction of the deep-frozen animals followed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-Sephadex A-25 and SP-Sephadex C-25 and by hydroxylapatite chromatography . The slightly acidic inhibitor (isoelectric point 5.9) is a small protein consisting of 48 amino-acid residues without tryptophan and phenylalanine . The single chain molecule contains two methionines and no free sulfhydryl group but six cysteines presumably forming disulfide bonds . Reaction with cyanogen bromide abolishes the inhibitory properties . The inhibitor exhibits a rather narrow specificity for elastases . It strongly inhibits porcine pancreatic elastase in a permanent fashion with an equilibrium dissociation constant Ki of about 10(-10)M and somewhat weaker the elastase from human leucocytes with a Ki of about 10(-7)M . No obvious inhibition is observed of other serine proteinase such as bovine trypsin, bovine chymotrypsin, subtilisin from Bacillus subtilis and cathepsin G from human leucocytes when tested with synthetic substrates.

J Biol Chem, 1987 Jan 25, 262(3), 1016 - 21
Heavy riboflavin synthase of Bacillus subtilis . Primary structure of the beta subunit; Ludwig HC et al.; Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis . The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000) . beta subunits were isolated and cleaved with cyanogen bromide . Fragments were isolated and further digested with trypsin and staphylococcal protease . Peptides were isolated by high performance liquid chromatography . Sequences were determined by automated liquid-phase Edman degradation . The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus . The sequence shows no detectable homologies to other proteins . A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.

Vet Med Nauki, 1987, 24(4), 37 - 42
{Cultivation of cells in a medium with blood hydrolysate}; Veleva E et al.; Use was made of fresh blood of swine, diluted with an equal amount of distilled water to produce a protein hydrolysate . Enzyme hydrolysis was effected through extracellular alkaline protease produced by stain DI of Bacillus subtilis . The cell lines BHK-21, PK-15 and spzv were cultured in a nutrient medium containing 0.180-0.200 mg/cm3 alpha-amine nitrogen and relevant growth factors of nonprotein character . It was found that the cells cultured in this medium showed no differences with regard both to morphology and karyology as against the control cells which were treated with the classic medium of Eagle . It was also found that the medium could successfully be used instead of the imported nutrient media.

Boll Ist Sieroter Milan, 1987, 66(5), 391 - 4
Lymphocyte activation by B . subtilis spores; Fais S et al.; The in vitro effect of Bacillus Subtilis spores (BSs) on lymphocyte activation was investigated by analyzing the kinetics of expression of the activation antigens MLR3, MLR4 and HLA-DR in cultures of peripheral mononuclear cells . It was found that BSs over a wide range of concentrations are capable of inducing an increased expression of these three markers and that at high BSs concentration the effect is both quantitatively and qualitatively similar to that induced by the mitogens PHA and ConA . These results suggest that T cells may be involved in the response to Bss.

Gene, 1987, 61(2), 217 - 24
Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli; Carlsson P et al.; The 2-oxoglutarate dehydrogenase multienzyme complex is composed of three different subenzymes: 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide transsuccinylase (E2o), and dihydrolipoamide dehydrogenase (E3) . Bacillus subtilis E1o and E2o are encoded by the citK and citM genes, respectively . A 3.4-kb BamHI DNA fragment containing citK and citM markers was isolated from a library of B . subtilis DNA in Escherichia coli . Functional E2o was expressed from the cloned DNA both in B . subtilis and E . coli . E2o had an apparent Mr of 60,000 when expressed in E . coli . The B . subtilis E2o component complemented an E . coli E2o-defective mutant in vivo and in vitro . It is concluded that functional B . subtilis E2o can be produced in E . coli and can interact with E . coli and E1o and E3 to form an active chimeric enzyme complex.

Folia Microbiol (Praha), 1987, 32(6), 465 - 80
Protein turnover and proteolysis during sporulation of Bacillus subtilis; Sekar V et al.; A two-dimensional electrophoretic method was used to show that protein degradation occurs immediately after the end of exponential growth but that its occurrence is masked in the usual assay methods for a 2-h period and that degradation is apparently nonselective with respect to protein molar mass or charge . The results suggest that considerable reutilization of internal amino acids may occur during sporulation regardless of the size of the external chase . Finally, the levels of intracellular proteinase activities present even at the end of exponential phase growth, as measured in vitro, are sufficient to account for the maximum rates of protein degradation observed in vivo.

Gene, 1987, 57(2-3), 221 - 7
Various means of integration of the expressible human dihydrofolate reductase gene into the Bacillus subtilis genome; Prozorov AA et al.; Integration of expressible DNA corresponding to the human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis genome has been achieved in different ways . The clones obtained contained one to seven copies of this gene per genome equivalent and were resistant to trimethoprim . Clones produce a new protein coded by the integrated hDHFR gene . In all clones, the integrated DNA was stably maintained even under nonselective growth conditions.

Bioelectromagnetics, 1987, 8(3), 275 - 82
Low-level, magnetic-field-induced growth modification of Bacillus subtilis; Ramon C et al.; Experimental studies showed an increase in the growth of Bacillus subtilis mutant strain FJ7 above controls by exposing the bacterial culture to 800-Hz or 1-KHz magnetic fields with a 2-s-on/2-s-off period . The magnetic field strength was between 0.8 and 2.5 mT . Light microscopy and scanning electron microscopy demonstrated the morphology of controls to grow in a macrofiber of right-handed helix formation . In contrast, the field-exposed group showed little to no cohesion; the cells appeared to be homogeneously distributed throughout the sample . These results suggest that growth patterns of Bacillus subtilis can be altered as a result of magnetic-field-induced effects.

Antonie Van Leeuwenhoek, 1987, 53(2), 125 - 36
Studies on cellulase production by a Bacillus subtilis; Chan KY et al.; Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source . Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 degrees C and at an initial pH of 6.0 . CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation . The optimal temperature for CMCase activity was 65 degrees C, and the enzyme was highly stable up to 60 degrees C . CMCase synthesis was subjected to catabolite repression by glucose and cellobiose.

Zentralbl Mikrobiol, 1987, 142(1), 63 - 70
Characteristics of a soil-isolated Bacillus subtilis phage, GS1, and GS1-mediated plasmid transduction; Van Elsas JD et al.; A previously described soil-isolated Bacillus subtilis bacteriophage, GS1, was further characterized . It is a tailed phage with regular head morphology and a discrete base plate . GS1 belongs to morphological group A1 of Bacillus phages . Its tail measured 170 nm and the head width was 92 nm . GS1 produced turbid plaques on its natural host strain, but clear plaque variants occurred at high frequency . All attempts to transduce either erythromycin (Em) or tetracycline (Tc) resistance markers, present in soil-isolated phage-sensitive bacilli, failed . Erythromycin resistance plasmid pE194, introduced into B . subtilis by protoplast transformation, was transduced among B . subtilis strains at a frequency of 10(-6) to 10(-7).

Gene, 1987, 51(1), 1 - 11
The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168; Zuberi AR et al.; The nucleotide sequence of the second and third genes in the Bacillus subtilis spore germination locus, gerA, has been determined and the amino acid (aa) sequence was derived . Two open reading frames (ORFs), corresponding to genes II and III, encode 364-aa residue and 373-aa residue polypeptides, respectively . The gene II product, Mr 41,257, would contain long stretches of hydrophobic aa residues and may be a membrane protein; the gene III product, Mr 42,363, is relatively hydrophilic but possesses an apparent signal peptide for transfer across, and perhaps localisation on, a membrane . The ORFs for genes I and II overlap by eleven codons and the termination codon of gene II overlaps the initiation codon of gene III . Insertional inactivation experiments using integrational plasmids have indicated that the gerA locus is a single transcriptional unit . The expression of the gerA genes has been studied using a lacZ transcriptional fusion; they constitute a developmentally regulated operon.

Microbios, 1987, 49(200-201), 199 - 212
Characterization of intracellular deoxyribonucleases of Bacillus subtilis by SDS-polyacrylamide gel electrophoresis; Rama JM et al.; Electrophoresis in polyacrylamide gels containing DNA was used to study nucleolytic activities in Bacillus . Optimal conditions (cations, pH and substrate) and a general pattern of DNases of B . subtilis 168 were established, revealing the presence of twelve to fourteen defined bands with DNase activity . Fractionation of cell cultures showed that most of the nucleases were located in the bacterial cytosol, and only two activities were membrane-associated . The employment of plasmid DNA in the gels facilitated distinguishing between exo- and endonucleases, the latter being the most active bands . The DNase pattern of various strains of B . subtilis, and species of the genus Bacillus, was compared . Evident differences were observed although similarities exist.

Folia Microbiol (Praha), 1987, 32(2), 96 - 100
Formation of extracellular neutral proteinase and the stringent response in Bacillus subtilis; Riedel K et al.; The kinetics of extracellular neutral proteinase synthesis by an isogenic stringent (IS58) and a relaxed (IS56) strain of B . subtilis were compared . The specific enzyme formation rate by the stringent strain was higher than that of the relaxed one . Norvaline addition (1 mg/mL) induced the formation of pppGpp and ppGpp, respectively, as well as the appearance of extracellular neutral proteinase activities in cultures of the stringent strain IS58 and a strain with high proteinase production (ZF-178) only . These correlations support the suggestion that (p)ppGpp are involved in the regulation processes responsible for production of extracellular neutral proteinases by B . subtilis.

Arch Microbiol, 1987 Jan, 146(4), 353 - 7
Characterization of chromosome and plasmid transformation in Bacillus subtilis using gently lysed protoplasts; Akamatsu T et al.; Competent cells of Bacillus subtilis were transformed with DNA from gently lysed protoplasts . Significant linkages among markers separated by distances of approximately 2.3% of the total chromosome were found, which have not been detected for conventional transformation . In comparison to previous reports, enhanced plasmid transformation was observed {4.0 X 10(7) transformants per microgram DNA (one transformant per 5 X 10(4) molecules added)}, when competent cells were transformed with DNA from lysed protoplasts harboring pUB110.

Food Chem Toxicol, 1987 Jan, 25(1), 31 - 4
Bacterial tests as indicators for the detoxification of the mycotoxin penicillic acid by ammonia treatment; Speer M et al.; The detoxification of penicillic acid by reaction with ammonia was examined by means of a polymerase assay using two strains of Escherichia coli (pol A+ and pol A-1) and a recombination assay using two strains of Bacillus subtilis (rec+ and rec-) . A 100-fold surplus of ammonia added to penicillic acid abolished the cytotoxic and genotoxic effects of penicillic acid towards the bacteria under the test conditions . The study presents the possibility of detoxifying mycotoxins in feeds by ammonia treatment and demonstrates the suitability of bacterial assays as indicators for mycotoxins.

Folia Microbiol (Praha), 1987, 32(1), 82 - 4
Selective regeneration of Bacillus subtilis protoplasts transformed to kanamycin resistance; Jandova Z et al.; An HTY medium osmotically stabilized with 0.5 M D-glucitol was used for regeneration of Bacillus subtilis protoplasts . The application of glucitol as osmotic stabilizer allows simultaneous selection of cells resistant to kanamycin to be made since this antibiotic is not inactivated by glucitol when added to the regeneration medium.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 498 - 502
Induced mRNA stability in Bacillus subtilis; Bechhofer DH et al.; We have investigated the induced stability of mRNA encoded by the ermC gene in Bacillus subtilis . Induction of ermC gene expression by erythromycin is known to occur at the translational level . We show that this induction is accompanied by an increase in ermC mRNA half-life from about 2 min to about 40 min . Induced stabilization of ermC mRNA occurs independently of induced translation . The regulatory sequences required for stability are promoter-proximal and can confer induced stability on large mRNAs having diverse 3' ends . Translation of the ermC leader peptide and ribosome-stalling in the leader peptide sequence are necessary for induced stabilization.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 421 - 3
Thymine-containing dimers as well as spore photoproducts are found in ultraviolet-irradiated Bacillus subtilis spores that lack small acid-soluble proteins; Setlow B et al.; Dormant spores of a Bacillus subtilis mutant that lacks two major small, acid-soluble spore proteins are very sensitive to UV irradiation, which in spores generates about half the amount of thymine-containing dimers formed by comparable irradiation of vegetative cells . Irradiation of mutant spores also produces spore photoproducts, but again only about one-half the amount formed in comparably irradiated wild-type spores . These findings suggest that the high UV sensitivity of the mutant spores is due to the production of pyrimidine dimers, which are not found in UV-irradiated wild-type spores, and that the high level of small, acid-soluble proteins found in wild-type spores is directly involved in spore UV resistance by facilitating a conformational change in spore DNA, preventing pyrimidine dimer formation.

J Bacteriol, 1987 Jan, 169(1), 444 - 6
Construction and properties of an intracellular serine protease mutant of Bacillus subtilis; Band L et al.; An intracellular serine protease (ISP-1) mutant of Bacillus subtilis was created by introducing a frameshift into the coding region of the cloned gene . Intracellular protease activity in the mutant was very low, yet sporulation in both nutrient broth and minimal medium was normal . The rate of bulk protein turnover in the mutant was slightly slower than that in the wild-type strain . These results suggest that the gene for ISP-1 is not essential and that ISP-1 is not the major enzyme involved in protein turnover during sporulation.

J Bacteriol, 1987 Jan, 169(1), 434 - 7
Characterization and mapping of the Bacillus subtilis prtR gene; Yang M et al.; A gene from Bacillus natto encoding a 60-amino-acid peptide has been previously described that, when cloned on a high-copy plasmid in B . subtilis, enhances production of alkaline protease, neutral protease, and levansucrase . An identical gene was isolated from B . subtilis and caused a similar phenotype when placed on a high-copy plasmid . Genetic mapping localized this gene near metB, distant from other pleiotropic genes causing similar effects . Deletion of this gene from the B . subtilis chromosome had no obvious phenotypic effect.

J Bacteriol, 1987 Jan, 169(1), 324 - 33
Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis; Amory A et al.; The sacQ gene from Bacillus licheniformis was cloned and expressed in Bacillus subtilis . Deletion analysis shows that it encodes a 46-amino-acid polypeptide homologous to the B . subtilis sacQ gene product . The polypeptide, when it is overexpressed, activates the expression of a number of target genes in B . subtilis, all encoding secreted enzymes: alkaline protease, levansucrase, beta-glucanase(s), xylanase, and alpha-amylase . The maximum stimulations measured for alkaline protease and levansucrase were by a factor of 70 and 50, respectively, when the sacQ gene from B . licheniformis was present on a multicopy plasmid in B . subtilis . The sacQ genes from B . subtilis and B . licheniformis, cloned in the same multicopy plasmid, were compared under the same conditions . The sacQ gene from B . licheniformis was more efficient than the sacQ gene from B . subtilis in producing the hypersecretion phenotype . The sacQ structural genes from B . subtilis and B . licheniformis were placed under the control of the same inducible promoter . Hypersecretion was specifically obtained under conditions of full induction of the promoter . The target site of levansucrase regulation by sacQ was identified as a 440-base-pair fragment located in the 5' noncoding region of sacB, suggesting transcriptional control.

Gene, 1987, 53(2-3), 247 - 55
Phosphoribosylpyrophosphate synthetase of Bacillus subtilis . Cloning, characterization and chromosomal mapping of the prs gene; Nilsson D et al.; The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation . Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting in a DNA fragment of approx . 1.8 kb complementing the E . coli prs mutation . Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx . 40,000 . B . subtilis strains harbouring the prs gene in a multicopy plasmid contained up to nine-fold increased PRPP synthetase activity . The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B . subtilis chromosome by homologous recombination . The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA.

Gene, 1987, 52(2-3), 175 - 83
Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis; Vosman B et al.; With the aim of cloning genes involved in transformation of Bacillus subtilis, a set of transformation-deficient mutants was isolated by means of insertional mutagenesis with plasmid pHV60 (Vosman et al., 1986) . Analysis of these mutants showed that those mapping in the aroI region lacked the DNA-entry nuclease activity . Plasmid pHV60 derivatives, containing flanking chromosomal DNA fragments, were isolated from these mutants and were used to screen a library of B . subtilis chromosomal DNA in phage lambda EMBL4 . In Escherichia coli lysates, prepared with the phages that hybridized to the pHV60-based probe, a prominent nuclease activity could be detected . The nuclease encoded by the phage DNA had the same Mr as the B . subtilis DNA-entry nuclease and its activity was strongly stimulated by Mn2+, which is also characteristic for the B . subtilis DNA-entry nuclease . From these results it was concluded that the gene specifying the B . subtilis DNA-entry nuclease had been cloned . It was shown that the nuclease activity was specified by a 700-bp EcoRI-PstI fragment.

Genetika, 1987 Jan, 23(1), 14 - 20
{Amplification of plasmid DNA inserted into the Bacillus subtilis chromosome}; Khasanov FK et al.; Amplification of plasmid pGG10 inserted into the Bacillus subtilis chromosome is described . The possibility of the 3.2 kb fragment of eucaryotic (wheat) DNA to be amplified within the bacterial genome is shown . The models explaining this phenomenon are discussed.

Drugs Exp Clin Res, 1987, 13(9), 547 - 50
Determination of teicoplanin concentration in serum using a bioassay technique; Patton KR et al.; An accurate and sensitive bioassay for determining concentrations of teicoplanin in serum has been developed using modifications of procedures described for assaying vancomycin . A linear relationship (r = 0.9983) was obtained between the diameter of the zone of inhibition and log10 teicoplanin concentration over the range 0.25-32 micrograms/ml . The medium used was Antibiotic Medium No . 1 (Oxoid, UK) adjusted to pH 5.5-5.7 by the addition of hydrochloric acid and containing sodium chloride to a final concentration of 3% . The indicator organism used was a Bacillus subtilis ATCC 6633 (NCTC 10400) . Teicoplanin was assayed: (i) in the presence of beta-lactams and cephalosporins, including ceftazidime, by prior treatment of serum with broad-spectrum beta-lactamase mixtures (Genzyme Laboratories, UK); (ii) in the presence of aminoglycosides by prior treatment of serum with cellu-ion phosphate (100 mg/ml) followed by centrifugation; (iii) in the presence of sulphamethoxazole and/or trimethoprim by the addition of p-aminobenzoic acid and/or thymidine to the assay medium . Teicoplanin was assayed in the presence of either rifampicin or erythromycin by using a rifampicin-resistant, erythromycin-resistant clinical isolate of Staphylococcus aureus as indicator organism . The lower limit of sensitivity of this assay was 1 microgram/ml . The presence of undeclared, broad-spectrum antimicrobials was detected by screening serum samples for antimicrobial activity using an assay plate seeded with Escherichia coli NCTC 10418.

Gene, 1987, 56(2-3), 277 - 82
A Bacillus subtilis phage phi 29 transcription terminator is efficiently recognized in Streptomyces lividans; Pulido D et al.; A DNA fragment from the Bacillus subtilis phage phi 29, containing the bidirectional transcription terminator TD1, where the right early transcription and late viral transcription terminate, has been inserted in one orientation between the aminoglycoside phosphotransferase (APH) gene (neo) and the phi 29 main early and late promoters present in derivative constructs of the Streptomyces promoter-probe plasmid pIJ486 . The TD1 terminator is efficiently recognized in S . lividans and ends the transcription, started in vivo at the phi 29 promoters, at the same point as the B . subtilis RNA polymerase, resulting in a considerable reduction of the level of the APHII enzyme synthesized under the control of the phage promoters.

Free Radic Biol Med, 1987, 3(2), 111 - 8
Comparison of the inactivation of Bacillus subtilis transforming DNA by the potassium superoxide and xanthine-xanthine oxidase systems for generating superoxide; Ito A et al.; Potassium superoxide (KO2) and xanthine-xanthine oxidase (X-XO), which are known generating systems for the superoxide anion, have different inactivating actions on Bacillus subtilis transforming DNA in vitro . Superoxide dismutase and CuSO4 enhanced the inactivation for KO2, but not for X-XO . Mannitol, a hydroxyl radical scavenger, protected against the inactivation by X-XO, but not by KO2 . The results obtained with X-XO were consistent with the involvement of Fenton reactions, in which hydroxyl radical is the reactive species that ultimately causes damage . On the other hand, KO2-induced inactivation was partly due to the effect of H2O2 . Differences in inactivation between the KO2 and X-XO systems may result from the different rates of production of the superoxide anion.

Gene, 1987, 55(1), 95 - 103
Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis; Le Grice SF et al.; A 2.4-kb DNA fragment from the pol region of the human T-cell lymphotropic virus, encoding the protease, reverse transcriptase and a portion of the endonuclease N-terminus was stably introduced into a high-level Bacillus subtilis expression system under inducible control of the Escherichia coli lac regulatory elements . The major expression plasmid, pRTL11, contains a bacteriophage T5 promoter/lac operator element, which is controlled by lac repressor, supplied by the secondary plasmid, pBL1 . Upon IPTG induction, a 90-kDa polyprotein is synthesised and subsequently proteolytically cleaved to reveal 64-kDa and 52-kDa polypeptides . Partial purification reveals that reverse transcriptase activity co-migrates with these two polypeptides.

Gene, 1987, 51(2-3), 281 - 6
Method for blot-hybridization analysis of mRNA molecules from Bacillus subtilis; Ambulos NP Jr et al.; By modifying hybridization techniques which are currently available to analyze RNA molecules we have developed a sensitive and reproducible method for 'Northern' analysis of RNA from Bacillus subtilis . The use of a thin (1 mm) vertical 2% agarose-6% formaldehyde gel seems to allow more efficient transfer and higher resolution of RNA upon hybridization analysis than does the use of thicker horizontal slab gels . Our improved hybridization method results in greatly reduced background upon autoradiography regardless of whether or not 32P-labelled nick-translated probes or probes synthesized on M13 vectors were purified from the unincorporated radionucleotides.

Nucleic Acids Res, 1986 Dec 22, 14(24), 9989 - 99
Nucleotide sequence and organization of dnaB gene and neighbouring genes on the Bacillus subtilis chromosome; Ogasawara N et al.; A region of the Bacillus subtilis chromosome containing dnaB, a gene essential for the initiation of chromosomal replication in B . subtilis, was cloned and nucleotide sequence of some 5000bp determined . The region consists of 4 open reading frames (ORFs) possibly comprising a single transcriptional unit . Two dnaB mutations, dnaB27 and dnaB19 were located within the first ORF which would give rise to a protein of 472 amino acids . The dnaB27 mutation involves codon at the position of 122 (replacement of Asp by Asn) close to a DNA binding domain and the dnaB19 codon 379 (replacement of Ala by Thr) close to a region rich in charged amino acids which may be alpha helical . The third ORF in the same transcriptional unit would produce a hydrophobic protein which might be involved in the DNA-membrane binding function of dnaB gene . No homologous Escherichia coli genes have been found.

EMBO J, 1986 Dec 20, 5(13), 3723 - 8
Requirements for the formation of plasmid-transducing particles of Bacillus subtilis bacteriophage SPP1; Alonso JC et al.; We had previously proposed that the production of concatemeric plasmid DNA in plasmid-transducing SPP1 particles is a consequence of phage-directed rolling-circle-type replication of plasmid DNA . The production of such DNA was greatly enhanced when DNA/DNA homology was provided between phage and plasmid DNAs (facilitation of transduction) . Here we present evidence that synthesis of concatemeric plasmid DNA can proceed after phage infection under conditions non-permissive for plasmid replication . We also propose that the naturally occurring homology between plasmid and phage is sufficient to account for the frequency of transduction observed in the absence of facilitating homology . Homology of greater than 47 bp gives the maximal facilitation of plasmid transduction . Recombination is not an essential part in the synthesis of concatemeric plasmid DNA.

J Biol Chem, 1986 Dec 15, 261(35), 16565 - 70
Interactions of Bacillus subtilis RNA polymerase with subunits determining the specificity of initiation . Sigma and delta peptides can bind simultaneously to core; Hyde EI et al.; The Bacillus subtilis RNA polymerase sigma 43 subunit and the phage SP82 encoded 28-kDa peptide are responsible for the binding of RNA polymerase to early and middle SP82 promoters, respectively . The delta peptide enhances the specificity of the interaction of B . subtilis RNA polymerase with these promoters . We have used sedimentation experiments to determine the effect of each of the three specificity factors, delta, sigma, and the 28-kDa peptide, on the binding of the other two factors to RNA polymerase core and the effect of NaCl on these binding equilibria . We show that sigma 43 and the 28-kDa peptide can each bind to RNA polymerase core at the same time as delta . Sigma 43 and the 28-kDa peptide have similar affinities to core at 0.1 M NaCl, but the 28-kDa peptide binds to core-delta more strongly than sigma 43 . The implications of these findings with respect to the replacement of sigma 43 by the 28-kDa peptide and the mechanism of promoter search by B . subtilis RNA polymerase are discussed.

J Mol Biol, 1986 Dec 5, 192(3), 557 - 65
Nucleotide sequences that define promoters that are used by Bacillus subtilis sigma-29 RNA polymerase; Rather PN et al.; There are at least five different forms of RNA polymerase holoenzyme in Bacillus subtilis . These enzymes differ in their sigma subunit and their specificity for promoter utilization . One form of RNA polymerase (E sigma 29) that contains a 29,000 Mr sigma appears in B . subtilis about two hours after the initiation of endospore formation . The determination of the nucleotide sequences that govern utilization of promoters by E sigma 29 has been limited by the small number of cloned promoters that are recognized by E sigma 29 . We have determined the nucleotide sequence of a recently isolated promoter (G4) that is used exclusively by E sigma 29 both in vitro and in vivo . The start-point of transcription was identified by S1 nuclease mapping and dinucleotide priming experiments and the probable promoter element was sequenced . We compared the sequence with that of six promoters that are used to varying degrees in vitro by E sigma 29 and found these sequences to be highly conserved at the -10 and near the -35 regions of these promoters . Single base substitutions were generated at positions -12, -15 and -36 of the G4 promoter and assayed for their influence on utilization by E sigma 29 in in-vitro competition experiments . The effects of these mutations in G4 on its use by E sigma 29 support a model in which E sigma 29 utilizes its cognate promoters by interacting with unique nucleotide sequences at the -10 region and near the -35 region of these promoters.

J Gen Microbiol, 1986 Dec, 132 ( Pt 12), 3451 - 7
Biophysics of pole formation of gram-positive rods; Koch AL et al.; During pole formation in Bacillus subtilis the inner and outer surfaces of the nascent pole are enlarged by almost exactly the same extent . This means that the stress is almost uniformly distributed throughout the polar wall . This differs from the situation in the cylindrical side wall, where most of the stress is exerted in the outer portions of the intact wall . Because the stress is shared more uniformly, the maximum strain in any part of the polar wall is reduced, compared with the maximum strain within the side wall . The lowered stress may account, in part, for the resistance of the polar wall to hydrolysis by autolytic enzymes under certain conditions . The shape of the newly completed pole is significantly different from the spherical shape that the hydrostatic pressure would tend to produce . It does, however, achieve the shape that maximizes the polar volume under the restrictions arising due to expansion along the circumference not being possible near the junction of cylindrical and polar wall.

J Gen Microbiol, 1986 Dec, 132 ( Pt 12), 3441 - 9
Normal pole formation during total inhibition of wall synthesis of Bacillus subtilis; Koch AL et al.; Previous work has shown that the side wall of a Gram-positive rod is initially laid down as a compact layer inside the older wall . It is then stretched as it comes to bear tension due to the osmotic pressure inside the cell . If the polar wall is likewise capable of a degree of expansion, then no new murein need be added while the planar cross-wall splits and converts into two poles . In Bacillus subtilis mutant strain FJ6, which is deficient in autolytic enzymes, pole formation can be caused by addition of exogenous muramidase (10 micrograms hen egg white lysozyme ml-1 for 10 min at 35 degrees C) . This strain grows as long filaments with many completed cross-walls, but enzymic treatment caused the formation of many new poles of normal morphology as judged by thin section electron microscopy . Fully separated poles of normal appearance were also found when more than 100 times the MIC (1 microgram ml-1) of vancomycin was added to block wall growth totally and rapidly 10 min before the addition of lysozyme . We conclude, therefore, that no new murein is needed in the conversion of the flat septum into poles and that the unstressed cross-wall is capable of the necessary expansion.

Chemioterapia, 1986 Dec, 5(6), 408 - 10
Effects of an adjunctive treatment with Bacillus subtilis for food allergy; Ciprandi G et al.; The authors evaluated the clinical efficacy of an adjunctive treatment with spores of Bacillus subtilis in 20 adult patients with urticaria-angioedema syndrome from food allergy . The patients treated with B . subtilis showed a significant reduction in frequency and severity of clinical features in respect to the patients who received no treatment . Bacillus subtilis spores may increase S-IgA synthesis or protect gastroenteric mucosa.

Chemioterapia, 1986 Dec, 5(6), 404 - 7
In vitro effects of Bacillus subtilis on the immune response; Ciprandi G et al.; The authors evaluated the in vitro effects of Bacillus subtilis on the following parameters of the immune response: mitogenic T cell proliferation (by PHA and OKT3) and mitogenic-induced lymphokine production (IL-2 and IFN-gamma) . The spores of Bacillus subtilis did not influence the immune response, while its vegetative forms enhanced mitogenic-induced T cell proliferation . Both spores and vegetative forms did not modify lymphokine production.

Proc Natl Acad Sci U S A, 1986 Dec, 83(24), 9438 - 42
New RNA polymerase sigma factor under spo0 control in Bacillus subtilis; Carter HL 3rd et al.; In Bacillus subtilis transcription of spoVG is activated within minutes after the initiation of sporulation . Mutations in several spo0 genes prevent the activation of spoVG transcription . We have found a sigma-like protein that is capable of directing core RNA polymerase to use the spoVG promoter in an in vitro run-off transcription assay . This sigma-like protein was not found to be associated with RNA polymerase in a spo0A or spo0B mutant but was present in a spo0H mutant . We suggest that one role of the spo0A gene product in transcription of spoVG is the modulation of RNA polymerase activity by this sigma-like protein.

J Bacteriol, 1986 Dec, 168(3), 1243 - 9
Characterization of heat shock in Bacillus subtilis; Arnosti DN et al.; We characterized the general properties of the heat shock response in Bacillus subtilis W168, B . subtilis JH642, and an spo0A mutant by using pulse-labeling of bacterial proteins and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The transfer of cells from 37 to 50 degrees C repressed synthesis of most cellular proteins and led to the induction of at least 26 distinct heat shock proteins after about 3 min . Ethanol (4% {vol/vol}) induced a similar set of proteins, but somewhat more slowly . Synthesis of the majority of heat shock proteins at 50 degrees C returned to a steady-state level 20 to 40 min after the shock . Although no B . subtilis heat shock protein has yet been extensively characterized, three of these proteins were found to be immunologically related to the Escherichia coli heat shock proteins Dnak, Lon, and GroEL . Synthesis of both sigma 28 and sigma 43 proteins was sharply reduced during heat shock . Although a spo0A amber mutation blocks transcription from promoters used by at least two minor B . subtilis sigma factors, it did not alter the kinetics or general properties of the heat shock response.

J Bacteriol, 1986 Dec, 168(3), 1133 - 41
Translational autoregulation of ermC 23S rRNA methyltransferase expression in Bacillus subtilis; Denoya CD et al.; ermC specifies an rRNA methyltransferase that confers resistance to erythromycin . The expression of this determinant is induced by the addition of erythromycin . The induction mechanism has been shown to operate posttranscriptionally, and its mechanism has been elucidated . We now show that synthesis of the ermC gene product in Bacillus subtilis is also autoregulated by a mechanism operating on the level of translation . The synthesis of methyltransferase was shown to be gene dosage compensated by Western blot analysis . Several mutants were analyzed that specify altered ermC gene products and are deregulated . Analysis of mutants and of the wild-type strain by Northern blotting demonstrated that autoregulation is posttranscriptional . We suggest a translational repression model in which the ermC methyltransferase binds to its own mRNA, at a region that resembles the methylation target site on 23S rRNA . The overall control of ermC expression is discussed in light of these multiple regulatory mechanisms.

J Bacteriol, 1986 Dec, 168(3), 1128 - 32
Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor; Himeno T et al.; Bacillus licheniformis penicillinase genes, penP and penI, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T . Imanaka, T . Tanaka, H . Tsunekawa, and S . Aiba, J . Bacteriol . 147:776-186, 1981) . The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis . B . subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized . Of about 150,000 colonies, two penI(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48 degrees C for penicillinase . By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment . The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced . A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found . Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB60E24), the coding region was concluded to be the penI gene . A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG) . A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction . A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penI promoter . The regulation of penP and penI by the repressor is discussed.

Eur J Biochem, 1986 Dec 1, 161(2), 479 - 89
Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23; Yokoyama K et al.; The stepwise formation and characterization of linkage unit intermediates and their functions in ribitol teichoic acid biosynthesis were studied with membranes obtained from Staphylococcus aureus H and Bacillus subtilis W23 . The formation of labeled polymer from CDP-{14C}ribitol and CDP-glycerol in each membrane system was markedly stimulated by the addition of N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine (ManNAc-GlcNAc) linked to pyrophosphorylyisoprenol . Whereas incubation of S . aureus membranes with CDP-glycerol and ManNAc-{14C}GlcNAc-PP-prenol led to synthesis of (glycerol phosphate) 1-3-ManNAc-{14C}GlcNAc-PP-prenol, incubation of B . subtilis membranes with the same substrates yielded (glycerol phosphate)1-2-ManNAc-{14C}GlcNAc-PP-prenol . In S . aureus membranes, (glycerol phosphate)2-ManNAc-{14C}GlcNAc-PP-prenol as well as (glycerol phosphate)3-ManNAc-{14C}GlcNAc-PP-prenol served as an acceptor for ribitol phosphate units, but (glycerol phosphate)-ManNAc-{14C}GlcNAc-PP-prenol did not . In B . subtilis W23 membranes, (glycerol phosphate)-ManNAc-{14C}GlcNAc-PP-prenol served as a better acceptor for ribitol phosphate units than (glycerol phosphate)2-ManNAc-{14C}GlcNAc-PP-prenol . In this membrane system (ribitol phosphate)-(glycerol phosphate)-ManNAc-{14C}GlcNAc-PP-prenol was formed from ManNAc-{14C}GlcNAc-PP-prenol, CDP-glycerol and CDP-ribitol . The results indicate that (glycerol phosphate)1-3-ManNAc-GlcNAc-PP-prenol and (glycerol phosphate)1-2-ManNac-GlcNAc-PP-prenol are involved in the pathway for the synthesis of wall ribitol teichoic acids in S . aureus H and B . subtilis W23 respectively.

Genetika, 1986 Dec, 22(12), 2750 - 7
{RecE-independent recombination of plasmids in Bacillus subtilis cells}; Bashkirov VI et al.; The pUB110 and pE194 plasmid cointegrates have been isolated and examined in rec+ and recE4 strains of Bacillus subtilis . Cointegrates were shown to be formed by recombination at the specific site present on both parental plasmids as a short region of homology designated RSA . The RSA consists of 63 nucleotides in pE194 and 49 in pUB110; the length of its fully conserved core segment is 10 nucleotides . All cointegrates examined were formed by single crossover event taking place within the core segment, and as a result they have identical nucleotide sequences of recombination junctions . No conversion of mismatched base pairs to nucleotide sequences originally belonging to one of the parental plasmids was found . Though the action of RecE gene did not affect the frequency of cointegrate formation, it was reduced in rec149 host by one order of magnitude . Cointegrates retained their stability during transformation.

J Virol, 1986 Dec, 60(3), 874 - 9
In vivo transcription of bacteriophage phi 29 DNA: transcription initiation sites; Barthelemy I et al.; The initiation sites of the RNA transcripts synthesized in vivo in Bacillus subtilis infected with bacteriophage phi 29 have been mapped by S1 protection experiments . Nine transcription initiation sites were localized along the entire phi 29 genome, close to previously reported B . subtilis and Escherichia coli RNA polymerase-binding sites . Eight of these sites corresponded to early transcription and only one corresponded to late transcription . By using 5'-end-labeled RNA, four of the early sites and the late one were shown to be the main sites where initiation of transcription occurs in vivo in the phi 29 genome.

Genetika, 1986 Nov, 22(11), 2583 - 92
{Substitution of RNA-polymerase sigma-subunits and transcription regulation}; Zograf IuN; Recent data on regulation of gene activity in bacteria by substitution of RNA polymerase sigma subunits are reviewed . The htpR gene which controls the switch-on of the Escherichia coli heat-shock protein synthesis codes for sigma 32 subunit . sigma 32-containing RNA polymerase transcribes the heat-shock genes in vitro from specific promoters of no use for RNA polymerase containing the major sigma 70 subunit . Several minor sigma subunits have been found in Bacillus subtilis vegetative cells, in addition to the major sigma 55 subunit, differing in the specificity of promoter recognition . Many B . subtilis genes are controlled by tandemly located promoters recognized by RNA polymerases carrying different sigma subunits . sigma 29 subunit is encoded by spoIIG gene and is probably involved in the regulation of sporulation . Specific sigma subunits for transcribing "middle" or "late" genes are encoded by a number of phages.

J Gen Microbiol, 1986 Nov, 132 ( Pt 11), 3025 - 35
Cloning and sequencing of a gene from Bacillus amyloliquefaciens that complements mutations of the sporulation gene spoIID in Bacillus subtilis; Turner SM et al.; A segment of DNA from Bacillus amyloliquefaciens, which complemented a mutant sporulation gene, spoIID68, in Bacillus subtilis, was cloned into a derivative of the temperate bacteriophage phi 105 . The segment of DNA included an entire structural gene and complemented the mutation spoIID298, in addition to spoIID68, in B . subtilis . The nucleotide sequence of the gene from B . amyloliquefaciens was determined and compared with that of the B . subtilis gene; 74% homology was found in the coding region . Amino acid primary sequences derived from the nucleotide sequences of the two genes were also compared . The gene from B . amyloliquefaciens coded for a protein of 344 amino acid residues, one more than the protein coded by the corresponding gene from B . subtilis . Comparison of the primary amino acid sequences of the two genes showed that 78% of the residues were completely conserved and 8% were semi-conserved . Variation, however, was not random, i.e . some segments were much more highly conserved than others . Both proteins had a hydrophobic region at the N-terminus.

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