Bacillus subtilis

Mol Microbiol, 1993 Mar, 7(5), 705 - 17
Initiation of mRNA decay in Bacillus subtilis; DiMari JF et al.; Ribosome stalling in the leader region of ermC mRNA results in a 10-15-fold increase in ermC mRNA half-life in Bacillus subtilis . Fusion of the ermC 5' regulatory region to several B . subtilis coding sequences resulted in induced stability of the fusion RNAs, showing that the ermC 5' region acts as a general '5' stabilizer' . RNA products of an ermC-lacZ transcriptional fusion were inducibly stable in the complete absence of translation and included a small RNA that is likely to be a decay product arising by blockage of a 3'-to-5' exoribonuclease activity . Insertion of sequences that encode endonucleolytic cleavage sites into the ermC coding sequence resulted in cleavage products whose stability depended on the nature of their 5' and 3' ends . It can be concluded from this study that initiation of mRNA decay in B . subtilis generally occurs at or near the 5' terminus.

Mol Microbiol, 1993 Mar, 7(5), 631 - 6
Regulation of peptide antibiotic production in Bacillus; Marahiel MA et al.; In Bacillus species, starvation leads to the activation of a number of processes that affect the ability to survive during periods of nutritional stress . Activities that are induced include the development of genetic competence, sporulation, the synthesis of degradative enzymes, motility, and antibiotic production . The genes that function in these processes are activated during the transition from exponential to stationary phase and are controlled by mechanisms that operate primarily at the level of transcription initiation . One class of genes functions in the synthesis of special metabolites such as the peptide antibiotics tyrocidine and gramicidin S as well as the cyclic lipopeptide surfactin . These genes include the grs and tyc operons in Bacillus brevis, which encode gramicidin S synthetase and tyrocidine synthetase, respectively, and the srfA operon of Bacillus subtilis which encodes the enzymes of the surfactin synthetase complex . Peptide antibiotic biosynthesis genes are regulated by factors as diverse as the early sporulation gene product Spo0A, the transition-state regulator AbrB, and gene products (ComA, ComP, and ComQ) required for the initiation of the competence developmental pathway.

Nucleic Acids Res, 1993 Feb 25, 21(4), 935 - 40
The main early and late promoters of Bacillus subtilis phage phi 29 form unstable open complexes with sigma A-RNA polymerase that are stabilized by DNA supercoiling; Rojo F et al.; Most Escherichia coli promoters studied so far form stable open complexes with sigma 70-RNA polymerase which have relatively long half-lives and, therefore, are resistant to a competitor challenge . A few exceptions are nevertheless known . The analysis of a number of promoters in Bacillus subtilis has suggested that the instability of open complexes formed by the vegetative sigma A-RNA polymerase may be a more general phenomenon than in Escherichia coli . We show that the main early and late promoters from the Bacillus subtilis phage phi 29 form unstable open complexes that are stabilized either by the formation of the first phosphodiester bond between the initiating nucleoside triphosphates or by DNA supercoiling . The functional characteristics of these two strong promoters suggest that they are not optimized for a tight and stable RNA polymerase binding . Their high activity is probably the consequence of the efficiency of further steps leading to the formation of an elongation complex.

J Biol Chem, 1993 Feb 25, 268(6), 4504 - 10
Lysine 106 of the putative catalytic ATP-binding site of the Bacillus subtilis SecA protein is required for functional complementation of Escherichia coli secA mutants in vivo; Klose M et al.; The SecA protein is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane . The secA gene from Bacillus subtilis was cloned and expressed in E . coli under the control of the lac or trc promoter . The temperature-sensitive growth and secretion defects of various E . coli secA mutants were complemented by the B . subtilis SecA protein, provided the protein was expressed at moderate levels . Under overproduction conditions, no complementation was observed . One of the main features of the SecA protein is the translocation ATPase activity which, together with the protonmotive force, drives the movement of proteins across the plasma membrane . A putative ATP-binding motif can be identified in the SecA protein resembling the consensus Walker A type motif . Replacement of a lysine residue at position 106, which corresponds to an invariable amino acid residue, in the consensus motif by asparagine (K106N) resulted in the loss of the ability of the B . subtilis SecA protein to complement the growth and secretion defects of E . coli secA mutants . In addition, the presence of the K106N SecA protein interfered with protein translocation, most likely at an ATP-requiring step . We conclude that lysine 106 is part of the catalytic ATP-binding site of the B . subtilis SecA protein, which is required for protein translocation in vivo.

Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 101 - 7
Purification and characterization of the HU-like protein HPB9 from the Bacillus subtilis nucleoid; Le Hegarat F et al.; The Bacillus subtilis HPB9 is the major heat-stable and acid-soluble protein associated with the nucleoid isolated at low ionic strength . The abundance of the protein in the cell is estimated to about 20,000 monomers per cell (Salti et al . (1985) J . Gen . Microbiol . 131, 581-590) . The protein cross reacts specifically with the antiserum against the Bacillus globigii HBg . Moreover, HPB9 is able to introduce negative supercoiling in a relaxed covalently closed circular DNA, in the presence of topoisomerase I as demonstrated by one and two-dimensional electrophoresis . These results indicate that the nucleoid associated protein HPB9 is an HU-like protein and could be involved in the DNA compaction.

FEMS Microbiol Lett, 1993 Feb 15, 107(1), 121 - 5
Heme b (protoheme IX) is a precursor of heme a and heme d in Bacillus subtilis; Hansson M et al.; Bacillus subtilis can synthesise cytochromes containing a-, b-, c- and d-type heme . The biosynthetic pathways of these heme prosthetic groups were investigated by using strains blocked in uroporphyrinogen III synthesis from porphobilinogen or in heme b (protoheme IX) synthesis from uroporphyrinogen III . The results strongly suggest that heme a and heme d are both synthesised from heme b (protoheme IX) . They also indicate that B . subtilis contains a novel ferrochelatase involved in the synthesis of siroheme.

Arch Biochem Biophys, 1993 Feb 15, 301(1), 129 - 37
Kinetic and ligand binding evidence for two heme A-based terminal oxidases in plasma membranes from Bacillus subtilis; Hill BC et al.; Detergent-solubilized plasma membranes from Bacillus subtilis have been characterized for their cytochrome oxidase content . Triton X-100-solubilized membranes show high O2 turnover with ascorbate plus TMPD . Reduced-oxidized difference spectroscopy of ascorbate-TMPD-reduced membranes reveals the presence of cytochrome c and cytochrome a . An additional, b-type cytochrome appears when the membranes are reduced with dithionite . Time-resolved difference spectra taken during reduction by ascorbate-TMPD reveal two kinetic forms of heme A-containing cytochromes . There is a high-turnover form that is rapidly reduced upon anaerobiosis, and a second type which is only slowly reduced upon anaerobiosis . The slowly reduced oxidase is distinguished by an alpha-band blue-shifted to 600 nm relative to the 603-nm position observed for high-turnover oxidase . Addition of CO to ascorbate-TMPD-reduced membranes gives a spectrum typical of ferrocytochrome a3-CO, and the intensity corresponds to the total ferrocytochrome a3 concentration . Photolysis of ascorbate-TMPD-reduced, CO-bound membranes indicates that both species are photosensitive with similar rates of recombination . Addition of CO to dithionite-reduced membranes shows an additional CO reactive center that has a spectrum characteristic of cytochrome o . Cyanide blocks complete reduction of high-turnover oxidase by ascorbate plus TMPD, but does not appear to effect slowly reduced oxidase . These results indicate the presence of two different types of cytochrome aa3 oxidase in plasma membranes of B . subtilis.

Gene, 1993 Feb 14, 124(1), 99 - 103
Synthesis of the Bacillus subtilis histone-like DNA-binding protein HBsu in Escherichia coli and secretion into the periplasm; Groch N et al.; A synthetic gene encoding the histone-like DNA-binding protein, HBsu, of Bacillus subtilis was cloned in-frame behind the coding region of the OmpA signal peptide of Escherichia coli . The gene encoding the fusion protein is under control of both the lpp promoter and the lac promoter-operator . Upon induction of gene expression, mature HBsu is secreted into the periplasm . The OmpA signal peptide is correctly removed, resulting in the production of authentic-length HBsu protein . The observed in vitro DNA-binding ability is taken as evidence for the correct folding and assembly of homodimeric HBsu protein . A normally intracellular protein can thus be secreted from E . coli in high yield and with full functionality . By analogy, every histone-like protein or mutant forms thereof may be produced heterologously in E . coli and may be purified without being contaminated by the homologous E . coli HU protein.

Biochim Biophys Acta, 1993 Feb 13, 1156(2), 173 - 80
Anomalies in cell wall turnover associated with the growth temperature of Bacillus subtilis; Wu TL et al.; Cell wall turnover appeared to be anomalously fast in Bacillus subtilis when the cells were grown at temperatures below 29 degrees C . Turnover rates k(generation-1), of exponential cultures at 25 degrees were approximately double those of cells grown at 37 degrees C . When autolysin levels were assayed in cell walls, it was found that the enzyme activities were constant between 25 degrees C and 40 degrees C, suggesting that there was no greater synthesis of autolysin at the lower temperature . Analyses of walls for individual components, extent of aminosugar substitution and extent of crosslinking, did not reveal significant differences between samples obtained from 25 degrees C or 37 degrees C cultures . The N-acetylmuramoyl-L-alanine amidase was stable over the temperature range studied . Lysis of cells, induced by carbonylcyanide-m-chlorophenylhydrazone, occurred at a faster rate for cells obtained at 25 degrees C than for cells obtained at 37 degrees C . In addition, the lysis of cells by hen egg white lysozyme was slightly faster when the cells were obtained from 25 degrees C cultures than from 37 degrees C cultures . It is possible the autolysin(s) responsible for cell wall turnover are cold-activated.

Hum Mol Genet, 1993 Feb, 2(2), 97 - 106
Cloning of the X-linked glycerol kinase deficiency gene and its identification by sequence comparison to the Bacillus subtilis homologue; Sargent CA et al.; cDNA clones from a human adult testis cDNA library were isolated and sequenced as part of a programme to produce expressed sequence tags (ESTs) . ESTs were used routinely to search DNA and protein sequence databases . One clone (142) showed 60% identity to the Bacillus subtilis glycerol kinase gene at both the DNA and amino acid sequence levels . Analysis of DNA from somatic cell hybrids carrying deleted X chromosomes, has shown that clone 142 detects homologous sequences between Xp21.2-p22.1 (the interval containing the locus responsible for glycerol kinase deficiency--GKD) . These sequences are deleted in two patients with GKD . Clone 142 also detects homologous sequences on Xq and at several autosomal loci . The sequences of clone 142 and two further cDNA clones isolated from a human foetal brain cDNA library are presented.

Int J Immunopharmacol, 1993 Feb, 15(2), 87 - 92
Expression of activation markers on peripheral-blood lymphocytes following oral administration of Bacillus subtilis spores; Caruso A et al.; This study was undertaken to assess the capability of Bacillus subtilis spores to modify the peripheral-blood lymphocyte (PBL) subsets or determine the de novo expression of activation markers . The data we obtained show that spores of B . subtilis are able to increase the expression of certain cell activation markers and that such activation is dose-dependent . In fact, doses of 2 x 10(9) spores did not give rise to changes in any of the parameters evaluated, while doses of 6 x 10(9) increased the HLA-DR antigen expression on T-lymphocytes . At the highest dosage used (12 x 10(9), B . subtilis spores caused the appearance of cells bearing the CD25 and CD71 activation markers . Therefore, such cell activation markers may prove useful for monitoring the activity of B . subtilis spores, and possibly of other immunomodulating agents, in the course of clinical research.

Yeast, 1993 Feb, 9(2), 189 - 99
The complete sequence of a 19,482 bp segment located on the right arm of chromosome II from Saccharomyces cerevisiae; Doignon F et al.; We report here the sequence of a 19,482 bp DNA segment of chromosome II of Saccharomyces cerevisiae . The fragment contains 16 open reading frames (ORFs) covering 74% of the sequence . Four predicted products present homology with known proteins . The ORF YBR1732 exhibits a strong homology to serine hydroxymethyl transferase; the best score is 53.1% identity in 458 amino acids overlap with the serine hydroxymethyl transferase from rabbit liver . YBR1724, which shows homology with riboflavin synthase of Bacillus subtilis, is probably the RIB5 gene implied in riboflavine synthesis and mapped in this region . YBR1733 is homologous to rab protein and YBR1728 is presumably a GTPase activating protein.

Mol Microbiol, 1993 Feb, 7(4), 611 - 21
Characterization of mutations in divIB of Bacillus subtilis and cellular localization of the DivIB protein; Harry EJ et al.; Four temperature-sensitive mutations in the divIB gene of Bacillus subtilis have been localized to the region corresponding to the C-terminal half of the 263-residue DivIB protein . Antiserum was raised to the 80% C-terminal portion lying on one side of a putative transmembrane (hydrophobic) segment, and used to examine aspects of the nature and localization of the DivIB protein in the cell . A single DivIB species of a size equal to the full-length protein encoded by the divIB gene was detected in wild-type cells . Cell fractionation studies established that DivIB is associated preferentially with the cell envelope (membrane plus cell wall), with approximately 50% being released into solution upon treatment of cells with lysozyme under conditions that yield protoplasts . Of the remaining 50%, approximately half remained firmly associated with the membrane fraction . On the basis of the 'positive-inside rule' of von Heijne (1986) it is suggested that the topology of membrane-bound DivIB is such that the long C-terminal portion is directed to the outside and the smaller N-terminal portion to the inside of the cell . DivIB in protoplasts was rapidly degraded by proteinase K under conditions where there was no general proteolysis of the cytoplasmic proteins . This is consistent with its absence from the cytoplasm, and with the predicted membrane topology . Septum positioning in a divIB null mutant, which grows as filaments at temperatures of 30 degrees C and below, was found to be normal . It appears that DivIB is needed for achieving the appropriate rate of initiation of septum formation at normal division sites . It is proposed that the C-terminal portion of DivIB, localized on the exterior surface of the membrane and in juxtaposition to the peptidoglycan, normally interacts with another protein (or proteins) to initiate septum formation.

Mol Microbiol, 1993 Feb, 7(4), 601 - 10
The minCD locus of Bacillus subtilis lacks the minE determinant that provides topological specificity to cell division; Lee S et al.; A key event of the sporulation process in Bacillus subtilis is the asymmetric cell division that divides the developing cell into two unequal compartments . To examine the function of vegetative cell division genes in this developmental division, we isolated and characterized the B . subtilis counterpart to the Escherichia coli minicell operon minB, which governs correct placement of the division septum . Starting from the closely linked spoIVF locus, we used walking methods to isolate the region of the B . subtilis chromosome proximate to the divIVB minicell locus . DNA sequence analysis found two open reading frames whose predicted products had significant identity to the E . coli MinC cell division inhibitor and the MinD ATPase activator of MinC, and disruption of minCD function generated a minicell phenotype in B . subtilis . Notably, no homologue to the E . coli MinE topological specificity element was found in the B . subtilis minCD region . The B . subtilis min genes were part of an operon transcribed from a major promoter more than 2.5 kb upstream from minC . An internal promoter immediately upstream from minC was dependent on RNA polymerase containing sigma-H and was active at the onset of sporulation . However, neither minC nor minD function was absolutely required for sporulation and, by implication, for asymmetric septum formation.

Mol Microbiol, 1993 Feb, 7(3), 337 - 42
Transition-state regulators: sentinels of Bacillus subtilis post-exponential gene expression; Strauch MA et al.; When Bacillus subtilis encounters a nutrient-depleted environment, it expresses a wide variety of genes that encode functions in alternative pathways of metabolism and energy production . Expression of these genes first occurs during the transition from active growth into stationary phase and is controlled by a class of proteins termed transition-state regulators . In several instances, a given gene is redundantly controlled by two or more of these regulators and many of these regulators control genes in numerous different pathways . The AbrB, Hpr and Sin proteins are the best-studied examples of these regulatory molecules . Their role is to prevent inappropriate and possibly detrimental functions from being expressed during exponential growth when they are not needed . They serve as elements integrating sporulation with ancillary stationary-phase phenomena and appear to participate in the timing of early sporulation events and in fine-tuning the magnitude of gene expression in response to specific environmental conditions.

FEMS Microbiol Lett, 1993 Feb 1, 106(3), 287 - 93
Cloning and characterization of heat-inducible promoters of Bacillus subtilis; Volker U et al.; Heat-inducible DNA fragments of Bacillus subtilis were cloned with two different promoter probe vectors . The increased synthesis of the reporter enzymes seemed to be due to a transient increase in the transcription of the encoding genes . The structure of the heat-sensitive promoters resembles the consensus sequence of promoters recognized by the vegetative form of RNA polymerase of B . subtilis . Our results support data in literature that the heat shock response of B . subtilis is regulated by a different mechanism than in Escherichia coli, where alternative sigma factors direct the transcription of heat shock genes.

J Appl Bacteriol, 1993 Feb, 74(2), 119 - 26
The production of antifungal volatiles by Bacillus subtilis; Fiddaman PJ et al.; A strain of Bacillus subtilis which produces an antibiotic metabolite was also found to produce a volatile compound(s) which was antifungal to Rhizoctonia solani and Pythium ultimum . Growth of the fungi was severely impaired in the presence of the volatiles and physiological abnormalities of the hyphae were observed, including hyphal distortion and vacuolation . A range of media were tested for volatile production and potato dextrose agar (PDA) was found to be the most active . Temperature had a considerable effect on antifungal volatile activity with the greatest inhibition occurring at 30 degrees C . Addition of iron (III) chloride to Sabouraud's glucose agar (SGA) also enhanced the antifungal effect . The volatiles were found to be water soluble and remained active when trapped in SGA.

J Gen Microbiol, 1993 Feb, 139 ( Pt 2), 361 - 70
DNA sequence of the murE-murD region of Bacillus subtilis 168; Daniel RA et al.; The sequence of a 4.4 kbp region of DNA from Bacillus subtilis 168, lying between sporulation genes spoVD and spoVE, has been determined as part of the B . subtilis genomic sequencing programme . The region contains three genes with high sequence similarity to the murE, mraY and murD genes of Escherichia coli . The products of these genes are likely to catalyse various steps in the formation of the precursors for peptidoglycan synthesis in B . subtilis . The regions at 133 degrees on the standard genetic map of the B . subtilis chromosome, and in the 2 min region of the E . coli genetic map, are now shown to contain a large cluster of functionally related genes . Although the linear order of the genes in the cluster is conserved, three genes that are present in the E . coli chromosome, and which are likely to be essential for peptidoglycan synthesis in both organisms, are absent from this region of the B . subtilis chromosome . In general, the B . subtilis cluster differs from that of E . coli in having more extensive intergenic regions, with less potential for translational coupling.

J Gen Microbiol, 1993 Feb, 139 ( Pt 2), 349 - 59
The glpP and glpF genes of the glycerol regulon in Bacillus subtilis; Beijer L et al.; The Bacillus subtilis glpPFKD region contains genes essential for growth on glycerol or glycerol 3-phosphate (G3P) . The nucleotide sequence of glpP encoding a regulatory protein and the previously unidentified glpF encoding the glycerol uptake facilitator was determined . glpF is located immediately upstream of glpK and the two genes were shown to constitute one operon which is transcribed separately from glpP . A sigma A-type promoter and the transcriptional start point for glpFK were identified . In the 5' untranslated leader sequence (UTL) of glpFK mRNA a conserved inverted repeat is found . The repeat is believed to be involved in the control of expression of glpFK by termination/antitermination of transcription, a control mechanism previously suggested for the regulation of glpD encoding G3P dehydrogenase . Expression of glpFK and glpD requires the inducer G3P and the glpP gene product . A 2.9 kb B . subtilis chromosomal DNA fragment containing the glpP open reading frame was cloned to give plasmid pLUM7 . pLUM7 contains a functional glpP gene as shown by its ability to complement various glpP mutants . Immediately upstream of glpP an open reading frame is found (ORF1) . Disrupting ORF1 by plasmid integration in the B . subtilis chromosome does not affect the ability to grow on glycerol as sole carbon and energy source . With the present report all B . subtilis glp genes located at 75 degrees on the chromosomal map have been identified.

J Gen Microbiol, 1993 Feb, 139 ( Pt 2), 343 - 7
Expression of Bacillus subtilis neutral protease gene (nprE) in Saccharomyces cerevisiae; Wang LF et al.; Expression in the yeast Saccharomyces cerevisiae of the intact nprE gene of Bacillus subtilis, which encodes the pre-pro-NprE neutral protease precursor, resulted in intracellular accumulation of unprocessed precursor without detectable secretion or processing of the expressed gene product . When sequences specifying the signal peptide of yeast invertase were fused upstream of sequences encoding the mature NprE enzyme, nprE gene products were secreted into the culture medium . The secreted protein products were, however, highly, glycosylated and biologically inactive.

Genes Dev, 1993 Feb, 7(2), 283 - 94
Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor; Ireton K et al.; Multiple physiological and environmental signals are needed to initiate endospore formation in Bacillus subtilis . One key event controlling sporulation is activation of the Spo0A transcription factor . Spo0A is a member of a large family of conserved regulatory proteins whose activity is controlled by phosphorylation . We have isolated deletion mutations that remove part of the conserved amino terminus of Spo0A and make the transcription factor constitutively active, indicating that the amino terminus normally functions to keep the protein in an inactive state . Expression of an activated gene product is sufficient to activate expression of several sporulation genes in the absence of signals normally needed for initiation of sporulation . Our results indicate that nutritional, cell density, and cell-cycle signals are integrated through the phosphorylation pathway that controls activation of Spo0A.

Orig Life Evol Biosph, 1993 Feb, 23(1), 37 - 52
Responses of Bacillus subtilis spores to space environment: results from experiments in space; Horneck G; Onboard of several spacecrafts (Apollo 16, Spacelab 1, LDEF), spores of Bacillus subtilis were exposed to selected parameters of space, such as space vacuum, different spectral ranges of solar UV-radiation and cosmic rays, applied separately or in combination, and we have studied their survival and genetic changes after retrieval . The spores survive extended periods of time in space--up to several years--, if protected against the high influx of solar UV-radiation . Water desorption caused by the space vacuum leads to structural changes of the DNA; the consequences are an increased mutation frequency and altered photobiological properties of the spores . UV-effects, such as killing and mutagenesis, are augmented, if the spores are in space vacuum during irradiation . Vacuum-specific photoproducts which are different from the 'spore photoproduct' may cause the synergistic response of spores to the simultaneous action of UV and vacuum . The experiments provide an experimental test of certain steps of the panspermia hypothesis.

J Bacteriol, 1993 Feb, 175(4), 1038 - 42
The missing link in phage lysis of gram-positive bacteria: gene 14 of Bacillus subtilis phage phi 29 encodes the functional homolog of lambda S protein; Steiner M et al.; In most bacteriophages of gram-negative bacteria, the phage endolysin is released to its murein substrate through a lesion in the inner membrane . The lesion is brought about by a second phage-encoded lysis function . For the first time, we present evidence that the same strategy is elaborated by a phage of a gram-positive bacterium . Thus, there appears to be an evolutionarily conserved lysis pathway for most phages whether their host bacterium is gram negative or gram positive . Phage phi 29 gene 14, the product of which is required for efficient lysis of Bacillus subtilis, was cloned in Escherichia coli . Production of protein 14 in E . coli resulted in cell death, whereas production of protein 14 concomitantly with the phi 29 lysozyme or unrelated murein-degrading enzymes led to lysis, suggesting that membrane-bound protein 14 induces a nonspecific lesion in the cytoplasmic membrane.

J Bacteriol, 1993 Feb, 175(3), 741 - 9
Stability and asymmetric replication of the Bacillus subtilis 168 chromosome structure; Itaya M; Chromosomal DNAs from a number of strains derived from Bacillus subtilis 168 were digested with restriction endonucleases NotI or SfiI, and the locations of chromosomal alterations were compared with the recently constructed standard NotI-SfiI restriction map (M . Itaya and T . Tanaka, J . Mol . Biol . 220:631-648, 1991) . In general, the chromosome structure of B . subtilis 168 was found to be stable, as expected from the genetic stability of this species . DNA alterations, typically deletions, are formed in three limited loci on the chromosome . One of these alterations was characterized as a spontaneous deletion formed between rrn operons, and another occurred as a result of prophage SP beta excision . I found that oriC and terC are not located on precisely opposite sides of the chromosome . Replication in the counter clockwise direction was 196 kb longer than replication in the clockwise direction . The characteristic of length difference is not changed by deletion formation.

J Bacteriol, 1993 Feb, 175(3), 647 - 54
Identification of a putative Bacillus subtilis rho gene; Quirk PG et al.; Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product . The insertional site was near the beginning of the open reading frame, which was located in a region of the B . subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented . The predicted B . subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628 . The growth of the mutant strain was less than that of the wild type on defined medium at 30 degrees C . On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30 degrees C . On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30 degrees C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30 degrees C . To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B . subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.

J Bacteriol, 1993 Feb, 175(3), 892 - 7
Altered regulation of the glnRA operon in a Bacillus subtilis mutant that produces methionine sulfoximine-tolerant glutamine synthetase; Schreier HJ et al.; A Bacillus subtilis mutant that produced glutamine synthetase (GS) with altered sensitivity to DL-methionine sulfoximine was isolated . The mutation, designated glnA33, was due to a T.A-to-C.G transition, changing valine to alanine at codon 190 within the active-site C domain . Altered regulation was observed for GS activity and antigen and mRNA levels in a B . subtilis glnA33 strain . The mutant enzyme was 28-fold less sensitive to DL-methionine sulfoximine and had a 13.0-fold-higher Km for hydroxylamine and a 4.8-fold-higher Km for glutamate than wild-type GS did.

J Bacteriol, 1993 Feb, 175(3), 795 - 801
High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation; Kuroda A et al.; Transcription of the major Bacillus subtilis autolysin gene (cwlB) was investigated . Deletion of the region upstream of the gene cluster lppX-cwbA-cwlB led to a loss of promoter activity . Primer extension analysis suggested that the cwlB operon is transcribed by E sigma D and E sigma A, the former transcripts being predominants at the exponential growth phase . Expression of the lppX-lacZ fusion gene was reduced by about 90% in a sigD-null mutant . A sin (flaD1) mutation caused a severe defect in transcription of the lppX-cwbA-cwlB operon . The sin (flaD1) mutation also reduced expression of a sigD-lacZ fusion gene constructed in the B . subtilis chromosome . Since the sigD-null mutant exhibits motility and autolysin deficiencies and filamentation, similar phenotypes in the sin (flaD1) mutant may be caused by reduction in expression of the sigma D protein.

Biosci Biotechnol Biochem, 1993 Feb, 57(2), 260 - 4
Identification of the cellulose-binding domain of a Bacillus subtilis endoglucanase distinct from its catalytic domain; Park JS et al.; The endoglucanase (BSC) from Bacillus subtilis IFO 3034, which shows no ability to hydrolyze microcrystalline cellulose, was found to bind to Avicel . Ninety-eight amino acids-truncation at the COOH-terminus of BSC did not abolish the carboxymethyl cellulose (CMC)-hydrolyzing ability, but removed the Avicel-binding ability . These data suggested the presence of an Avicel-binding domain at the COOH-terminus of BSC, despite its inability to hydrolyze crystalline cellulose . A mutant enzyme with Phe at the 131st His, generated by site-directed mutagenesis, had no enzymatic activity with CMC as the substrate, as predicted from hydrophobic cluster analysis, while the cellulose-binding ability of the mutant enzyme still remained . Similarly, the mutation at the 169th Glu severely affected the enzyme activity, but not the cellulose-binding ability . All these data clearly show that BSC is composed of the catalytic domain at its NH2-terminal portion and the cellulose-binding domain at its COOH-terminal portion, and that the two domains are independently functional in the absence of the other.

Appl Microbiol Biotechnol, 1993 Feb, 38(5), 581 - 5
Scale-up of purine nucleoside fermentation from a shaking flask to a stirred-tank fermentor; Sumino Y et al.; Scaling-up purine nucleoside fermentation by a mutant strain of Bacillus subtilis from a shaking flask to a stirred-tank fermentor was attempted . The dimensions and the operating conditions of the stirred tank were determined in order to satisfy the optimum conditions of O2 transfer and power consumption per unit volume for the shaking flask . When the purine nucleoside fermentation was carried out in the stirred-tank fermentor under these conditions, in which the temperature simulated that in the shaking flask, the total amount of purine nucleosides produced was almost the same as that in the shaking flask, but the accumulation ratio of guanosine to total nucleosides was different from that in the flask . Since urea could not be utilized so efficiently in the stirred-tank fermentor, the NH4+ concentration and the pH of the culture broth were lower than those in the shaking-flask culture during fermentation . The activity of inosine monophosphate dehydrogenase and the accumulation ratio were significantly affected by the NH+4 concentration . When the pH of the stirred-tank culture was maintained at 6.9 by ammonia water to keep the NH+4 level higher, the ratio was improved to the same level as that observed in the shaking-flask culture . The fermentation heat calculated from the shaking-flask data and its pattern of change were similar to those in the stirred-tank fermentor.

FEMS Microbiol Lett, 1993 Feb 1, 106(3), 347 - 56
Heterologous recognition in vivo of promoter sequences from the Streptomyces coelicolor dagA gene; Parro V et al.; The Streptomyces coelicolor dagA gene that codes for an extracellular agarase was cloned in the closely related bacterium S . lividans and transferred to the distantly related low G+C Gram-positive bacterium Bacillus subtilis and to the far more distantly related Gram-negative bacterium Escherichia coli . S1 nuclease mapping experiments identified a putative fifth promoter from which transcription of the dagA gene can take place, and accurately mapped the transcription termination site . The transcription terminator was specific for the Streptomyces strains and could terminate transcription initiated by promoters other than those of dagA . The agarase gene is efficiently transcribed in B . subtilis and E . coli, although pulse-chase experiments failed to detect the synthesis of agarase in these two bacteria.

J Bacteriol, 1993 Feb, 175(4), 933 - 40
Two developmentally controlled promoters of Streptomyces coelicolor A3(2) that resemble the major class of motility-related promoters in other bacteria; Tan H et al.; Experiments were designed to allow isolation of Streptomyces coelicolor promoters that depend on the whiG sporulation gene, which encodes a putative sigma factor important in the sporulation of aerial hyphae . The strategy, based on earlier evidence that sigma WhiG is limiting for sporulation (K . F . Chater, C . J . Burton, K . A . Plaskitt, M . J . Buttner, C . Mendez, and J . Helmann, Cell 59:133-143, 1989) was to seek DNA fragments that inhibit sporulation in aerial hyphae when present at a high copy number . In a suitable Sau3AI-generated library of DNA from S . coelicolor A3(2), two inserts were found to inhibit sporulation . Both inserts caused expression of the adjacent xylE reporter gene present in the vector in a developmentally normal strain of S . coelicolor, but there was no xylE expression in an otherwise isogenic whiG mutant . S1 nuclease protection experiments were done with RNAs isolated from these plasmid-bearing strains or from the wild-type strain lacking either recombinant plasmid . In each case, an apparent transcription start site was found upstream of an apparent open reading frame (ORF) and just downstream of sequences that resemble consensus features of promoters for motility-related genes in Bacillus subtilis and coliform bacteria . Such promoters depend on sigma factors (sigma D and sigma F, respectively) particularly similar to the deduced whiG gene product . Each of the putative whiG-dependent promoters is within an ORF that is upstream of, and potentially translationally coupled to, the putative whiG-dependent ORF (although use of one of the promoters would necessitate the use of a different start codon, further downstream) . Thus, in unknown circumstances, the whiG-dependent ORFs may be expressed from a more remote promoter as part of a complex transcription unit.

Gene, 1993 Jan 15, 123(1), 39 - 44
Application of the mini-mu phage for the isolation of lac transcriptional fusions in Bacillus subtilis genes; Gardiol D et al.; A cassette containing a selectable cat gene and the lacZ gene without its own promoter has been incorporated into the mini-Mu bacteriophage genome . This mini-Mu derivative, referred to as mMu-Bs, can be used in Escherichia coli for the generation of lacZ transcriptional fusions to Bacillus subtilis genes cloned into plasmids . The resultant fusions can be analyzed in B . subtilis either as multicopy plasmids or as a single copy integrated via a Campbell-like recombination into the wild-type locus of the cloned fragment.

J Biol Chem, 1993 Jan 15, 268(2), 1424 - 9
Purification and properties of the RecR protein from Bacillus subtilis 168; Alonso JC et al.; Genetic evidence suggests that the Bacillus subtilis recR gene product is involved in DNA repair and recombination . To assign a biochemical function to the recR gene product, the RecR protein was labeled and purified by monitoring the radioactive label . NH2-terminal protein sequence analysis of RecR was consistent with the deduced amino acid sequence of the recR gene . The RecR protein (molecular mass of 25 kDa, isoelectric point 5.4) bound single- and double-stranded DNA in a filter binding assay . RecR-DNA complex formation is enhanced by the presence of a damage in the DNA substrate . The RecR-DNA complex formation proceeds in the absence of divalent cations and nucleotide cofactors, but is markedly stimulated by ATP and divalent cations . In our experimental conditions the apparent equilibrium constants of the optimized RecR-DNA complexes are 3 x 10(-7) M and 9 x 10(-7) M for damaged and undamaged DNA, respectively . The binding reaction is cooperative . Electron microscopy studies show that the presence of divalent cations increases the rate of RecR protein self-assembly . Addition of ATP or dATP promotes the organization of discrete series of quaternary structures on DNA, but ATP gamma S inhibits the DNA binding activity . A possible mechanism for the RecR function in DNA repair is discussed.

FEMS Microbiol Lett, 1993 Jan 15, 106(2), 183 - 6
Injury and repair in biocide-treated spores of Bacillus subtilis; Williams ND et al.; Bacillus subtilis NCTC 8236 spores exposed to appropriate concentrations of test biocides (glutaraldehyde, two iodine and two chlorine preparations) were able to repair injury if subsequently held in nutrient broth at 37 degrees C but not in broth at 22 degrees C, sterile filtered water at 4, 22 or 37 degrees C or germination medium at 37 degrees C . Repair appeared to occur primarily during outgrowth and was initiated soon for iodine-treated spores and latest for glutaraldehyde-treated ones.

Biochem Biophys Res Commun, 1993 Jan 15, 190(1), 192 - 200
Molecular cloning of human amidophosphoribosyltransferase; Iwahana H et al.; The cDNA of human amidophosphoribosyltransferase (EC 2.4.2.14, ATase), which is the supposed regulatory allosteric enzyme of de novo purine nucleotide biosynthesis, has been cloned from human hepatoma (HepG2) cDNA library . The predicted open reading frame encodes a protein of 517 amino acids with a deduced molecular weight (Mr) of 57,398, which is consistent with the molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the ATase subunit purified from human placenta . The derived amino acid sequence exhibits 93, 82, 41, 37, and 33% identity with the sequences of rat, chicken, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae ATases, respectively . Southern blot analysis suggested that the ATase gene exists as multiple copies . ATase mRNA (3.5 kb) is ubiquitously expressed in various human tissues . Comparison with rat and chicken ATases showed that two cysteine residues for an iron-sulfur cluster were conserved . Four consensus phosphorylation sites for cAMP-dependent protein kinase were found.

Biochemistry, 1993 Jan 12, 32(1), 32 - 7
Mapping of the binding interfaces of the proteins of the bacterial phosphotransferase system, HPr and IIAglc; Chen Y et al.; Enzyme IIAglc and HPr are central regulatory and phosphocarrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of bacteria . During phosphoryl transfer from phosphoenolpyruvate to glucose, phosphate is transferred from HPr to enzyme IIAglc . In order to characterize the binding interfaces of the two proteins during phosphate transfer, 15N-edited and 15N-filtered NMR experiments have been recorded for the complex of enzyme IIAglc and HPr from Bacillus subtilis . Uniformly 15N-labeled enzyme IIAglc and nonlabeled HPr were used in these studies . Residues which undergo significant chemical shift changes upon complex formation have been identified for both proteins . The binding interfaces of the two proteins, suggested by the observed chemical shift changes, involve predominantly hydrophobic surfaces near the active site His-15 of HPr and the phosphoryl acceptor His-83 of IIAglc.

J Mol Biol, 1993 Jan 5, 229(1), 235 - 8
Crystallization and preliminary X-ray crystallographic analysis of alpha-amylase from Bacillus subtilis; Chang C et al.; Large crystals of alpha-amylase from Bacillus subtilis have been obtained at room temperature using polyethylene glycol 6000 as precipitant . They grow to typical dimensions of 0.25 mm x 0.3 mm x 2.0 mm in five days . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 85.46 A, b = 166.5 A and c = 332.7 A . The asymmetric unit seems to contain eight molecules of alpha-amylase, with crystal volume per protein mass (Vm) of 2.69 A3/Da and solvent content of 54.3% by volume . Despite a very long c-axis, the crystals diffracted to about 2.2 A Bragg spacing using the rotating anode X-rays and were resistant to damage by X-rays . Thus they are suitable for structure determination by X-ray methods at high resolution . X-ray diffraction data have been collected to 3.4 A Bragg spacing from a native crystal.

J Biol Chem, 1993 Jan 5, 268(1), 678 - 85
Cloning of the cta operon from alkaliphilic Bacillus firmus OF4 and characterization of the pH-regulated cytochrome caa3 oxidase it encodes; Quirk PG et al.; We have cloned and sequenced the DNA of alkaliphilic Bacillus firmus OF4 encompassing the cta operon that encodes a pH-regulated cytochrome caa3 oxidase . The gene organization is identical with that of the homologous Bacillus subtilis caa3 oxidase locus (van der Oost, J., von Wachenfeld, C., Hederstedt, L . & Saraste, M . (1991) Mol . Microbiol . 5, 2063-2072) . The deduced amino acid sequences of the four putative structural subunits (CtaC-F) indicate substantial similarity to caa3-type oxidases from other Bacillus species and to other members of the family of mitochondrial-type aa3 oxidases . A marked paucity of basic residues was noted in the cytochrome c-containing domain of CtaC, which faces the highly alkaline external milieu . We have also purified the enzyme as a three-subunit complex, with possible trace amounts of a fourth subunit . N-terminal sequence analysis of the two largest subunits confirmed them to be encoded by the cloned cta genes . An additional, minor caa3 component with distinctive chromatographic properties was noted during purification . Analysis of mRNA with a ctaD probe revealed an abundant 4-kilobase message of the right size to encode CtaC-F . The cellular content of this message varied with growth pH . Cells grown at pH 10.5 contained 2 to 2.5 times more message than those grown at pH 7.5, in good correspondence with the relative amounts of caa3 oxidase found in the cells . The ctaB gene, immediately upstream from the ctaC-F genes, was found to be transcribed onto a low abundance 5-kilobase message, which is likely also to encode CtaC-F . Levels of this message were not affected by growth pH.

Microbios, 1993, 73(297), 237 - 47
A continuous, simple and rapid method for the detection, extraction and identification of residual antibacterial agents in meat; Kondo F et al.; Most antibacterial agents produced larger inhibition zones and showed lower detectable concentrations on minimal medium (MM) seeded with Bacillus subtilis than on Mueller-Hinton medium . After simple extraction, using a small amount of acetonitrile, from an agar block inside the inhibitory zone produced by each antibacterial agent, identification was carried out by reverse-phase high-performance liquid chromatography (HPLC) . It is recommended that for inspection of residues MM is superior as a bioassay medium . The continuous, simple and rapid method described may be useful for routine laboratory testing of residual antimicrobial agents in food.

Folia Microbiol (Praha), 1993, 38(1), 22 - 4
Sporulation and synthesis of extracellular proteinases in Bacillus subtilis are more temperature-sensitive than growth; Jansova E et al.; Bacillus subtilis 115 grew in a medium with amino acids and glucose with the maximum specific growth rates mu of 1.20-1.10/h in the temperature range of 45-48 degrees C . Activity of the extracellular neutral proteinase excreted by 1.3 mg/mL dry mass during 8 h of the postexponential and stationary growth phases decreased from its maximum value of 0.23 TU/mL at 40 degrees C to 0.13 and 0.06 TU/mL at 45 and 48 degrees C, respectively . Formation of the extracellular serine proteinase decreased even more - from 0.18 TU/mL at 40 degrees C to 0.06 and 0.03 TU/mL at 45 and 48 degrees C, respectively . Sporulation, expressed as the portion of sporangia with refractile spores at the 6th h of the stationary phase decreased from 46% at 40 degrees C to 17 and 3% at 45 and 48 degrees C, respectively.

Antonie Van Leeuwenhoek, 1993 Jan, 63(1), 45 - 53
The growth kinetics of B . subtilis; Koch AL; There has been considerable discussion by Kubitschek and Cooper concerning the growth rate of cells of E . coli throughout the cell cycle . Consequently, it is relevant to test Kubitschek's linear model against the exponential model espoused by Cooper (and many others) with another organism and another technique . Burdett et al . measured, by electron microscopy and computer analysis of the microphotographs, the distribution of lengths of a population of cells of Bacillus subtilis grown in 0.4% succinate in a minimal medium . The data were fitted to the extended Collins-Richmond method of Kirkwood & Burdett which subdivided the cell cycle into several phases . I have taken their results and compared them with the linear and exponential growth models for the entire cell cycle after applying correction to the data for the shape of completed and forming poles; i.e., to put the data on a cell-volume basis instead of a cell-length basis . Most of the correction involves no arbitrary assumptions . The conclusion is that global volume growth rate is nearly proportional to cell volume; i.e . growth of Bacillus subtilis is nearly exponential for almost every cell in the growing culture.

Jpn J Antibiot, 1993 Jan, 46(1), 1 - 7
{Microbiological determination of netilmicin using a thin-layer chromatography scanner}; Tsuda Y et al.; We presented a new colorimetric bioassay of aminoglycoside antibiotics, which were represented by netilmicin (NTL) in this study, based on the discoloration of thymolphthalein (TP) in paper (indicator-disc) by carbon dioxide produced by Bacillus subtilis . To evaluate the amount of the carbon dioxide, the following experiment was carried out . One milliliter of B . subtilis suspension containing 4.5 x 10(7) colony forming units/ml, 1 ml of nutrient broth, 0.9 ml of 0.1 M phosphate buffer (pH 8.0) and 0.1 ml NTL sample solution were added to an incubation container, which was then placed in a water-bath (37 degrees C) for 3 hours . The oxygen concentration in the head space of flask was determined using gas-chromatograph . The dose-response curves showed good correlation between amounts of NTL and carbon dioxide produced by B . subtilis . The indicator-disc containing TP and sodium hydroxide was placed into the Reacti-flask and then incubated in the same manner as described above . After incubation, concentration of blue colored TP was determined using a TLC scanner . The discoloration of blue color to white showed the proportionality between NTL concentrations and the degrees of discoloration of TP . The method can accurately measure NTL levels down to 2.5 micrograms/ml in water using 0.1 ml samples, and should be adequate for rapid bioassay.

J Biochem (Tokyo), 1993 Jan, 113(1), 101 - 5
Zinc protease of Bacillus subtilis var . amylosacchariticus: construction of a three-dimensional model and comparison with thermolysin; Tsuru D et al.; The active site structure of the Zn-containing neutral protease from Bacillus subtilis var . amylosacchariticus (BANP) was predicted by computer-aided modeling on the basis of the three-dimensional structure of thermolysin (TLN) . As expected from the high homology in amino acid sequence of the two enzymes, the overall folding of BANP was very similar to that of TLN . Glu144, Tyr158, and His228 of BANP were located near the active site Zn ion, to which three amino acid residues, His143, His147, and Glu167, were coordinated . This model is supported by the previous results that chemical modifications of Tyr158 and photooxidation of His228 of BANP markedly affect the proteolytic activity of the enzyme . Interestingly, BANP was found to be significantly less sensitive to metalloprotease inhibitors such as phosphoramidon and talopeptin . From a comparison of the enzyme-inhibitor complex models between BANP and thermolysin, it is suggested that replacement of Thr129 in TLN by Phe130 in BANP is related to difference in inhibitor sensitivity between BANP and TLN.

J Gen Microbiol, 1993 Jan, 139 ( Pt 1), 31 - 7
Functional analysis of the outB gene of Bacillus subtilis; Caramori T et al.; Three additional alleles of the outB gene of Bacillus subtilis, whose activity is required for spore outgrowth, were identified . The nucleotide sequence of three mutant genes was determined . Analyses of dominance-recessivity showed that the wild-type allele is dominant over the mutant ones . When the outB gene was placed under the control of the inducible spac-1 promoter, the presence of IPTG was necessary to obtain normal growth . The results suggested that the outB gene is required for growth of B . subtilis . Expression of outB from the sporulation promoter spoIID negatively affected subsequent spore outgrowth, without altering vegetative growth and sporulation.

FEMS Microbiol Lett, 1993 Jan 1, 106(1), 105 - 10
Conformation of Escherichia coli outer membrane protein OmpA produced in Bacillus subtilis: influence of lipopolysaccharide; Puohiniemi R et al.; The conformation of the outer membrane protein OmpA of Escherichia coli produced in Bacillus subtilis and solubilized in Sarkosyl was studied by measuring its ability to bind OmpA-specific phage K3 and to inhibit F-mediated conjugation . The partially purified protein was inactive in both of these assays . Refolding of the protein in the presence of lipopolysaccharide resulted in preparations with full phage-binding and conjugation-inhibiting capacity, indicating the formation of surface-exposed loops of OmpA of native conformation . The finding is of importance for the potential use of outer membrane proteins of Gram-negative bacteria as vaccines.

Appl Environ Microbiol, 1993 Jan, 59(1), 296 - 303
Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system; Klein C et al.; Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) which is produced by Bacillus subtilis ATCC 6633 . Upstream from the structural gene of subtilin, spaS, three genes (spaB, spaT, and spaC) which are involved in the biosynthesis of subtilin have been identified (C . Klein, C . Kaletta, N . Schnell, and K.-D . Entian, Appl . Environ . Microbiol . 58:132-142, 1992) . By using a hybridization probe specific for these genes, the DNA region downstream from spaS was isolated . Further subcloning revealed a 5.2-kb KpnI-HindIII fragment on which two open reading frames, spaR and spaK, were identified approximately 3 kb downstream from spaS . The spaR gene encodes an open reading frame of 220 amino acids with a predicted molecular mass of 25.6 kDa . SpaR shows 35% similarity to positive regulatory factors OmpR and PhoB . The spaK gene encodes an open reading frame of 387 amino acids with a predicted molecular mass of 44.6 kDa and was highly similar to histidine kinases previously described (PhoM, PhoR, and NtrB) . Hydrophobicity blots suggested two membrane-spanning regions . Thus, spaR and spaK belong to a recently identified family of environmentally responsive regulators . These results indicated a regulatory function of spaR and spaK in subtilin biosynthesis . Indeed, batch culture experiments confirmed the regulation of subtilin biosynthesis starting in the mid-logarithmic growth phase and reaching its maximum in the early stationary growth phase . Gene deletions within spaR and spaK yielded subtilin-negative mutants, which confirms that subtilin biosynthesis is under the control of a two-component regulatory system.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Biochem, 1993 Jan, 51(1), 75 - 82
Three-dimensional structures of the central regulatory proteins of the bacterial phosphotransferase system, HPr and enzyme IIAglc; Chen Y et al.; Enzyme IIA and HPr are central regulatory proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase (PTS) system . Three-dimensional structures of the glucose enzyme IIA domain (IIAglc) and HPr of Bacillus subtilis and Escherichia coli have been studied by both X-ray crystallography and Nuclear Magnetic Resonance (NMR) Spectroscopy . Phosphorylation of HPr of B . subtilis and IIAglc of E . coli have also been characterized by NMR spectroscopy . In addition, the binding interfaces of B . subtilis HPr and IIAglc have been identified from backbone chemical shift changes . This paper reviews these recent advances in the understanding of the three-dimensional structures of HPr and IIAglc and their interaction with each other.

J Cell Biochem, 1993 Jan, 51(1), 55 - 61
The phosphorelay signal transduction pathway in the initiation of Bacillus subtilis sporulation; Hoch JA; The formation of spores in Bacillus subtilis is a developmental process under genetic control . The decision to either divide or sporulate is regulated by the state of phosphorylation of the SpoOA transcription factor . Phosphorylated SpoOA (SpoOA approximately P) is both a repressor and an activator of transcription depending on the promoter it is affecting . SpoOA approximately P is the end product of the phosphorelay, a signal transduction system linking environmental information to the activation of sporulation . Activation or deinhibition of two ATP-dependent kinases, KinA and KinB, to phosphorylate the SpoOF secondary messenger initiates the phosphorelay . SpoOF approximately P is the substrate for the SpoOB protein, a phosphoprotein phosphotransferase which transfers the phosphate group to SpoOA . The SpoOA approximately P formed from this pathway orchestrates transcription events during the initial stage of spore development through direct effects on a variety of promoters and through the use of other transcription factors, termed transition state regulators, whose activity it controls . Because commitment to sporulation has serious cellular programming consequences and is not undertaken capriciously, the phosphorelay is subject to a variety of complex controls on the flow of phosphate through its components.

Antimicrob Agents Chemother, 1993 Jan, 37(1), 128 - 9
Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter; Neyfakh AA et al.; The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis . NorA provides B . subtilis with resistance to the same drugs and to a similar extent as the B . subtilis multidrug transporter protein Bmr does . NorA and Bmr share 44% sequence similarity . Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

Genes Dev, 1993 Jan, 7(1), 139 - 48
SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction; Bai U et al.; SinR, a 111-amino-acid DNA-binding protein, is a pleiotropic regulator of several late growth processes in Bacillus subtilis . It acts as a developmental switch, positively regulating genes for competence and motility and repressing aprE and stage II sporulation genes . It is encoded by the second gene in a two gene operon, but previous results have also indicated that these two genes are differently regulated . We show in this discussion that the product of sinI, the first open reading frame (ORF) of this operon, interferes with the function of SinR . In vivo experiments have demonstrated that overexpression of sinI results in phenotypes that are observed in cells with a null mutation of sinR . A chromosomal in-frame deletion of sinI gives rise to a phenotype associated with higher levels of SinR . Thus, SinI acts as an antagonist to SinR . In vitro experiments have shown that the interaction between these two proteins is a direct one . SinI prevents SinR from binding to its target sequence on aprE, and the two proteins form a complex that can be immunoprecipitated with antibodies to either SinR or SinI.

Proc Natl Acad Sci U S A, 1993 Jan 1, 90(1), 128 - 32
MtrB from Bacillus subtilis binds specifically to trp leader RNA in a tryptophan-dependent manner; Otridge J et al.; MtrB regulates transcription attenuation of the Bacillus subtilis trp operon . We have shown that MtrB, either from B . subtilis or overexpressed in Escherichia coli, binds specifically to RNA from the leader region of the trp operon by a gel mobility-shift assay . This binding is tryptophan dependent . MtrB binds to a transcript terminated at the trp attenuator (-2 to +138) or a read-through transcript (-2 to +318) . MtrB does not bind antisense trp leader RNA or single-stranded trp leader DNA . These results support the model in which attenuation is controlled by tryptophan-activated MtrB influencing the secondary structure of the leader region transcript to form a terminator structure.

J Bacteriol, 1993 Jan, 175(2), 528 - 40
Sporulation gene spoIIB from Bacillus subtilis; Margolis PS et al.; We have cloned and characterized the sporulation gene spoIIB from Bacillus subtilis . In extension of previous nucleotide sequence analysis, our results show that the order of genes in the vicinity of spoIIB is valS folC comC spoIIB orfA orfB mreB mreC mreD minC minD spoIVFA spoIVFB L20 orfX L24 spoOB obg pheB pheA . All 20 genes have the same orientation; the direction of transcription is from valS to pheA . We show that spoIIB is a 332-codon-long open reading frame whose transcription is under sporulation control . The deduced amino acid sequence of the spoIIB gene product, a 36-kDa polypeptide, is highly charged and contains a stretch of uncharged amino acids that could correspond to a transmembrane segment . Surprisingly, mutations in spoIIB, including an in vitro-constructed null mutation, cause only a mild impairment of spore formation in certain otherwise wild-type bacteria . However, when combined with mutations in another sporulation gene, spoVG, mutations in spoIIB cause a severe block in spore formation at the stage (stage II) of septum formation . (As with spoIIB mutations, mutations in spoVG cause little impairment in sporulation on their own.) The nature of the spoIIB spoVG mutant phenotype is discussed in terms of the events involved in the maturation of the sporulation septum and in the activation of sporulation transcription factors sigma F and sigma E.

J Bacteriol, 1993 Jan, 175(2), 503 - 9
Two tRNA gene clusters associated with rRNA operons rrnD and rrnE in Bacillus subtilis; Rudner R et al.; Sequence analysis of cloned rescued DNA fragments from a Bacillus subtilis strain with an inserted recombinant plasmid in ribosomal operon rrnE revealed the presence of two tRNA genes for Met and Asp at the 3' end of the operon . Probing chromosomal DNA from a strain carrying a plasmid inserted in rrnD with a fragment containing the genetically unassigned cluster of 16 tRNA genes revealed that the cluster is located immediately following the rrnD operon . Our findings show that all 10 rrn operons in B . subtilis are associated with tRNA gene clusters.

Arch Microbiol, 1993, 159(6), 574 - 8
The menaquinol oxidase of Bacillus subtilis W23; Lemma E et al.; The quinol oxidase appears to be mainly responsible for the oxidation of the bacterial MKH2 in Bacillus subtilis W23 growing with either glucose or succinate . The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol . Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all . After fourtyfold purification the isolated enzyme contained 5.3 mumol cytochrome aa3 per gram of protein and negligible amounts of cytochrome b and c . The turnover number based on cytochrome aa3 was about 10(3) electrons.s-1 at pH 7 and 37 degrees C . The preparation consisted mainly of a M(r) 57,000 and a M(r) 36,000 polypeptide . The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B . subtilis strain 168 (Santana et al . 1992), in that asp-14 predicted by qoxA was missing in the M(r) 36,000 polypeptide.

Arch Microbiol, 1993, 160(2), 158 - 61
Type selective inhibition of microbial fatty acid synthases by thiolactomycin; Arimura N et al.; The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g . Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g . Saccharomyces cerevisiae enzyme) . We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus . Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin . E . coli synthase, as expected, was strongly inhibited by thiolactomycin . Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E . coli synthase, it is largely insensitive to thiolactomycin.

Biol Pharm Bull, 1993 Jan, 16(1), 81 - 3
Detection of tetracycline resistance protein encoded by Bacillus subtilis plasmid pNS1; Aoki T et al.; Tetracycline resistance protein (TET) of Bacillus subtilis plasmid pNS1 was detected by immunoblot analysis using a specific antibody to TET-chloramphenicol acetyltransferase (CAT) fusion protein . In two-dimensional electrophoresis, one major spot which seemed to be the pNS1-encoded TET (pNS1-TET), was detected by immunostaining . Its molecular weight and isoelectric point were approximately 52 kDa and 6.2, respectively . Judging from the nucleotide sequence, the pNS1-TET is a very hydrophobic, 50 kDa protein . Therefore, the 52 kDa protein is thought to be an intact form of the pNS1-TET produced by B . subtilis cells.

Microbios, 1993, 74(299), 121 - 9
Conditions suitable for the recovery of biocide-treated spores of Bacillus subtilis; Williams ND et al.; Various factors were studied in order to determine the optimum conditions for the recovery of Bacillus subtilis spores treated with two iodine preparations, two chlorine-releasing agents or glutaraldehyde . The composition of the recovery medium was not usually important except that counts on brain heart infusion agar were significantly lower than on other media for iodine-treated spores . The addition to recovery media of soluble starch, charcoal, D-glucose or yeast extract usually had no discernible beneficial effect on colony counts . Maximum counts of survivors were obtained after an incubation period of 3 days and an optimum incubation temperature of 30 or 37 degrees C . Germination and outgrowth of biocide-exposed spores were more sensitive to changes in incubation temperature than were control spores.

Ukr Biokhim Zh, 1993 Jan-Feb, 65(1), 104 - 6
{Effect of an electromagnetic field on subtilisin from Bacillus subtilis 316 m}; Povaliaeva IV et al.; The influence of electromagnetic field on the subtilisin of Bacillus subtilis strain 316 M preparation has been studied . It is the increase 30-60% of the proteolytic activity during 60-100 min treatment was observed . The effect of activation is stable for a month at +4 degrees C . The increase of the proteolytic activity is connected with conformational changes of the enzyme molecule, with the decrease of pI from 11.4 to 9.2 in particular.

Appl Biochem Biotechnol, 1993 Jan-Feb, 38(1-2), 83 - 92
Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM no . 64; Kembhavi AA et al.; The proteolytic activity produced by a Bacillus subtilis isolated from a hot spring was investigated . Maximum protease production was obtained after 38 h of fermentation . Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production . Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5-11 . The partially purified enzyme was stable at 60 degrees C for 30 min . Calcium ions were effective in stabilizing the enzyme, especially at higher temperature . The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h . The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000 . The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.

Microbios, 1993, 74(298), 29 - 37
Studies on bacillomycin D biosynthesis by Bacillus subtilis; Tenoux I et al.; The biosynthesis of bacillomycin D, an antibiotic containing a beta-amino fatty acid and a peptide moiety with asparagine, glutamic acid, serine, proline, threonine, and tyrosine, was studied by incubating the Bacillus subtilis producer with various 14C-labelled precursors . Sodium acetate was incorporated into beta-amino fatty acids of bacillomycin D, and asparagine was the best precursor of the peptidic moiety . The kinetics of the incorporation of radioactive substrates into bacillomycin D and into beta-amino fatty acids show that the lipid and the peptide moieties of the antibiotic were synthesized at the same stage of growth of the bacteria . Comparing the effects of different inhibitors on the incorporation of radioactive precursors, the bacillomycin D and beta-amino fatty acids biosyntheses are discussed in relation to the biosyntheses of proteins, lipids and with sporulation.

Res Microbiol, 1993 Jan, 144(1), 25 - 33
rRNA gene restriction patterns as an epidemiological marker in nosocomial outbreaks of Staphylococcus aureus infections; Meugnier H et al.; rRNA gene restriction patterns (ribotyping) were compared with phage typing, serotyping, enterotoxins and exfoliatin production in the analysis of 26 Staphylococcus aureus strains isolated from two different nosocomial outbreaks . Total DNA was cleaved by EcoRI restriction endonuclease . After agarose gel electrophoresis and Southern transfer, the hybridization of the membranes was done with radiolabelled 16S rRNA gene from Bacillus subtilis inserted into a plasmid vector . Six to 13 fragments were visualized . A core of common fragments was discerned for all strains tested . A full correlation between ribotyping and conventional markers was observed in only one of the outbreaks studied . In both outbreaks, ribotyping proved helpful in characterizing otherwise untypable strains.

Arch Microbiol, 1993, 160(6), 486 - 91
Anti-SOS effects induced in Bacillus subtilis by a phi 105 mutant prophage; Rubinstein CP et al.; The presence of the mutant prophage phi 105cts23 in Bacillus subtilis strains strongly affected several biological parameters including the viability of protoplasts and the establishment of plasmid pC194 . A defective inducibility of the prophage after treatments that de-repress the SOS-like response were also observed . Although these alterations suggested a Rec-deficient phenotype, homologous recombination was not impaired in these lysogenic derivatives . In fact, chromosomal DNA transformation in these competent cells was more efficient than in cells carrying the wild type prophage: cell death due to prophage induction upon competence development was lower than expected . Alterations in the response to SOS-inducing agents and to osmotic stress correlated with the presence of this particular mutant prophage or the cloned thermosensitive repressor at the permissive temperature . The induction of an anti-SOS effect is discussed.

Microbiol Immunol, 1993, 37(10), 809 - 12
Purification of Bacillus subtilis spore coat protein by electrophoretic elution procedure and determination of NH2-terminal amino acid sequences; Abe A et al.; Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure . Solubilized coat protein components were separated on SDS-PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined . Two purified polypeptides with molecular weights of about 40 kDa were obtained; each of them was in very closed size on SDS-PAGE, both retaining antigenic activity against anti-spore coat protein serum on immunoblot analysis . The N-terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.

Annu Rev Microbiol, 1993, 47, 441 - 65
Regulation of the phosphorelay and the initiation of sporulation in Bacillus subtilis; Hoch JA; The initiation of sporulation of bacteria is a complex cellular event controlled by an extensive network of regulatory proteins that serve to ensure that a cell embarks on this differentiation process only when appropriate conditions are met . The major signal-transduction pathway for the initiation of sporulation is the phosphorelay, which responds to environmental, cell cycle, and metabolic signals, and phosphorylates the Spo0A transcription factor activating its function . Signal input into the phosphorelay occurs through activation of kinases to phosphorylate a secondary-messenger protein, Spo0F . Spo0F-P serves as a substrate for phosphoprotein phosphotransferase, Spo0B, which phosphorylates Spo0A . The pathway is regulated by transcriptional control of its component proteins and by regulating phosphate flux through the pathway . This is accomplished by several regulatory proteins, and by activated Spo0A, which regulates transcription of genes for its own synthesis . Spo0A-P indirectly controls the transcription of numerous genes by regulating the level of other transcription regulators and directly activates the transcription of several regulatory proteins and sigma factors required for progression to the second stage of sporulation . Although the pathway and regulatory proteins have been identified, the signals and effectors for these regulators remain a mystery.

Yi Chuan Xue Bao, 1993, 20(4), 362 - 73
{Effect of ribosomal protein mutation on the expression of alkaline protease gene in Bacillus subtilis}; Zhang W et al.; Altogether 19 strains belong to 13 species of ribosomal protein mutants of Bacillus subtilis were tested in vitro transcription--translation system for their influence on the translation of alkaline protease gene (apr) . It was found that 10 species (13 strains) of ribosomal protein mutants did affect the translation of apr mRNA . Streptomycin-dependent (Str-D) ribosomes almost did not translate the apr mRNA . Str-D inhibited the expression of apr gene at the translational level, but had no influence on neutral protease gene . There is a secondary structure complex at the translation initiation region of apr mRNA . When one of the secondary structure was destroyed by site directed mutagenesis, the translation efficiency was enhanced by 7.3 to 9.1 folds . The higher order structure of Str-D and Str-R ribosome were different and so were the affinity of Str-D and Str-R 30S subunits to the 5' end of apr mRNA . These results suggest that Str-D ribosomes could not translate apr mRNA because of the secondary structure complex, low initiation strength of apr mRNA, and alteration of the higher order structure of the Str-D ribosomes.

Nucleic Acids Symp Ser, 1993, (29), 145 - 6
Construction of the Bacillus subtilis chromosome physical map and the strategy for mapping newly isolated genes in one membrane filter for hybridization; Itaya M; A complete physical map of the Bacillus subtilis 168 chromosome was constructed . The merging of this physical map is expected not only to provide important insights into the organization and rearrangement of genes of this species but also to be a powerful means for the genome analysis . One of the most practical aspects is rapid and accurate mapping of newly isolated genes using a single membrane filter for hybridization . This protocol proved that not only unique genes but also multiple homologous genes dispersed on the chromosome can be physically mapped.

Crit Rev Biotechnol, 1993, 13(3), 173 - 93
Use of microbial spores as a biocatalyst; Murata K; Endospores of a bacterium Bacillus subtilis and ascospores of a yeast Saccharomyces cerevisiae contained almost all the activities for the same enzymes as vegetative cells . The biotechnological potential of spores was studied by selecting adenosine 5'-triphosphatase and alkaline phosphatase in bacterial and yeast spores, respectively, as model enzymes . The activity of both enzymes was efficiently expressed when the spores were treated by physical (sonication or electric field pulse) and chemical (organic solvents or detergents) methods . The yeast spores were immobilized in polyacrylamide gel without any appreciable loss of activity . The immobilized spores were packed in a column and used successfully for the continuous reactions of alkaline phosphatase and glyoxalase I . The microbial spores were confirmed to be promising as a biocatalyst for the production of useful chemicals in bioreactor systems.

Vaccine, 1993, 11(9), 970 - 3
Assessment of non-protein impurities in potential vaccine proteins produced by Bacillus subtilis; Himanen JP et al.; The levels of non-protein impurities at different stages of purification of model vaccine proteins produced by Bacillus subtilis were assessed with special emphasis on peptidoglycan-wall teichoic acid and lipoteichoic acid . Intracytoplasmically produced proteins were purified by disrupting the lysozyme protoplasts using osmotic shock, depositing the inclusion bodies by low-speed centrifugation, and washing them with detergent . By this procedure most of the cell envelope-derived impurities could be removed . The final product contained less than 1% (w/w) of neutral sugars, fatty acids, phosphate, hexosamine, diaminopimelic acid and glycerol . A secreted protein was purified from the culture supernatant by successive ion-exchange and adsorption chromatography . The cell envelope-derived impurities were efficiently removed by the cation-exchanger, and the final product contained only minute amounts of non-protein components . The amounts of non-protein components such as peptidoglycan and lipoteichoic acid in proteins produced in either mode were shown to be negligible in relation to their potentially harmful biological effects.

Acta Microbiol Hung, 1993, 40(2), 141 - 9
Antibacterial antagonism between fusidic acid and ciprofloxacin; Uri JV; A routine laboratory disk susceptibility testing of a resistant Staphylococcus aureus strain showed that around the ciprofloxacin disk, placed by chance in proximity to a fusidic acid disk, the inhibition zone was truncated . Follow-up of this observation by a planned disk approximation method showed that there is a real antagonism between these two antibacterial agents . The antagonism was observed while testing S . aureus isolates including the standard ATCC 25923 strain, with Bacillus subtilis ATCC 6633 spores and also with a mutant Escherichia coli made fusidic acid susceptible . The antagonistic property was found structure-specific, only associated with those fluoroquinolones containing the cyclopropyl substituent at the N1-position: ciprofloxacin, enrofloxacin, sparfloxacin and WIN 57273 . Fluoroquinolones without this substituent such as enoxacin, norfloxacin, pefloxacin and ofloxacin were not antagonized by fusidic acid, the steroidal Gram-positive active antibiotic.

Microbiol Immunol, 1993, 37(12), 935 - 41
Quantitative analysis of polymyxin B released from polymyxin B-treated dormant spores of Bacillus subtilis and relationship between its permeability and inhibitory effect on outgrowth; Fujita-Ichikawa Y et al.; Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination . When about 2.8 x 10(8) cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 micrograms/ml of polymyxin B were released into the liquid medium during germination . Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl2 solution released 4.0 micrograms/ml of the antibiotic . The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 micrograms/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination . Young vegetative cells were less sensitive to the antibiotic than germinated spores . In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.

Sci Prog, 1993-94, 77 ( Pt 1-2), 113 - 30
Spore formation in Bacillus subtilis; Yudkin M; Bacillus subtilis is the best studied of the bacteria that make heat-resistant spores in response to starvation . The process of sporulation results in the formation of a cell type which is quite different in morphology from that of bacteria during normal growth . Sporulation requires the activation, in an ordered sequence, of many genes that are kept silent during vegetative growth . It also requires that these genes be activated differentially in two sister cells that are genetically identical . The sophisticated mechanisms responsible for both the temporal and the spatial regulation of gene expression are now understood in a fair amount of detail . They are likely to provide models which will make a valuable contribution to studies of development and differentiation in higher cells.

Biotechnology (N Y), 1993 Jan, 11(1), 71 - 6
Efficient production of a functional single-chain antidigoxin antibody via an engineered Bacillus subtilis expression-secretion system; Wu XC et al.; We have applied a Bacillus subtilis expression-secretion system to produce a functional antidigoxin SCA (single-chain antibody consisting of VL-linker-VH) and the individual variable domains of light (VL) and heavy (VH) chains . The secreted antidigoxin SCA can be affinity purified in one step by applying the culture supernatant directly to a ouabain-Sepharose column . N-terminal sequence determination indicated that the protein has the expected N-terminus with the signal peptide properly processed . Affinity and ligand specificity studies demonstrated that the engineered antidigoxin SCA has almost identical properties as those of the parental monoclonal antibody . The use of B . subtilis WB600, an engineered, six-extracellular protease-deficient strain, is vital for the production of antidigoxin SCA in high quality and quantity (5 mg/liter in a shake flask culture) . All the secreted SCAs are biologically active . The ability to produce secreted SCAs by the B . subtilis expression system provides a simple and efficient means to analyze the binding properties of engineered antibodies generated through rational design or site-directed mutagenesis.

Mol Gen Genet, 1993 Jan, 236(2-3), 374 - 8
Leader region of the gene encoding DNA polymerase III of Bacillus subtilis; Sanjanwala B et al.; Previously we cloned and sequenced the polC gene of Bacillus subtilis and identified regions corresponding to various catalytic domains of DNA polymerase III, the enzyme it encodes . In the present study, by using primer extension, we have identified the transcription start site and a 139 nucleotide leader region upstream of the first codon . This region contains a DnaA box in the non-transcribed DNA strand . An RNA transcript of the leader would contain a sequence that could form a 29 bp stem-loop secondary structure followed by a strong terminator sequence, rich in uracil, before the ribosome binding site . Plasmids were constructed containing either the intact leader region or deletion mutations of the leader, fused to the Escherichia coli lacZ gene in an expression vector . Single copies of the fusions were then integrated into the B . subtilis genome by transformation . Studies of the expression of beta-galactosidase by the transformed cells supported the idea that the leader region is important in regulating polC gene expression.

FASEB J, 1993 Jan, 7(1), 188 - 95
Gap-scan deletion analysis of Bacillus subtilis RNase P RNA; Waugh DS et al.; We carried out an exhaustive deletion analysis of Bacillus subtilis ribonuclease P (RNase P) RNA, seeking sequences that are essential for its catalytic activity . A collection of partially deleted RNase P RNA genes was used to construct templates for synthesis, by in vitro transcription, of circularly permuted RNA molecules that lack various wild-type sequences . The mutant RNAs were assayed for catalytic activity in vitro, using a precursor of B . subtilis tRNAAsp as a substrate . Gap-scan deletion analysis revealed that most of the RNase P RNA sequence is important for activity; only two substantive deletions did not dramatically inhibit pre-tRNA processing in vitro . One part of the molecule (nucleotides 225-270) seemed particularly sensitive to deletion, but considering a collection of mutants with overlapping deletion gaps, it was possible to remove every residue in the RNase P RNA without completely abolishing its catalytic activity . Thus, the catalytic mechanism of RNase P does not depend absolutely on a single, particular nucleotide or local sequence for activity.

Proc Natl Acad Sci U S A, 1993 Jan 1, 90(1), 133 - 7
Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein; Babitzke P et al.; We have reconstituted Bacillus subtilis trp attenuation in vitro . Purification of the mtrB gene product (TRAP) to near homogeneity allowed us to demonstrate that addition of this protein plus L-tryptophan to template, RNA polymerase, and nucleoside triphosphates caused transcription termination in the trpEDCFBA leader region . TRAP acts by binding to the nascent transcript and preventing formation of an RNA antiterminator structure, thereby allowing terminator formation and transcription termination . Oligonucleotides complementary to segments of the antiterminator were used to demonstrate that formation of this RNA hairpin was responsible for transcription read-through . TRAP was found to be a 60-kDa multimeric protein composed of identical 6- to 8-kDa subunits, and its elution profile on a chromatographic column did not change in the presence of tryptophan.

Acta Microbiol Pol, 1993, 42(2), 127 - 36
Instability of hybrid plasmids in Bacillus subtilis; Wojcik K et al.; We studied the mechanisms responsible for instability of hybrid derivatives of staphylococcal R plasmids in B . subtilis cells . Four pBCH plasmids were constructed from staphylococcal chloramphenicol-resistance plasmid pC756 ligated with pBR322 or pUC8 vectors, and two pAEC plasmids contained pC756 plasmid, erm gene from S . aureus pE3692 and pBR322 plasmid vector . The level of instability of these recombinant plasmids was clearly related to their sizes, the larger derivatives were segregationally highly unstable . The four hybrid pBE plasmids constructed from erythromycin-resistant pE3692 plasmid or its deletion derivative and pBR322 vector were poorly maintained in B . subtilis rec+ strain in comparison with the B . subtilis (recE4) strain . Moreover, the absence in pBE-hybrids of a part of palA sequence led to considerable reduction of plasmid stability in B . subtilis cells . However, the clones of B . subtilis harbouring pBE plasmids integrated into the bacterial chromosome presented much higher stability of the plasmid erm gene than the clones maintaining autonomously replicating plasmids.

Nucleic Acids Symp Ser, 1993, (29), 163 - 4
Origin of 16S and 23S rRNAs and the E . coli str operon, as derived from tandem tRNA repeats; Ohnishi K; The bases 1175-1542 (3'-term.) and 94-510 of the E . coli (EC) 16S rRNA are both homologous to the {5S rRNA-tRNA Asn-tRNA(Ser)-tRNA(Glu)-tRNA(Val)-tRNA(Met)} region of the Bacillus subtilis (BSU) trrnD operon, and to the S12 and S7 r-protein-encoding region (S12/S7 region) in the EC str operon . The 2570-2906 (3'-term.) of the EC 23S rRNA is also homologous to the trrnD . These rRNAs had evolved from a str-like pre-mRNA.

J Mol Biol, 1992 Dec 20, 228(4), 1255 - 8
Crystallization and preliminary X-ray studies of the pectate lyase from Bacillus subtilis; Jenkins J et al.; The pectate lyase (EC 4.2.2.9) from Bacillus subtilis has been crystallized . Crystals of form 1, grown by the hanging drop method using polyethylene glycol as precipitant, diffract to at least 2.4 A resolution . They belong to the spacegroup P2(1) with a = 132.9 A, b = 41.2 A, c = 156.8 A and beta = 114.9 degrees with probably four molecules in the asymmetric unit . A second crystal form grown from 2-methyl-2,4-pentandiol also belongs to the spacegroup P2(1) with a = 55.0 A, b = 88.1 A, c = 50.2 A and beta = 109.0 degrees . These crystals diffract to at least 2.0 A and have one molecule in the asymmetric unit . Both crystal forms are suitable for the determination of high-resolution structures.

Eur J Biochem, 1992 Dec 15, 210(3), 881 - 91
Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy; van Nuland NA et al.; We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments {van Nuland, N . A . J., van Dijk, A . A., Dijkstra, K., van Hoesel, F . H . J., Scheek, R . M . & Robillard, G . T . (1992) Eur . J . Biochem, 203, 483-491} to the side-chain 1H,15N and 13C resonances . From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE 1H-15N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features . A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure . The average root-mean-square positional difference for the C alpha atoms is less than 0.12 nm . The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography {Herzberg, O., Reddy, P., Sutrina, S., Saier, M . H., Reizer, J . & Kapafia, G . (1992) Proc . Natl, Acad . Sci . USA 89, 2499-2503).

FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 233 - 41
Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: fusions with secretable peptides; Simonen M et al.; The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis . We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase . In spite of this, no secretion of the fusion proteins was detected in B . subtilis . With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences . Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane . Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only . We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B . subtilis before the start of translocation, which we postulate to be a late post-translational event.

Biochem J, 1992 Dec 15, 288 ( Pt 3), 1045 - 51
Site-directed mutagenesis and substrate-induced inactivation of beta-lactamase I; Thornewell SJ et al.; The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied . Both the wild-type enzyme and mutants have been used . The kinetics follow a branched pathway of the type recently analysed {Waley (1991) Biochem . J . 279, 87-94} . The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation . A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine . Spontaneous oxidation yielded the disulphide . The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased . A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e . the first alpha-helix . The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected . These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

J Biol Chem, 1992 Dec 15, 267(35), 24989 - 94
Sorbitol dehydrogenase from Bacillus subtilis . Purification, characterization, and gene cloning; Ng K et al.; Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering . As a first step to clone gutB, B . subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized . It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit . Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit . Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established . Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning . A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced . Sequence alignment indicated that the deduced amino acid sequence of B . subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human . In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified . Sequence information related to the structure-function relationships of the enzyme is discussed.

FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 91 - 100
Molecular biology of Bacillus subtilis cytochromes; von Wachenfeldt C et al.; Bacillus subtilis cells must have cytochromes for growth and can synthesize cytochromes of a-, b-, c-, d-, and o-types . After a long lag, our knowledge of the structure, genetics and specific role for these cytochromes is now growing exponentially as the result of recent research . This progress is reviewed here and includes, for example, the discovery of two different cytochrome a systems and genes required for their biogenesis.

FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 217 - 20
Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis; Beijer L et al.; Glycerol and glycerol 3-phosphate uptake in Bacillus subtilis does not involve the phosphotransferase system . In spite of this, B . subtilis mutants defective in the general components of the phosphotransferase system, EnzymeI or Hpr, are unable to grow with glycerol as sole carbon and energy source . Here we show that a Hpr mutant can grow on glycerol 3-phosphate and that glycerol 3-phosphate, but not glycerol, can induce glpD encoding glycerol-3-phosphate dehydrogenase . Induction of glpD also requires the glpP gene product which is a regulator of all known glp genes . Thus the phosphotransferase system general components do not interfere with the overall regulation of the glp regulon . Revertants of a Hpr mutant which can grown on glycerol carry mutations closely linked to the glp region at 75 degrees on the B . subtilis chromosomal map . This region contains the glpP, the glpFK and the glpD operons . The glpFK operon encodes the glycerol uptake facilitator (glpF) and glycerol kinase (glpK) . The present results demonstrate that one of these genes, or their gene products, is the target for phosphotransferase system control of glycerol utilisation . Furthermore we conclude that utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in B . subtilis.

FEBS Lett, 1992 Dec 7, 314(1), 77 - 80
Complex formation between glutamyl-tRNA synthetase and glutamyl-tRNA reductase during the tRNA-dependent synthesis of 5-aminolevulinic acid in Chlamydomonas reinhardtii; Jahn D; The formation of a stable complex between glutamyl-tRNA synthetase and the first enzyme of chlorophyll biosynthesis glutamyl-tRNA reductase was investigated in the green alga Chlamydomonas reinhardtii . Apparently homogenous enzymes, purified after previously established purification protocols were incubated in various combinations with ATP, glutamate, tRNA(Glu) and NADPH and formed complexes were isolated via glycerol gradient centrifugation . Stable complexes were detected only after the preincubation of glutamyl-tRNA synthetase, glutamyl-tRNA reductase with either glutamyl-tRNA or free tRNA(Glu), ATP and glutamate, indicating the obligatory requirement of aminoacylated tRNA(Glu) for complex formation . The further addition of NADPH resulting in the reduction of the tRNA-bound glutamate to glutamate 1-semialdehyde led to the dissociation of the complex . Once complexed to the two enzymes tRNA(Glu) was found to be partially protected from ribonuclease digestion . Escherichia coli, Bacillus subtilis and Synechocystis 6803 tRNA(Glu) were efficiently incorporated into the protein-RNA complex . The detected complexes provide the chloroplast with a potential channeling mechanism for Glu-tRNA(Glu) into chlorophyll synthesis in order to compete with the chloroplastic protein synthesis machinery.

J Mol Biol, 1992 Dec 5, 228(3), 840 - 9
Fate of the SpoIIID switch protein during Bacillus subtilis sporulation depends on the mother-cell sigma factor, sigma K; Halberg R et al.; Sporulation of Bacillus subtilis involves the differentiation of two cell types, the mother cell and the forespore . Two key regulators of mother-cell gene expression are SpoIIID, a DNA-binding protein that activates or represses transcription of many different genes, and sigma K, a subunit of RNA polymerase that directs the enzyme to transcribe genes encoding proteins that form the spore coat . Previous studies showed that SpoIIID is needed to produce sigma K, but suggested that SpoIIID represses sigma K-directed transcription of genes encoding spore coat proteins . Here we show that a feedback loop connects the levels of sigma K and SpoIIID, such that production of sigma K leads to a decrease in the level of SpoIIID . The existence of the feedback loop was demonstrated by using antibodies prepared against SpoIIID to measure the level of SpoIIID during sporulation of wild-type cells, mutants defective in sigma K production, and a mutant engineered to produce sigma K earlier than normal . The feedback loop operates at the level of synthesis and/or stability of spoIIID mRNA, as demonstrated by measuring the level of spoIIID mRNA during sporulation of wild-type cells and mutants defective in sigma K production . Our results suggest that a rise in the level of sigma K during the stage (IV) of spore cortex formation causes a decrease in the level of SpoIIID, which, at least in part, establishes the switch to the stage V (spore coat formation) pattern of mother-cell gene expression.

J Biol Chem, 1992 Dec 5, 267(34), 24819 - 23
An atomic model for protein-protein phosphoryl group transfer; Herzberg O; The high resolution crystal structures of two interacting proteins from the phosphoenolpyruvate:sugar phosphotransferase system, the histidine-containing phosphocarrier protein (HPr) and the IIA domain of glucose permease (IIA(Glc)) from Bacillus subtilis, provide the basis for modeling the transient binary complex formed during the phosphoryl group transfer . The complementarity of the interacting surfaces implies that no major conformational transition is required . The negatively charged phosphoryl group is buried in the interface, suggesting a key role for electrostatic interactions . It is proposed that the phosphoryl transfer is triggered by a switch between two salt bridges involving Arg-17 of the HPr . The first, prior to phosphoryl group transfer, is intramolecular, with the phosphorylated His-15 . The second, during the transfer, is intermolecular, with 2 aspartate residues associated with the active site of IIA(Glc) . Such alternating ion pairs may be mechanistically important in other protein-protein phosphotransfer reactions.

J Biol Chem, 1992 Dec 5, 267(34), 24508 - 15
Cytochrome b560 (QPs1) of mitochondrial succinate-ubiquinone reductase . Immunochemistry, cloning, and nucleotide sequencing; Yu L et al.; Mitochondrial succinate-ubiquinone reductase is composed of two parts, a water-soluble succinate dehydrogenase and a two-polypeptide membrane-anchoring protein fraction (QPs) . The larger polypeptide of QPs is believed to be associated with cytochrome b560 (QPs1) . The structure of QPs1 was studied by immunochemistry and molecular cloning and sequencing . Antibodies against QPs1 were raised in rabbits, purified, and characterized by enzyme-linked immunosorbent assay and Western blotting . The purified antibodies inhibited 75% of the reconstitutive activity of QPs and reacted with both submitochondrial particles (SMP) and mitoplasts . The binding of these antibodies to SMP was greatly increased when succinate dehydrogenase was removed from SMP by alkaline treatment, indicating that QPs1 is a transmembranous protein and that some of its specific epitopes are covered by succinate dehydrogenase . Anti-QPs1 antibodies were used to screen one cDNA clone encoding QPs1 from a bovine heart cDNA lambda gt11 expression library . The cDNA insert is 946 base pairs with an open reading frame of 396 base pairs that encodes for 132 amino acid residues . The molecular weight of QPs1, calculated from the deduced amino acid sequence, is 14,320 . Although the apparent molecular weight of QPs1, estimated by high resolution SDS-polyacrylamide gel electrophoresis, is approximately 11,000, the existence of a presequence was ruled out by mass spectrometric analysis of protein fragments . QPs1 is a very hydrophobic protein . Three probable membrane-spanning segments were revealed by a hydropathy plot of the sequence . QPs1 has a higher sequence similarity to the sdhC peptide of Escherichia coli than to the sdhC peptide (cytochrome b558) of Bacillus subtilis . Like the bacterial proteins, QPs1 has 2 conserved histidines at positions 34 and 90 . The conserved nature and similar location of these 2 histidines, on the matrix-side surface of the membrane, suggest that they are involved in heme ligation of cytochrome b560.

Science, 1992 Dec 4, 258(5088), 1633 - 6
Production and initial characterization of bionites: materials formed on a bacterial backbone; Mendelson NH; The addition of soluble metal salts of calcium, iron, or copper to cultures of Bacillus subtilis grown in web form nucleated precipitation at the surface of the bacterial cell walls . The mineralized cell filaments can be drawn into a fiber that when dried consists of a bacterial thread backbone carrying an inorganic solid . The ratios of organic to inorganic components (by weight) in the stiff brittle materials, called bionites, were: 1.08 for fe(2)bactonite, 1.8 for calbactonite, 2.3 for fe(3)bactonite, and 5 for cu(2)bactonite . X-ray photoelectron spectra suggest that the fe(3)bactonite contains Fe2O3, that calbactonite contains calcium carbonate, and that cu(2)bactonite contains CuCl (Cu I) . Acid-base reactions of the bionites are compatible with these identifications . Burning out the organic phase of the febactonites yields a black magnetic material, presumably magnetite . The burnt cubactonite appears to yield elemental Cu(s) . Calbactonite upon hydration was able to retain a genetically engineered enzymatic activity.

Mol Gen Genet, 1992 Dec, 236(1), 60 - 4
Intramolecular homologous recombination in Bacillus subtilis 168; Alonso JC et al.; Plasmid resolution from a phage::plasmid chimera was used to measure directly intramolecular recombination in Bacillus subtilis . The system is based on a sigma-replicating plasmid (pC194) cloned into a dispensable region of the lytic bacteriophage SPP1 . The plasmid, which confers chloramphenicol resistance, is resolved when SPP1::pC194 phages infect B . subtilis cells, provided the chimera carries a functional, intact copy of the plasmid repH gene . Intramolecular homologous recombination was independent of the RecA and RecL-RecR functions, but dependent on RecF, RecB, RecG, RecP, RecH and AddAB functions . These results are consistent with the hypothesis that B . subtilis has multiple pathways for genetic recombination and allow us to tentatively place the recB and recG genes into a new epistatic group epsilon.

FEMS Microbiol Lett, 1992 Dec 1, 78(2-3), 277 - 80
The nature and site of biocide-induced sublethal injury in Bacillus subtilis spores; Williams ND et al.; Spores of Bacillus subtilis NCTC 8236 exposed at 22 degrees C to test biocides (alkaline glutaraldehyde, an iodophor, Lugol's solution, sodium hypochlorite and sodium dichloroisocyanurate) demonstrated varying degrees of injury to stressing agents (sodium hydroxide, sodium lauryl sulphate, polymyxin B sulphate or cetylpyridinium chloride) incorporated into a recovery agar medium . This injury to stressing agents was expressed mainly during outgrowth.

J Gen Microbiol, 1992 Dec, 138 ( Pt 12), 2609 - 18
Structure and function of the spoIIIJ gene of Bacillus subtilis: a vegetatively expressed gene that is essential for sigma G activity at an intermediate stage of sporulation; Errington J et al.; The spo-87 mutation is one of two sporulation mutations originally used to define the spo0J locus of Bacillus subtilis . We now show that it blocks sporulation after completion of prespore engulfment (stage III) . Surprisingly, the operon is expressed vegetatively, probably from a sigma A-dependent promoter, and its expression is shut down at the transcriptional level at about the onset of sporulation . DNA sequencing reveals that the locus defined by spo-87, which we now designate spoIIIJ, consists of a bicistronic operon . However, only the first gene is essential for sporulation; the function of the second cistron is cryptic . The predicted SpoIIIJ product has an M(r) of 29,409 . It probably forms a lipoprotein and is rich in basic and hydrophobic amino acids . Mutations in spoIIIJ abolish the transcription of prespore-specific genes transcribed by the sigma G form of RNA polymerase but not transcription of the spoIIIG gene encoding sigma G . The SpoIIIJ product could be involved in a signal transduction pathway coupling gene expression in the prespore to events in the mother cell, or it could be necessary for essential metabolic interactions between the two cells.

Appl Environ Microbiol, 1992 Dec, 58(12), 4016 - 25
Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the NH2-terminal region of the enzyme; Fujiwara S et al.; Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is produced mainly by Bacillus strains . CGTase from Bacillus macerans IFO3490 produces alpha-cyclodextrin as the major hydrolysis product from starch, whereas thermostable CGTase from Bacillus stearothermophilus NO2 produces alpha- and beta-cyclodextrins . To analyze the cyclization characteristics of CGTase, we cloned different types of CGTase genes and constructed chimeric genes . CGTase genes from these two strains were cloned in Bacillus subtilis NA-1 by using pTB523 as a vector plasmid, and their nucleotide sequences were determined . Three CGTase genes (cgt-1, cgt-5, and cgt-232) were isolated from B . stearothermophilus NO2 . Nucleotide sequence analysis revealed that the three CGTase genes have different nucleotide sequences encoding the same amino acid sequence . Base substitutions were found at the third letter of five codons among the three genes . Each open reading frame was composed of 2,133 bases, encoding 711 amino acids containing 31 amino acids as a signal sequence . The molecular weight of the mature enzyme was estimated to be 75,374 . The CGTase gene (cgtM) of B . macerans IFO3490 was composed of 2,142 bases, encoding 714 amino acids containing 27 residues as a signal sequence . The molecular weight of the mature enzyme was estimated to be 74,008 . The sequence determined in this work was quite different from that reported previously by other workers . From data on the three-dimensional structure of a CGTase, seven kinds of chimeric CGTase genes were constructed by using cgt-1 from B . stearothermophilus NO2 and cgtM from B . macerans IFO3490 . We examined the characteristics of these chimeric enzymes on cyclodextrin production and thermostability . It was found that the cyclization reaction was conferred by the NH2-terminal region of CGTase and that the thermostability of some chimeric enzymes was lower than that of the parental CGTases.

J Bacteriol, 1992 Dec, 174(24), 8148 - 51
Properties of purified sporlets produced by spoII mutants of Bacillus subtilis; Magill NG et al.; A number of abortively disporic spoII mutants of Bacillus subtilis released their forespore compartments (termed stage II sporlets) after mother cell lysis during sporulation in nutrient exhaustion or resuspension media . Stage II sporlets were viable and contained levels of ATP and a number of enzymes similar to those in cells 2 to 3 h after sporulation . However, stage II sporlets carried out essentially no macromolecular synthesis, a result suggesting that they were in a quiescent state . The nucleoid of these quiescent stage II sporlets was significantly condensed relative to that in the original vegetative cells, as was previously found to take place 1 to 2 h after initiation of sporulation (B . Setlow, N . Magill, P . Febbroriello, L . Nakhimousky, D . E . Koppel, and P . Setlow, J . Bacteriol . 173:6270-6278, 1991) . Stage II sporlets may be a useful model system for analysis of forespore properties early in stage II of sporulation.

J Bacteriol, 1992 Dec, 174(24), 7954 - 62
Stabilization of phosphorylated Bacillus subtilis DegU by DegR; Mukai K et al.; The production of Bacillus subtilis extracellular proteases is under positive and negative regulation . The functional role of degR, one of the positive regulators, was studied in relation to the degS and degU gene products, which belong to the bacterial two-component regulatory system . Studies with a translational fusion between the Escherichia coli lacZ and the Bacillus subtilis subtilisin (aprE) genes indicated that the stimulatory site of DegR lay upstream of position -140, with the region upstream of position -200 being the major target . It was also found that degS and degU were epistatic to degR . These results suggested some relationship among the degR, degS, and degU gene products . The DegR protein was purified to homogeneity, and its in vitro effect on the phosphorylation reaction involving DegS and DegU was studied . For this purpose, a soluble-extract system in which the formation and dephosphorylation of DegU-phosphate could be examined was devised . The addition of DegR to the soluble-extract system enhanced the formation of DegU-phosphate . The enhancing effect was found to be due to the protection of DegU-phosphate from dephosphorylation . From these results, it was concluded that the positive effect of DegR on the production of the extracellular proteases is brought about by the stabilization of DegU-phosphate, which in turn may result in the stimulation of transcription of the exoprotease genes.

Proc Natl Acad Sci U S A, 1992 Dec 1, 89(23), 11401 - 5
Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts; Nuez B et al.; Transcription from the late promoter, PA3, of Bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4 . A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process . Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA . Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA . These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter . In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.

Gene, 1992 Dec 1, 122(1), 187 - 92
A series of shuttle vectors for Bacillus subtilis and Escherichia coli; Bruckner R; A series of shuttle vectors for Bacillus subtilis and Escherichia coli was developed . These are derived from one basic construct composed of parts of the Gram+ plasmid pUB110 and the Gram- plasmid pBR322 . They contain multiple cloning sites flanked by transcriptional terminators . In one plasmid, a vegetative B . subtilis promoter drives transcription of inserted genes . For the construction of operon and gene fusions, the cat of pUB112 and the lacZ gene of E . coli were employed as reporter genes . With these vectors, cloning and expression of genes as well as probing of regulatory signals can be performed in B . subtilis and E . coli.

Biosci Biotechnol Biochem, 1992 Dec, 56(12), 1995 - 2002
Isolation and characterization of the groES and groEL genes of Bacillus subtilis Marburg; Tozawa Y et al.; The complete set of groES and groEL gene homologues from Bacillus subtilis Marburg 168 was identified, cloned, and characterized . The nucleotide sequence indicated the presence of two open reading frames corresponding to the groES and groEL genes . The presumptive GroES and GroEL proteins were calculated to be polypeptides of 10,175 and 57,175 Da, respectively, and showed extensive sequence similarities with the known GroES and GroEL proteins of Escherichia coli and Mycobacterium tuberculosis . A heat-inducible transcript initiated upstream of the groES coding region was identified by primer-extension analysis of in vivo transcripts, indicating that the two genes consist of an operon . At least six heat-shock inducible proteins were identified in the cell extract of heat treated B . subtilis . Two proteins of 10 and 60 kDa overproduced in B . subtilis cells carrying a multi-copy groES and groEL plasmid were demonstrated to correspond to two out of the six heat-shock inducible proteins . The groES and groEL genes of B . subtilis were physically mapped on the 60 degrees region of a 360 degrees map and genetically mapped at the position of 40% linkage with the purB locus using PBS1 transduction of the groEL genes tagged with a chloramphenicol resistance (chlr) marker.

J Biochem (Tokyo), 1992 Dec, 112(6), 828 - 33
Inactivation of Bacillus subtilis glutamine synthetase by metal-catalyzed oxidation; Kimura K et al.; Instability of Bacillus subtilis glutamine synthetase in crude extracts was attributed to site-specific oxidation by a mixed-function oxidation, and not to limited proteolysis by intracellular serine proteases (ISP) . The crude extract from B . subtilis KN2, which is deficient in three intracellular proteases, inactivated glutamine synthetase similarly to the wild-type strain extract . To understand the structural basis of the functional change, oxidative modification of B . subtilis glutamine synthetase was studied utilizing a model system consisting of ascorbate, oxygen, and iron salts . The inactivation reaction appeared to be first order with respect to the concentration of unmodified enzyme . The loss of catalytic activity was proportional to the weakening of subunit interactions . B . subtilis glutamine synthetase was protected from oxidative modification by either 5 mM Mn2+ or 5 mM Mn2+ plus 5 mM ATP, but not by Mg2+ . The CD-spectra and electron microscopic data showed that oxidative modification induced relatively subtle changes in the dodecameric enzyme molecules, but did not denature the protein . These limited changes are consistent with a site-specific free radical mechanism occurring at the metal binding site of the enzyme . Analytical data of the inactivated enzyme showed that loss of catalytic activity occurred faster than the appearance of carbonyl groups in amino acid side chains of the protein . In B . subtilis glutamine synthetase, the catalytic activity was highly sensitive to minute deviations of conformation in the dodecameric molecules and these subtle changes in the molecules could be regarded as markers for susceptibility to proteolysis.

Rev Saude Publica, 1992 Dec, 26(6), 379 - 83
{Ethylene oxide sterilization . I . Influence of the sporulation medium on the resistance of spores of Bacillus subtilis var . niger}; Pinto Tde J et al.; Some elements influencing the resistance of spores used in ethylene oxide sterilization process control are standardized . Spores of Bacillus subtilis var . niger were produced in chemically defined liquid and solid sporulation media to a total of 12 lots; after standardization of the number of spores, they were challenged by sub-lethal cycles, followed by a lethality study . According to the statistical model applied, there were no differences between the resistance of spores produced in chemically defined liquid and those produced in solid sporulation media . The advantage of the solid sporulation media consists in the larger production of spores.

Appl Environ Microbiol, 1992 Dec, 58(12), 3837 - 44
The membrane-induced proton motive force influences the metal binding ability of Bacillus subtilis cell walls; Urrutia Mera M et al.; Bacillus subtilis 168 is a gram-positive bacterium whose cell wall contains the highly electronegative polymers peptidoglycan (chemotype A1 gamma) and glycerol-based teichoic acid to produce a surface with a net negative charge with high metal binding capacity . During metabolism, a membrane-induced proton motive force continuously pumps protons into the wall fabric . As a result, a competition between protons and metal ions for anionic wall sites occurs, and less metal is bound in living cells than in nonliving cells or those in which the plasma membrane has been uncoupled . This was shown by using two metallic ions, UO2(2+) and Sc3+, on control cells, cells uncoupled with either carbonyl cyanide m-chlorophenylhydrazone or NaN3, or cells killed by gamma radiation . Transmission electron microscopy, energy-dispersive X-ray spectroscopy, and inductively coupled plasma atomic-emission spectroscopy showed that more metal was retained in the walls of nonliving cells and those with deenergized membranes than in their living counterparts.

Boll Chim Farm, 1992 Dec, 131(11), 401 - 8
Studies on the antibiotic resistance of Bacillus subtilis strains used in oral bacteriotherapy; Mazza P et al.; Bacillus subtilis antibiotic resistant strains used in oral bacteriotherapy were tested for the resistance to new therapeutically useful antibiotics . Chromosomal mutations originally selected in these strains proved to confer resistances also to these antibiotics . To investigate the stability of the antibiotic resistance markers present in B . subtilis O/C, T, N/R, SIN strains, we considered resistances to Chloramphenicol, Tetracycline, Rifampicin and Streptomycin . Resistances to Tetracycline, Rifampicin and Streptomycin were stably maintained for at least 200 generations in the absence of selective pressure . Chloramphenicol resistance proved to be inducible, showing a progressive loss when the resistant strain was grown in the absence of the antibiotic and a return to its original resistance levels after growth in the presence of the antibiotic . By in vitro and in vivo experiments we demonstrated the absence of homologous transfer of resistance markers among the resistant strains.

Wei Sheng Wu Xue Bao, 1992 Dec, 32(6), 394 - 9
{The expression of 130kDa mosquitocidal protein gene of Bacillus thuringiensis subsp . israelensis in Bacillus subtilis}; Zhang X et al.; Two recombinant plasmid pFZ1 and pFZ2 containing Bti 130kDa mosquitocidal protein gene in opposite insertion orientation were constructed . The expression of 130kDa mosquitocidal protein of Bti in Bacillus subtilis was confirmed by western blotting . The mosquito-larvicidal activity against the larvae of Aedes albopictus was shown by the bioassay.

Biochimie, 1992 Dec, 74(12), 1047 - 51
Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A; Maget-Dana R et al.; Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis . Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant . The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A . This synergistic effect seems to be in relation with interactions between iturin A and surfactin . Iturin A adsorbs to and penetrates into surfactin monolayers . Iturin A and surfactin are miscible and interact specifically in mixed monolayers.

Can J Microbiol, 1992 Dec, 38(12), 1270 - 3
Promotion of apple tree growth and fruit production by the EBW-4 strain of Bacillus subtilis in apple replant disease soil; Utkhede RS et al.; A field trial was conducted near Kelowna, British Columbia, to determine the effect of biological treatments alone and in combination with formalin fumigation in apple replant disease soil . The response was measured by the increase in cross-sectional trunk area, total shoot growth, and fruit yield of McIntosh apple trees on M.26 rootstock . The postplanting drench application of strain EBW-4 of Bacillus subtilis alone was consistently effective in increasing cross-sectional trunk area for 5 years, total shoot growth for 4 years, and fruit yield for 3 years . The biological agent EBW-4 of B . subtilis in combination with formalin fumigation was also effective in promoting total shoot growth and cross-sectional trunk area . The application of formalin fumigation alone was effective in increasing shoot growth for 2 years and cross-sectional trunk area for 1 year only . This treatment did not increase fruit yield for 3 years . The consistent performance of strain EBW-4 of B . subtilis during 1986-1991 indicates that this bacterium has the potential for biological control of replant disease under orchard conditions in the Okanagan Valley of British Columbia.

Am J Infect Control, 1992 Dec, 20(6), 291 - 300
Significant factors in the disinfection and sterilization of flexible endoscopes; Vesley D et al.; BACKGROUND: Many nosocomial infection outbreaks have been linked to improper disinfection of the flexible endoscopes used in hospitals and clinics . The objective of this study was to evaluate the efficacy of scope disinfection with glutaraldehyde and hydrogen peroxide in manual and mechanical protocols . METHODS: Bacillus subtilis and Pseudomonas cepacia were the test organisms . Each channel in two different endoscopes was seeded and evaluated separately . Residual chemical germicide levels in the channels and in the work environment were also measured . RESULTS: Parametric analyses were carried out on log transformations of number of colony-forming units recovered . Repeated measures analysis demonstrated that both the type of disinfectant and the method of washing were significant factors for disinfection . CONCLUSIONS: Hydrogen peroxide proved to be more efficacious than glutaraldehyde for killing or removing B . subtilis in a 10-minute contact period . Automatic disinfection was more efficacious than manual disinfection for killing or removing B . subtilis in a 10-minute contact period . The channel being disinfected also proved to be a significant factor, with carbon dioxide and elevator channels the most difficult to disinfect consistently.

J Bacteriol, 1992 Dec, 174(24), 8163 - 5
Acholeplasma laidlawii has tRNA genes in the 16S-23S spacer of the rRNA operon; Nakagawa T et al.; We amplified the 16S-23S rRNA intergenic spacer region of Acholeplasma laidlawii PG8 by polymerase chain reaction (PCR) and obtained two specific PCR products in different sizes . We have sequenced both PCR products and found that one of them has sequence homologous to the spacer tRNA genes in Bacillus subtilis . This is the first evidence of tRNA genes between the 16S-23S rRNA intergenic spacer regions in members of the class Mollicutes.

J Biol Chem, 1992 Nov 25, 267(33), 23942 - 9
The carboxyl terminus of RAP30 is similar in sequence to region 4 of bacterial sigma factors and is required for function; Garrett KP et al.; Transcription factor beta gamma (RAP30/74) from rat liver was previously shown in biochemical studies to control the binding of RNA polymerase II to promoters by a mechanism analogous to that utilized by bacterial sigma factors, by decreasing the affinity of polymerase for nonpromoter sites on DNA and by increasing the affinity of the enzyme for the preinitiation complex (Conaway, R . C., Garrett, K . P., Hanley, J . P., and Conaway, J . W . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 6205-6209) . By constructing and analyzing mutants of beta gamma, we have identified a novel functional domain located in the carboxyl terminus of the gamma (RAP30) subunit . This domain shares sequence similarity with region 4 of bacterial sigma factors; in particular, it exhibits striking similarity to the carboxyl-terminal regions 4.1 and 4.2 of SpoIIIC (Bacillus subtilis sigma k) . Evidence from biochemical studies argues that a mutant gamma (RAP30), lacking amino acid sequences similar to sigma homology region 4.2, is able to assemble with the beta (RAP74) subunit to form a mutant beta gamma (RAP30/74) with impaired ability to interact with RNA polymerase II.

J Biol Chem, 1992 Nov 25, 267(33), 23782 - 8
Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580; Kakudo S et al.; A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized . The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis . This protease, which we propose to call BLase (glutamic acid-specific protease from B . licheniformis ATCC 14580), was characterized enzymatically . Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds . Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme . The findings clearly indicate that BLase can be classified as a serine protease . To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B . licheniformis ATCC 14580, and the nucleotide sequence was determined . Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues . The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity . Its key physical and chemical characteristics were the same as those of the wild-type enzyme . BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K . (1992) Eur . J . Biochem . 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.

Biochim Biophys Acta, 1992 Nov 23, 1112(1), 29 - 38
A patch-clamp study of Bacillus subtilis; Szabo I et al.; In patch-clamp experiments on giant protoplasts of the Gram-positive bacterium Bacillus subtilis, membrane stretch resulted in an initial transient collapse of the membrane resistance, after which stretch-activated, voltage modulated, high-conductance channels could be observed . The channel open probability increased exponentially with applied suction and positive voltage, as a result of variations of both the mean open and the mean closed times . The substate structure and other characteristics of the electrical activity suggested the presence of a family of pores exhibiting cooperative behavior . A role in osmotic protection is suggested . In the intact bacteria, the pores may be part of an unidentified envelope apparatus, having other functions as well.

Biochim Biophys Acta, 1992 Nov 20, 1160(2), 188 - 92
Nucleophile specificity in alpha-chymotrypsin- and subtilisin-(Bacillus subtilis strain 72) catalyzed reactions; Gololobov MYu et al.; Nucleophilic properties of amino-acid amides were studied systematically in acyl-transfer reactions catalyzed by alpha-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal-L-Ala-L-Ala-L-PheOMe as the acyl-group donor . In alpha-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: D-AlaNH2 < GlyNH2 < L-AlaNH2 < L-SerNH2 < L-ThrNH2 < L-HisNH2 < L-ValNH2 < L-LeuNH2 < L-TrpNH2 < L-MetNH2 < L-NvaNH2 < L-PheNH2 < L-IleNH2 < L-TyrNH2 < L-ArgNH2 . In reactions catalyzed by subtilisin 72, the reactivities increase as follows: L-LeuNH2 < L-IleNH2 < L-ThrNH2 < L-ArgNH2 < L-TrpNH2 < L-NvaNH2 < L-ValNH2 < L-MetNH2 < L-AlaNH2 < L-SerNH2 < D-AlaNH2 < GlyNH2 . In alpha-chymotrypsin-catalyzed reactions, hydrophobic interactions are entirely responsible for the differences between the reactivity of the nucleophiles for amides of all the amino-acids tested with the exception of D-AlaNH2, L-ArgNH2 and L-TyrNH2 . In reactions catalyzed by subtilisin 72, amino-acid side-chain characteristics and the nucleophile reactivities are not related . The data obtained show the low selectivity of the S1' subsite of subtilisin 72 and high specificity of this subsite in alpha-chymotrypsin.

Gene, 1992 Nov 2, 121(1), 63 - 9
Genetic suppression analysis of sigma E interaction with three promoters in sporulating Bacillus subtilis; Diederich B et al.; Genetic evidence suggests that the sigma (sigma) subunit of RNA polymerase determines the specificity of promoter utilization, by making sequence-specific contacts with DNA . We examined the effects of two single amino acid(aa) substitutions in sigma E on the utilization of mutated derivatives of three different promoters in sporulating Bacillus subtilis . We found allele-specific suppression of mutations in all three promoters by each aa substitution in sigma E . These results provide strong evidence that sigma E interacts with each of these promoters in vivo . Moreover, the specificity of suppression of the mutations by the aa substitutions in sigma E lead us to speculate that the Met124 of sigma E closely contacts two adjacent bp in the -10 region of the promoters.

J Bacteriol, 1992 Nov, 174(21), 6815 - 21
Effects of amino acid substitutions in the -10 binding region of sigma E from Bacillus subtilis; Jones CH et al.; The sigma subunit of bacterial RNA polymerase is required for specific binding to promoters . One region in most sigma factors makes sequence-specific contacts at the -10 region of its cognate promoters . To test the role of the amino acids in this -10 binding region, we examined the effects of 49 single-amino-acid substitutions in sigma E from Bacillus subtilis . We assayed the effect of each amino acid substitution on spore formation because sigma E is essential for endospore formation in B . subtilis . Our results showed that substitutions at several positions, including the highly conserved aromatic amino acid at position 102, had little or no detectable effect . Substitutions at another position, position 117, produced dominant negative mutations; we suggest that these mutations allow RNA polymerase containing the mutant sigma factor to bind specifically to promoters but prevent transcription initiation . Of the recessive defective alleles, those that produced substitutions at positions 113, 115, and 120 produced the most defective sigma factors . These results suggest that the residues at or near these positions in wild-type sigma E play important roles in sigma E function.

FEBS Lett, 1992 Nov 9, 312(2-3), 132 - 8
Tandem translation of Bacillus subtilis initiation factor IF2 in E . coli . Over-expression of infBB.su in E . coli and purification of alpha- and beta-forms of IF2B.su; Hubert M et al.; The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E . coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su . In this work we have cloned infBB.su in E . coli cells . Two proteins of molecular mass identical to the B . subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure . The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene . The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe . The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon . This is a new example of the unusual tandem translation in E . coli of an open mRNA reading frame.

J Biol Chem, 1992 Nov 5, 267(31), 22512 - 21
Characterization of the Escherichia coli uracil-DNA glycosylase.inhibitor protein complex; Bennett SE et al.; The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein was characterized and shown to form a stable complex with Escherichia coli uracil-DNA glycosylase (Ung) . As determined by mass spectrometry, the Ugi protein had a molecular weight of 9,474 . We confirmed this value by sedimentation equilibrium centrifugation and determined that Ugi exists as a monomeric protein in solution . Amino acid analysis performed on both Ugi and Ung proteins was in excellent agreement with the amino acid composition predicted from the respective nucleotide sequence of each gene . The Ung.Ugi complex was resolved from its constitutive components by nondenaturing polyacrylamide gel electrophoresis and shown to possess a 1:1 stoichiometry . Analytical ultracentrifugation studies revealed that the Ung.Ugi complex had a molecular weight of 35,400, consistent with the complex containing one molecule each of Ung and Ugi . The acidic isoelectric points of the protein species were 6.6 (Ung) and 4.2 (Ugi), whereas the Ung.Ugi complex had an isoelectric point of 4.9 . Dissociation of the Ung.Ugi complex by SDS-polyacrylamide gel electrophoresis revealed no apparent alteration in the molecular weight of either polypeptide subsequent to binding . Furthermore, when the Ung.Ugi complex was treated with urea and resolved by urea-polyacrylamide gel electrophoresis, both uracil-DNA glycosylase and inhibitor activities were recovered from the dissociated complex . Thus, the complex seems to be reversible . In addition, we demonstrated that the Ugi interaction with Ung prevents enzyme binding to DNA and dissociates uracil-DNA glycosylase from a preformed DNA complex.

Gene, 1992 Nov 2, 121(1), 137 - 42
A novel Bacillus subtilis expression vector based on bacteriophage phi 105; Gibson RM et al.; We have developed a novel expression vector based on the bacteriophage phi 105, and employed it for the production of mutant beta-lactamases in Bacillus subtilis . Expression of the beta-lactamase-encoding gene was low when cloned into the prophage under the control of its own promoter . However, expression was considerably elevated when the gene was inserted into the phage genome in the same orientation as phage transcription . A defective phi 105 vector was constructed with a deletion removing a region needed for cell lysis, and with a mutation in the immunity repressor, rendering it temperature sensitive . Production of beta-lactamase could then be induced by a shift in temperature and without concomitant cell lysis, facilitating purification of the protein from the culture supernatant . This phage has considerable potential for development as a vector for controllable production of heterologous proteins in B . subtilis.

Mol Gen Genet, 1992 Nov, 235(2-3), 333 - 9
Characterization of the secretion efficiency of a plant signal peptide in Bacillus subtilis; Ribbe J et al.; The ability of the Bacillus subtilis secretion machinery to interact with a heterologous signal peptide was studied using a plant (wheat alpha-amylase) signal peptide . The plant signal peptide was capable of mediating secretion of Escherichia coli alkaline phosphatase and B . amyloliquefaciens levansucrase from B . subtilis . This secretion was dependent on the plant signal peptide, as deletion of five amino acids from the hydrophobic core resulted in a block of secretion . Attempts to improve the efficiency of the plant signal peptide in B . subtilis were made by increasing the length of the hydrophobic core from 10 to 16 residues by insertion of 2, 4, 5 or 6 amino acids . None of the alterations improved the secretion efficiency relative to the wild-type plant signal peptide.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10479 - 83
Supercoiled DNA wraps around the bacteriophage phi 29 head-tail connector; Turnquist S et al.; Supercoiled pBR322 DNA wraps around the outside of the isolated Bacillus subtilis bacteriophage phi 29 head-tail connector, the crux of the DNA packaging machine of the viral precursor capsid or prohead . The contour length of the supercoiled DNA, determined by EM, decreased by approximately 180 base pairs for each connector bound . Mass and radial density determinations by scanning transmission EM showed that the increased mass of the connector-DNA complex relative to the connector alone was equivalent to approximately 170 base pairs of DNA and was located around the outside of the connector . Topoisomerase I treatment of the complexes followed by deproteinization suggested that supercoils were restrained by the connectors . Connectors bound linear and open-circular plasmid DNAs inefficiently but were not wrapped by these DNAs . The wrapping of supercoiled DNA around the isolated phi 29 connector is hypothesized to reflect the initiation phase of the normal process of DNA packaging . Packaging substrates would be supercoiled, wrapped by the connector, linearized, and translocated by rotation of the connector relative to the viral capsid with the aid of ATP hydrolysis.

J Bacteriol, 1992 Nov, 174(22), 7194 - 201
Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis; Ives CL et al.; BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC . The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene . The small open reading frame has been designated bamHIC (for BamHI controlling element) . It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli . Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated . The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans . The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome . In B . subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.

J Bacteriol, 1992 Nov, 174(22), 7144 - 8
Activity of mutant sigma F proteins truncated near the C terminus; Min KT et al.; sigma F, the product of the spoIIAC gene of Bacillus subtilis, is homologous in amino acid sequence throughout most of its length with several other sigma factors of B . subtilis and Escherichia coli . However, 8 residues from the C terminus the homology abruptly breaks down, suggesting that the C-terminal tail of the protein may be dispensable . It is known that an amber mutation at the 11th codon (wild-type glutamine 245) from the C terminus abolishes the function of the sigma factor . We have now placed chain-terminating codons at the ninth codon (wild-type lysine 247), the eighth codon (wild-type valine 248), or the seventh codon (wild-type glutamine 249) from the C terminus . We have tested the resulting mutants for their capacity to sporulate and for their ability to transcribe from a promoter (spoIIIG) that is normally read by RNA polymerase bound to sigma F (E sigma F) . The results indicate that a mutant sigma F lacking the terminal 7 residues functions almost normally, which suggests that glutamine 249 is dispensable . By contrast, lysine 247 is crucial for the activity of sigma F: deletion of the 9 C-terminal residues totally inactivates the protein . When the terminal 8 residues were deleted, placing lysine 247 at the C terminus, the transcriptional activity of the factor is reduced by about 80%: we attribute this effect to neutralization of the positive charge of lysine 247 by formation of a salt bridge with the -COO- terminus.

J Bacteriol, 1992 Nov, 174(21), 6997 - 7002
Imprecise excision of plasmid pE194 from the chromosomes of Bacillus subtilis pE194 insertion strains; Conrad B et al.; Plasmid pE194 has been shown to be rescued by integration after cultivation of infected Bacillus subtilis recE4 cells at a restrictive high temperature . The plasmid is also spontaneously excised from the chromosome at a low frequency by precise or imprecise excision (J . Hofemeister, M . Israeli-Reches, and D . Dubnau, Mol . Gen . Genet . 189:58-68, 1983) . We have investigated nine excision plasmids, carrying insert DNA 1 to 6 kbp in length, either in a complete pE194 or in a partially deleted pE194 copy . Type 1 (additive) excision plasmids have the left- and right-junction DNAs preserved as 13-bp direct repeats (5'-GGGGAGAAAACAT-3') corresponding to the region between positions 864 and 876 in pE194 . In type 2 (substitutive) excision plasmids, a conserved 13-bp sequence remains only at the right junction while the left junction has been deleted during the excision process . The type 3 excision plasmid carries at each junction the tetranucleotide 5'-TCCC-3', present in pE194 between positions 1995 and 1998 . Although we isolated the excision plasmids from different integration mutants, the insert DNAs of eight independently isolated plasmids showed striking sequence homology, suggesting that they originated from one distinct region of the B . subtilis chromosome . Thus, we postulate that imprecise excision of pE194 occurs most frequently after its translocation from the original insertion site into a preferred excision site within the host chromosome . The imprecise excision from this site occurs at excision breakpoints outside the pE194-chromosome junctions in a chromosomal region which remains to be investigated further.

J Bacteriol, 1992 Nov, 174(21), 6763 - 70
Characterization of the Bacillus subtilis rpsD regulatory target site; Grundy FJ et al.; The Bacillus subtilis rpsD gene, which encodes ribosomal protein S4, is subject to autogenous regulation . Repression of rpsD expression by excess S4 protein was previously shown to be affected by mutations in the leader region of the gene . A large number of deletion and point mutations in the leader region were generated, and their effect on repression by S4 in vivo was tested . These studies indicated that the required region was within positions +30 to +190 relative to the transcription start point . Replacement of the rpsD promoter with a lac promoter derivative which is expressed in B . subtilis had no effect, indicating that repression by S4 occurs at a level subsequent to transcription initiation . The rpsD leader region was isolated from several Bacillus species . Members of the B . subtilis group, as defined by analysis of 16S rRNA sequence, contained a leader region target site very closely related in structure to that of B . subtilis, despite considerable primary sequence variation; the B . brevis rpsD leader contained some but not all of the structural features found in the regulatory target sites of the other Bacillus species . Very little similarity to the Escherichia coli alpha operon S4 target site was found at either the primary-sequence or the secondary-structure level . Mutagenic and phylogenetic data indicate that the secondary structure of the leader region regulatory target site contains two large stem-loop domains . The first of these helices has a side loop which is essential for autoregulation, is highly conserved among Bacillus rpsD genes, and is similar to a region of 16S rRNA important in S4 binding.

J Bacteriol, 1992 Nov, 174(21), 6729 - 42
The divIVB region of the Bacillus subtilis chromosome encodes homologs of Escherichia coli septum placement (minCD) and cell shape (mreBCD) determinants; Varley AW et al.; Mutation of the divIVB locus in Bacillus subtilis causes frequent misplacement of the division septum, resulting in circular minicells, short rods, and filaments of various sizes . The divIVB1 mutant allele maps to a region of the chromosome also known to encode sporulation (spo0B, spoIVF, spoIIB) and cell shape (rodB) determinants . This study reports the cloning and sequence analysis of 4.4 kb of the B . subtilis chromosome encompassing the divIVB locus . This region contains five open reading frames (ORFs) arranged in two functionally distinct gene clusters (mre and min) and transcribed colinearly with the direction of replication . Although sequence analysis reveals potential promoters preceding each gene cluster, studies with integrational plasmids suggest that all five ORFs are part of a single transcription unit . The first gene cluster contains three ORFs (mreBCD) homologous to the mre genes of Escherichia coli . We show that rodB1 is allelic to mreD and identify the rodB1 mutation . The second gene cluster contains two ORFs (minCD) homologous to minC and minD of E . coli but lacks a minE homolog . We show that divIVB1 is allelic to minD and identify two mutations in the divIVB1 allele . Insertional inactivation of either minC or minD or the presence of the divIVB region on plasmids produces a severe minicell phenotype in wild-type cells . Moreover, E . coli cells carrying the divIVB region on a low-copy-number plasmid produce minicells, suggesting that a product of this locus may retain some function across species boundaries.

J Bacteriol, 1992 Nov, 174(21), 6717 - 28
Identification of Bacillus subtilis genes for septum placement and shape determination; Levin PA et al.; The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells . We report the cloning of the wild-type allele of divIVB1 and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD . Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E . coli shape-determining genes mreB, mreC, and mreD . The B . subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodB1) that causes the production of aberrantly shaped cells under restrictive conditions . Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon . The possible involvement of min gene products in the switch from medial to polar placement of the septum during sporulation is discussed.

Curr Microbiol, 1992 Nov, 25(5), 279 - 82
Molecular cloning and expression of Bacillus subtilis bglS gene in Saccharomyces cerevisiae; Chen Y et al.; A 2.7-kb EcoRI DNA fragment carrying a Bacillus subtilis endo-beta-1,3-1, 4-glucanase gene (bglS) from the E . coli plasmid pFG1 was cloned into an Escherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH . The hybrid plasmid was used to transform Saccharomyces cerevisiae, and the bglS gene was expressed . Variation between levels of bglS gene expression in S . cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment . Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found in B . subtilis, but the expression level of bglS gene in S . cerevisiae (YCSH) was much lower than that in E . coli (YCSH).

J Biotechnol, 1992 Nov, 26(2-3), 331 - 6
Production of antimicrobials by Bacillus subtilis MIR 15; Perez C et al.; We report the isolation and characterization of a strain of Bacillus, designed MIR 15, which appears to produce and excrete antimicrobials active against Gram-negative bacteria, but not against fungi . B . subtilis MIR 15 varied its antimicrobial profiles and production with the cultivation temperature.

J Biotechnol, 1992 Nov, 26(2-3), 245 - 56
Improving the production of E . coli beta-lactamase in Bacillus subtilis: the effect of glucose, pH and temperature on the production level; Hemila H et al.; Bacillus subtilis has been considered a promising host for the production of foreign proteins . However, proteases released by the host organism can often cause rapid breakdown of secreted heterologous proteins . Here we report that the addition of 6% glucose and 100 mM potassium phosphate to the growth medium significantly reduces the degradation of E . coli TEM beta-lactamase secreted from B . subtilis, when applying an expression system based on B . amyloliquefaciens alpha-amylase . The yield of beta-lactamase was increased 10-20-fold when compared to the yield in Luria medium . The promoter of B . amyloliquefaciens alpha-amylase gene is repressed by glucose . However, here we show that the repression does not take place in a multicopy plasmid, thus enabling our approach to efficiently reduce the protease action by catabolite repression . We have also studied the role of pH and temperature on the beta-lactamase production in laboratory scale bioreactors . Low temperature and low pH are both favorable for a high level beta-lactamase production by the high copy plasmid construction.

Appl Microbiol Biotechnol, 1992 Nov, 38(2), 226 - 33
Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis; Kakudo S et al.; In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B . subtilis-Escherichia coli shuttle vectors . B . subtilis harbouring a simple recombinant plasmid containing the coding and the 5'-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium . As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B . subtilis, (2) the promoter and the leader sequences of the alpha-amylase gene or of alkaline protease gene from B . amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110 . By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33-120 mg/l . The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.

J Bacteriol, 1992 Nov, 174(21), 6852 - 6
Identification of tms-26 as an allele of the gcaD gene, which encodes N-acetylglucosamine 1-phosphate uridyltransferase in Bacillus subtilis; Hove-Jensen B; The temperature-sensitive Bacillus subtilis tms-26 mutant strain was characterized biochemically and shown to be defective in N-acetylglucosamine 1-phosphate uridyltransferase activity . At the permissive temperature (34 degrees C), the mutant strain contained about 15% of the wild-type activity of this enzyme, whereas at the nonpermissive temperature (48 degrees C), the mutant enzyme was barely detectable . Furthermore, the N-acetylglucosamine 1-phosphate uridyltransferase activity of the tms-26 mutant strain was much more heat labile in vitro than that of the wild-type strain . The level of N-acetylglucosamine 1-phosphate, the substrate of the uridyltransferase activity, was elevated more than 40-fold in the mutant strain at the permissive temperature compared with the level in the wild-type strain . During a temperature shift, the level of UDP-N-acetylglucosamine, the product of the uridyltransferase activity, decreased much more in the mutant strain than in the wild-type strain . An Escherichia coli strain harboring the wild-type version of the tms-26 allele on a plasmid contained increased N-acetylglucosamine 1-phosphate uridyltransferase activity compared with that in the haploid strain . It is suggested that the gene for N-acetylglucosamine 1-phosphate uridyltransferase in B . subtilis be designated gcaD.

Mikrobiol Zh, 1992 Nov-Dec, 54(6), 16 - 22
{The chitinases of aerobic sporulating bacteria isolated from different ecological sources}; Slabospitskaia AT et al.; The chitinolytic activity of 171 strains of 15 species of spore-forming aerobic bacteria isolated from different ecological sources has been studied . 85 strains of the studied bacilli (50%) hydrolyzed colloidal chitin in a different degree . Among the cultures isolated from human and animal organism 60% of strains were characterized by the presence of extracellular chinases, among the collection strains--37%, among those isolated from soil and from insects--40 and 43%, respectively . The cultures of Bacillus subtilis as well as B . coagulans, B . megaterium and some other possessed the highest activity on the liquid medium . Some strains have been chosen for further research aimed at their possible use for biotechnology.

Appl Microbiol Biotechnol, 1992 Nov, 38(2), 268 - 72
Rapid assessment of bacterial viability by flow cytometry; Diaper JP et al.; The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide {DiOC6(3)} and fluorescein diacetate (FDA) . Rh123 was found to clearly differentiate viable from non-viable bacteria . The methodology for staining bacteria with this dye was optimised . Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal . Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123 . The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.

Mol Microbiol, 1992 Nov, 6(22), 3375 - 83
The effect of ribosomal protein S1 from Escherichia coli and Micrococcus luteus on protein synthesis in vitro by E . coli and Bacillus subtilis; Farwell MA et al.; We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine-Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG) . These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E . coli and B . subtilis ribosomes . Translation of these nine constructions was determined with three types of ribosomes: E . coli containing ribosomal protein S1, E . coli depleted of S1, and B . subtilis which is naturally free of S1 . E . coli ribosomes were able to translate all nine transcripts with variable efficiencies . B . subtilis and S1-depleted E . coli ribosomes were similar to each other and differed from non-depleted E . coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts . Addition of S1 from either E . coli or Micrococcus luteus, a Gram-positive bacterium, enabled S1-depleted E . coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B . subtilis ribosomes . AUG was the preferred initiation codon for all ribosome types; however, B . subtilis ribosomes showed greater tolerance for the non-AUG codons than either type of E . coli ribosome . The presence of a strong or stronger SD sequence increased the efficiency by which E . coli ribosomes could utilize non-AUG codons.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10410 - 4
Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family; Aymerich S et al.; Induction of the Bacillus subtilis sacB gene and sacPA operon and Escherichia coli bgl operon is mediated by structurally homologous antiterminators encoded by the sacY, sacT, and bglG genes, respectively . When activated, these proteins prevent early transcription termination at terminators located in the leader regions of the three operons . BglG was previously shown to bind in vitro to an imperfectly palindromic 29-nucleotide RNA sequence located upstream of the terminator and partially overlapping with it {Houman, F., Diaz-Torres, M.R . & Wright, A . (1990) Cell 62, 1153-1163} . Similar motifs, here termed ribonucleic antiterminators (RATs), strongly conserved in sequence and in position, are found in the leader of both sacB and sacPA . Mutations were created in sacB RAT and tested in B . subtilis; this showed that sacB RAT is the target for SacY-mediated induction of sacB and that a stem-loop structure in the mRNA is required for regulatory function . Mutations increasing the similarity of the sacB RAT with those of sacPA or bgl rendered sacB inducible by SacT or BglG, respectively; most of these changes did not strongly affect induction by SacY, suggesting that the nucleotides at these variable positions act as negative specificity determinants.

FEBS Lett, 1992 Oct 26, 311(3), 246 - 50
Ion channels from the Bacillus subtilis plasma membrane incorporated into planar lipid bilayers; Alcayaga C et al.; Fusion of Bacillus subtilis plasma membrane vesicles with planar lipid bilayers induced the appearance of discrete current fluctuations characteristic of ion channels . These channels showed a wide range of conductances and kinetic behaviors . In 300 mM KCl, their conductances ranged from a few hundreds of pS to more than 1 nS, and most of them exhibited several sub-states . The channels poorly discriminated between small univalent anions and cations . Some of them showed voltage dependence and most of them presented a complex gating kinetics . The results are consistent with the hypothesis of the presence in the B . subtilis plasma membrane of pores composed of subunits that function cooperatively.

J Biol Chem, 1992 Oct 25, 267(30), 21705 - 11
Nuclear-encoded tobacco chloroplast ribosomal protein L24 . Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis; Elhag GA et al.; Using a Nicotiana tabacum leaf cDNA library in the expression vector lambda gt11, two cDNAs encoding the full-length precursor polypeptide (M(r) 20,696) of tobacco chloroplast ribosomal protein L24 were identified and sequenced . These cDNAs encode a mature protein of 146 amino acids (M(r) 16,418) with a transit peptide of 41 amino acids (M(r) 4,278) . The mature tobacco L24 protein has 78, 65, 45, and 35% sequence identity with ribosomal proteins L24 of pea, spinach, Bacillus subtilis, and Escherichia coli, respectively . The transit peptide of tobacco L24 is 54 and 57% identical with that of L24 chloroplast ribosomal proteins of pea and spinach, respectively . An expressed beta-galactosidase:L24 fusion protein, bound to nitrocellulose filters, was used as affinity matrix to purify monospecific antibody to L24 protein . Using this monospecific antibody protein L24 was identified among high performance liquid chromatography (HPLC)-purified tobacco chloroplast ribosome 50 S subunit proteins . The predicted amino terminus of the mature L24 protein was confirmed by partial sequencing of the HPLC-purified L24 protein . Northern blot analysis revealed a single mRNA band (0.85-0.90 kilobase) corresponding in size to full-length L24 cDNA . The presence of multiple genes for L24 is suggested by Southern blot hybridization and characterization of two cDNAs for L24 which only differ in their 3'-noncoding sequences.

Biochemistry, 1992 Oct 20, 31(41), 10054 - 60
Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis; Lauraeus M et al.; The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water . It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme . We have used optical and resonance Raman spectroscopies to study the B . subtilis oxidase in detail . The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600 . The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases . Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases . Possible explanations for the occurrence of these two modes are discussed . Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.

Biochem Biophys Res Commun, 1992 Oct 15, 188(1), 184 - 9
Effect of the weak Ca(2+)-binding site of subtilisin J by site-directed mutagenesis on heat stability; Jang JS et al.; The functional role of the negatively charged amino acid residue in subtilisin J from Bacillus stearothermophilus has been investigated by site-directed mutagenesis . Glu-195 located at the weak Ca2+-binding site was replaced with Gln to examine the role of Glu-195 in the heat stability of subtilisin J . Mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant . When the mutant enzyme was expressed at 37 degrees C in the presence of 2mM calcium chloride, the pattern of enzyme production was quite different from that of wild-type . The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability . The mutation was found to decrease the heat stability but not catalytic efficiency (kcat/Km) and optimal pH . These results demonstrate the important role of the negatively charged side chains at the weak Ca(2+)-binding site in the heat stability of subtilisin.

Gene, 1992 Oct 12, 120(1), 17 - 26
Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids; Gamel PH et al.; A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t . Unlike other vectors which have been reported to be acceptable for B.t., this new B.t . vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes) . The compatibility of this B.t . vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t . strain . When a cryIIIA gene of B.t . tenebrionis was cloned in this vector and introduced into B.t . kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes . The stability of this vector and its compatibility with native B.t . plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t . The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t . kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment . Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein . The deduced aa sequence of the ORF showed no homology to any published aa sequences . Deletion analysis indicated that the B.t . vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.

Protein Sci, 1992 Oct, 1(10), 1363 - 76
Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy; Wittekind M et al.; The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein . Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra . Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA . The calculated models were refined by dynamical simulated annealing using the program X-PLOR . The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88 . The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet . The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I . The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40 . If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.

J Mol Biol, 1992 Oct 5, 227(3), 648 - 57
Definition and polarity of action of DNA replication terminators in Bacillus subtilis; Smith MT et al.; The first stage in termination of chromosome replication in Bacillus subtilis involves arrest of the clockwise fork at the inverted repeat region (IRR), comprising the opposed IRI and IRII sequences, adjacent to the upstream region of the rtp gene, which encodes the replication terminator protein RTP . RTP binds to IRI and IRII . The ability of the IRR and its components to function as terminators, in conjunction with RTP, and their polarity of action have now been tested by the use of plasmids replicating in B . subtilis as unidirectional theta structures and into which potential terminator sequences were inserted in alternate orientations relative to fork movement . When the complete IRR was inserted into such plasmids and the new plasmids transferred into a B . subtilis strain overproducing RTP, it was able to block movement of a replication fork approaching from either direction . IRI and IRII were shown to function as polar terminators, each blocking movement of a fork when it approached from one particular direction but not the other . Furthermore, the polarity of action was in accordance with the IRR being able to operate as a replication fork trap . Thus, a fork approaching the IRR would pass through the first terminator encountered (IRI or IRII) and be halted by the second . The previously observed nonfunctioning of a particular orientation of chromosomal IRR as a fork arrest site probably reflects a limiting level of RTP in the cell . Interestingly, a 21 base-pair core sequence spanning a single RTP binding site within IRI (the 47 base-pair IRI contains 2 binding sites) was unable to arrest a fork approaching from either direction in the plasmid system . This suggests that both binding sites within an IR must be filled in order to function as an arrest site . It is possible that co-operative interaction between adjacent dimers within IRI or IRII provides the necessary conformation for causing fork arrest.

Mol Microbiol, 1992 Oct, 6(20), 2931 - 8
An inverted repeat preceding the Bacillus subtilis glpD gene is a conditional terminator of transcription; Holmberg C et al.; The Bacillus subtilis glpD gene, encoding glycerol-3-phosphate (G3P) dehydrogenase, is preceded by a promoter and an inverted repeat which is located between the promoter and the glpD coding region . The inverted repeat acts as a transcriptional terminator in vitro . Expression of glpD is induced by G3P in the presence of the glpP gene product . Full-length glpD transcripts can be detected only in glycerol-induced cells . The major glpD transcript is initiated from the glpD promoter but minor amounts of larger transcripts, possibly initiated at upstream glp promoters, can also be found . In uninduced cells short transcripts are present, corresponding to initiation at the glpD promoter and termination at the inverted repeat . Upon induction, these short transcripts disappear and are replaced by full-length glpD transcripts . The 3'-ends of full-length glpD transcripts were mapped to an inverted repeat located immediately downstream of glpD . These results show that glpD of B . subtilis is regulated by termination/antitermination of transcription.

Mol Microbiol, 1992 Oct, 6(20), 2909 - 17
cis-unsaturated fatty acids specifically inhibit a signal-transducing protein kinase required for initiation of sporulation in Bacillus subtilis; Strauch MA et al.; The initiation of sporulation in Bacillus subtilis is controlled by the Spo0A transcription factor which is activated by phosphorylation through a phosphorelay mechanism that is dependent upon the activity of one or more protein kinases . The enzymatic activity of one of these protein kinases, KinA, was found to be inhibited in vitro by certain fatty acids . The most potent inhibitors have at least one unsaturated double bond in the cis configuration and a chain length of 16-20 carbon atoms . Homologous isomers with a trans double bond are not inhibitory . Saturated straight- or branched-chain fatty acids are either much weaker inhibitors or have no effect . The inhibitors prevent autophosphorylation of KinA and are non-competitive with ATP . B . subtilis phospholipids were found to contain at least one as yet unidentified type of fatty acid that, when present in an unesterified form, inhibited KinA . The results suggest that the concentration of a specific unsaturated fatty acid may act as a signal linking the initiation of sporulation to the status of membrane synthesis and septation or some other specific membrane-associated activity.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2159 - 66
Analysis of the flagellin (hag) gene of alkalophilic Bacillus sp . C-125; Sakamoto Y et al.; Motility of the alkalophilic Bacillus sp . C-125, a flagellate bacterium, was demonstrated to be Na(+)- and pH-dependent . Flagellin protein from this strain was purified to homogeneity and the N-terminal sequence determined . Using the hag gene of Bacillus subtilis as a probe, the hag gene of Bacillus sp . C-125 was identified and cloned into Escherichia coli . Sequencing of this hag gene revealed that it encodes a protein of 272 amino acids (M(r) 29,995) . The predicted N terminal sequence of this protein was identical to that determined by N-terminal sequencing of the flagellin protein from strain C-125 . The alkalophilic Bacillus sp . C-125 flagellin shares homology with other known flagellins in both the N- and C-terminal regions . The middle portion, however, shows considerable differences, even from that of flagellin from the related species, B . subtilis.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2069 - 74
Bacillus subtilis SenS exerts its activity through a site in the 5' flanking region of the aprE promoter; McCready PM et al.; The Bacillus subtilis gene senS, when present in high copy number, stimulates the expression of several extracellular protein genes during the onset of stationary phase, e.g . aprE . A novel integration vector, pINT, was constructed for transcription expression studies; it employed a unique method of promoter insert production for fusion with the lacZ reporter gene . Deletions were made of the 5' flanking region of the aprE promoter to localize the site responsible for SenS-mediated enhancement activity . pINT was used translationally fuse aprE promoter deletion fragments with the lacZ reporter gene . A site between -177 and -415 with respect to the aprE start site of transcription was found to be required for the maximal SenS-mediated transcription increase from the aprE promoter . A multicopy vector containing the senS coding region without its native negative regulation was highly unstable in B . subtilis; this was due to the expressed senS insert.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2061 - 8
Cloning and characterization of genes induced by hydrogen peroxide in Bacillus subtilis; Hartford OM et al.; Transcriptional fusions of Bacillus subtilis DNA to the lacZ gene were screened for induction, initially by ethanol and then by hydrogen peroxide (H2O2) . Two fusions were identified which were induced late following treatment with sublethal concentrations of H2O2 (100 microM) . The oxy-1 promoter was induced 4-5-fold and mapped to 11 degrees while the oxy-2 promoter was induced 20-fold and mapped close to the right of the defective prophage PBSX, at about 120 degrees . The oxy-2 fusion was induced by mitomycin C as well as H2O2, which correlated with the induction of PBSX by these agents . This was probably not a transcriptional induction, but rather a consequence of the induction of PBSX replication extending into adjacent regions of the chromosome.

Biotechnol Appl Biochem, 1992 Oct, 16(2), 144 - 51
Interactions of bioactive lipopeptides, iturin A and surfactin from Bacillus subtilis; Thimon L et al.; The antifungal activity of iturin A and its interaction with erythrocyte membranes were enhanced in the presence of surfactin . The modification of the properties of iturin A was explained by the formation of mixed iturin A-surfactin micelles . Such mixed micelles were easily generated when both lipopeptides were in aqueous solutions in the absence of mineral salts but the formation of these micelles did not occur when the solutions contained a high molarity of mineral cations.

Am Ind Hyg Assoc J, 1992 Oct, 53(10), 660 - 7
Evaluation of eight bioaerosol samplers challenged with aerosols of free bacteria; Jensen PA et al.; The need to quantify airborne microorganisms in the commercial microbiology industry (biotechnology) and during evaluations of indoor air quality, infectious disease outbreaks, and agriculture health investigations has shown there is a major technological void in bioaerosol sampling techniques to measure and identify viable and nonviable aerosols . As commercialization of microbiology increases and diversifies, it is increasingly necessary to assess occupational exposure to bioaerosols . Meaningful exposure estimates, by using area or environmental samplers, can only be ensured by the generation of data that are both precise and accurate . The Andersen six-stage viable (microbial) particle sizing sampler (6-STG) and the Ace Glass all-glass impinger-30 (AGI-30) have been suggested as the samplers of choice for the collection of viable microorganisms by the International Aerobiology Symposium and the American Conference of Governmental Industrial Hygienists . Some researchers consider these samplers inconvenient for evaluating industrial bioprocesses and indoor or outdoor environments . Alternative samplers for the collection of bioaerosols are available; however, limited information has been reported on their collection efficiencies . A study of the relative sampling efficiencies of eight bioaerosol samplers has been completed . Eight samplers were individually challenged with a bioaerosol, created with a Collison nebulizer, of either Bacillus subtilis or Escherichia coli . The samplers were evaluated under controlled conditions in a horizontal bioaerosol chamber . During each experimental run, simultaneous samples were collected with a reference AGI-30 to verify the concentration of microorganisms in the chamber from run to run and day to day.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochem Photobiol, 1992 Oct, 56(4), 423 - 6
Application of time-resolved diffuse reflectance techniques in studies of reaction intermediates in suspensions of Bacillus subtilis; Berinstain AB et al.; Short lived reaction intermediates such as triplet states and free radicals can be detected in vivo using laser photolysis techniques with time-resolved diffuse reflectance detection . This novel approach is illustrated for bacterial suspensions of Bacillus subtilis.

Mol Gen Genet, 1992 Oct, 235(1), 89 - 96
Suppression of the growth and export defects of an Escherichia coli secA(Ts) mutant by a gene cloned from Bacillus subtilis; Muller J et al.; A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42 degrees C . A B . subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E . coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins . The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon . No similarity to csaA was found among DNA or protein sequences deposited in databases . In contrast to other homologous or heterologous suppressors of the E . coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E . coli secY24(Ts) or lep9(Ts) mutations . However, it restored the ability of a SecB-deficient mutant to grow on complex medium . It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.

J Dairy Sci, 1992 Oct, 75(10), 2681 - 6
Biochemical activities of Bacillus species isolated from flat sour evaporated milk; Kalogridou-Vassiliadou D; Forty strains of bacilli were isolated from flat sour evaporated milk and were characterized as Bacillus stearothermophilus (5 strains), Bacillus licheniformis (10 strains), Bacillus coagulans (15 strains), Bacillus macerans (5 strains), and Bacillus subtilis (5 strains) . The hydrolases of these strains were examined with the API ZYM system, and the ability of these strains to produce acid in milk incubated at different temperatures was also examined . Esterase, esterase lipase, lipase, valine aminopeptidase, phosphoamidase, beta-glucuronidase, and beta-glucosidase were the activities found in all strains tested; they were strain-dependent and ranged between 1 and 5 by the API ZYM test . The same strains produced various concentrations of acid in milk . These organisms may be responsible for the flat sour spoilage in evaporated milk.

FEMS Microbiol Lett, 1992 Oct 1, 76(1-2), 191 - 6
Maltose uptake and its regulation in Bacillus subtilis; Tangney M et al.; Extracts prepared from cultures of Bacillus subtilis, grown on maltose as the sole carbon source, lacked maltose phosphotransferase system activity . There was, however, evidence for a maltose phosphorylase activity, and such extracts also possessed both glucokinase and glucose phosphotransferase system activities . Maltose was accumulated by whole cells of B . subtilis by an energy-dependent mechanism . This uptake was sensitive to the effects of uncouplers, suggesting a role for the proton-motive force in maltose transport . Accumulation of maltose was inhibited in the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsI6 null-mutation . A strain carrying the temperature-sensitive ptsI1 mutation accumulated maltose normally at 37 degrees C but, in contrast to the wild-type, was devoid of maltose transport activity at 47 degrees C . The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.

FEMS Microbiol Lett, 1992 Oct 1, 76(1-2), 141 - 7
Cloning and sequencing of a plasmid-mediated erythromycin resistance determinant from Staphylococcus xylosus; Milton ID et al.; A 2.3-kb DNA fragment cloned from plasmid pCH200, the largest (52 kb) of four plasmids detected in Staphylococcus xylosus, was found to confer resistance to 14-membered ring macrolides in Bacillus subtilis and Staphylococcus aureus . DNA-sequence analysis of the fragment revealed the presence of an open-reading frame, the deduced product of which was identical to one of the two ATP-binding domains encoded by the macrolide/streptogramin-B-resistance gene msrA of Staphylococcus epidermidis . The observation that a polypeptide homologous to the C-terminus of MsrA is capable of mediating erythromycin resistance in the absence of the N-terminal region is of significance both to the evolution and functional activity of members of the ATP-binding transport super-gene family.

J Bacteriol, 1992 Oct, 174(20), 6326 - 35
Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures; Willimsky G et al.; A new class of cold shock-induced proteins that may be involved in an adaptive process required for cell viability at low temperatures or may function as antifreeze proteins in Escherichia coli and Saccharomyces cerevisiae has been identified . We purified a small Bacillus subtilis cold shock protein (CspB) and determined its amino-terminal sequence . By using mixed degenerate oligonucleotides, the corresponding gene (cspB) was cloned on two overlapping fragments of 5 and 6 kb . The gene encodes an acidic 67-amino-acid protein (pI 4.31) with a predicted molecular mass of 7,365 Da . Nucleotide and deduced amino acid sequence comparisons revealed 61% identity to the major cold shock protein of E . coli and 43% identity to a family of eukaryotic DNA binding proteins . Northern RNA blot and primer extension studies indicated the presence of one cspB transcript that was initiated 119 bp upstream of the initiation codon and was found to be induced severalfold when exponentially growing B . subtilis cell cultures were transferred from 37 degrees C to 10 degrees C . Consistent with this cold shock induction of cspB mRNA, a six- to eightfold induction of a cspB-directed beta-galactosidase synthesis was observed upon downshift in temperature . To investigate the function of CspB, we inactivated the cold shock protein by replacing the cspB gene in the B . subtilis chromosome with a cat-interrupted copy (cspB::cat) by marker replacement recombination . The viability of cells of this mutant strain, GW1, at freezing temperatures was strongly affected . However, the effect of having no CspB in GW1 could be slightly compensated for when cells were preincubated at 10 degrees C before freezing . These results indicate that CspB belongs to a new type of stress-inducible proteins that might be able to protect B . subtilis cells from damage caused by ice crystal formation during freezing.

J Bacteriol, 1992 Oct, 174(19), 6087 - 95
Transcription of the Bacillus subtilis sacX and sacY genes, encoding regulators of sucrose metabolism, is both inducible by sucrose and controlled by the DegS-DegU signalling system; Crutz AM et al.; The adjacent sacX and sacY genes are involved in sucrose induction of the Bacillus subtilis sacB gene by an antitermination mechanism . sacB, encoding the exoenzyme levansucrase, is also subject to regulation by the DegS-DegU signalling system . Using sacXY'-lacZ and sacX'-lacZ fusions, we show that the transcription of the sacX and sacY genes is both inducible by sucrose and regulated by DegU . sacX and sacY appear to constitute an operon, since the deletion of the sacX leader region abolished the expression of a sacXY'-lacZ fusion . The degU-dependent promoter was located by deletion analysis and reverse transcriptase mapping 300 nucleotides upstream from the sacX initiator codon . Sucrose induction of the sacX'-lacZ fusion requires either SacY or the homologous SacT antiterminator, which is involved in sucrose induction of the intracellular sucrase gene (sacPA operon) . Sequence analysis of the sacX leader region revealed (20 nucleotides downstream from the transcription start site) a putative binding site for these regulators; however, no structure resembling a rho-independent terminator could be found overlapping this site, unlike the situation for sacPA and sacB . Deletion of a segment of the leader region located 100 nucleotides downstream from this site led to constitutive expression of the sacXY'-lacZ and sacX'-lacZ fusions . These results suggest that the mechanism of sucrose induction of sacXY is different from that of sacPA and sacB.

Eur J Biochem, 1992 Oct 1, 209(1), 121 - 7
Gene cloning and characterization of a novel extracellular ribonuclease of Bacillus subtilis; Nakamura A et al.; An extracellular nuclease gene of Bacillus subtilis was cloned in the same organism by detecting the amplified enzyme activity, which was secreted from the transformant cells on an RNA-containing agar medium . An open reading frame encoding 289 amino acids was identified within the cloned fragment . The transcriptional initiation site was determined by nuclease S1 mapping and the promoter region showed similarity to the conserved recognition sequences for the E sigma A and/or E sigma E RNA polymerases . The production of the nuclease by the B . subtilis transformants greatly depends on the liquid medium used . SDS/PAGE analysis of the purified enzyme showed two adjoining bands of molecular mass about 32 kDa, and the NH2-terminal amino acid sequence analysis suggested that the NH2-terminal portion of the nuclease was subjected to a limited proteolysis after or during secretion . The nuclease was uniquely characterized as a Mg(2+)-activated ribonuclease which hydrolyzes RNA apparently nonspecifically into oligonucleotides with 5'-terminal phosphate . The deduced amino acid sequence of this enzyme shows no obvious similarity with other nuclease sequences.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2125 - 35
Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis; Volker U et al.; Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress . Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g . 52 degrees C) . In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations . A mild heat shock induced a cross-protection against lethal salt stress . The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress . Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli . The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.

Biochim Biophys Acta, 1992 Sep 24, 1132(2), 145 - 53
Readthrough of the Bacillus subtilis stop codon produces an extended enzyme displaying a higher polymerase activity; Chambert R et al.; It has been generally accepted that the structural sacB gene of Bacillus subtilis levansucrase encodes a 50,000 Da extracellular protein . However, examination of the DNA sequence of the sacB flanking regions shows a putative open reading frame coding for a 20 amino acid peptide downstream immediately following the terminal TAA stop codon . By site-directed mutagenesis we have changed this stop codon to a glutamine codon . This stop codon readthrough leads to the synthesis and secretion by B . subtilis of a levansucrase possessing an extended polypeptide chain . The extended levansucrase has a molecular weight of 53,000 with a new carboxyl-terminus, rich in basic and hydrophobic amino acids and possessing one cysteine residue . This enzyme synthesizes fructosyl polymer levan of higher molecular weight than the shorter levansucrase . The increase in molecular weight was achieved by increasing the number of branches . These results suggest that the C-terminal part of the enzyme plays a specific role in the degree of branching of the synthesized polymer . Moreover, the extended enzyme is able to form an active dimer from two polypeptide chains linked by an S-S bridge.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8808 - 12
Coupling between gene expression and DNA synthesis early during development in Bacillus subtilis; Ireton K et al.; Endospore formation in the bacterium Bacillus subtilis involves generation of two cell types, each with different developmental fates . Each cell type contains an active chromosome, and treatments that inhibit DNA synthesis at the beginning of development inhibit spore formation . We describe experiments demonstrating that gene expression early during sporulation is coupled to DNA synthesis . Expression of several genes that are induced early during sporulation, before the formation of two cell types, is inhibited when DNA synthesis is inhibited . Genes that are affected require the transcription factor encoded by spo0A for normal induction . Spo0A protein is normally activated early in development by a multicomponent phosphorylation pathway, or phospho-relay . Altered function mutations in spo0A that bypass the need for the phospho-relay allow early sporulation gene expression, even when DNA synthesis is inhibited . These results indicate that inhibition of DNA synthesis prevents activation of the Spo0A transcription factor by inhibiting a step in the phospho-relay . It seems likely that coupling early developmental gene expression to DNA synthesis is a general mechanism to prevent inappropriate or unnecessary gene expression.

J Biol Chem, 1992 Sep 15, 267(26), 18885 - 9
Crystallization and preliminary structural analysis of the replication terminator protein of Bacillus subtilis; Mehta PP et al.; The replication terminator protein (RTP) is a dimeric molecule that binds specific sequences within the replication terminus of the Bacillus subtilis chromosome and prevents the passage of replication forks . The gene for RTP has been expressed in Escherichia coli, and the protein has been purified in amounts sufficient for structural studies by nuclear magnetic resonance (NMR) and x-ray crystallography . One-dimensional NMR experiments show that the protein has a well-folded compact tertiary structure, as well as a high alpha-helical content . Circular dichroism (CD) studies confirm this finding and show that approximately 32% of the protein is alpha-helical . The terminator protein has been crystallized as monoclinic plates that diffract to better than 2.5 A and are suitable for high resolution structural analysis . Precession photographs show the space group to be C2 with unit cell dimensions a = 77 A, b = 53 A, c = 70 A, and beta = 90 degrees, and two molecules occupy the asymmetric unit . With a view to producing crystals of an RTP.DNA complex, gel-shift assays were performed to establish the shortest sequence of DNA that is required for tight binding to RTP . These clearly show that two turns of DNA are required, centered on an 8-base pair consensus sequence, to elicit relatively stable binding.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8011 - 5
Primary structure, import, and assembly of the yeast homolog of succinate dehydrogenase flavoprotein; Schulke N et al.; We have isolated a homolog for the flavoprotein subunit of succinate dehydrogenase {succinate:(acceptor) oxidoreductase, EC 1.3.99.1} from Saccharomyces cerevisiae and used the obtained peptide sequences to clone and characterize the corresponding gene . It contained an open reading frame of 1923 base pairs and encoded a protein of 640 amino acids (M(r), 70,238) that showed approximately 49% and approximately 28% identity with the Escherichia coli and Bacillus subtilis enzymes, respectively . All features of the FAD cofactor binding site were completely conserved . Comparison of the deduced protein sequence with the N-terminal sequence determined from the isolated protein revealed an N-terminal extension of 28 amino acids that presumably represents a mitochondrial signal sequence . After in vitro transcription and translation, the preprotein was efficiently imported into isolated yeast mitochondria, cleaved to its mature form, and assembled into the membrane-bound succinate dehydrogenase complex.

Clin Exp Immunol, 1992 Sep, 89(3), 384 - 9
The macrophage response to bacteria . Modulation of macrophage functional activity by peptidoglycan from Moraxella (Branhamella) catarrhalis; Keller R et al.; Moraxella (Branhamella) catarrhalis organisms have been shown to be particularly efficient in inducing in a pure population of bone marrow-derived mononuclear phagocytes secretory and cellular activities . In the present study, the ability of peptidoglycan from this Gram-negative organism to trigger a macrophage response was compared with that elicited by peptidoglycan from Staphylococcus aureus and Bacillus subtilis . The results show that the three peptidoglycans were similarly active in triggering the secretion of tumour necrosis factor and tumouricidal activity but differed considerably in their ability to induce the generation of nitrite in macrophages; in this respect, peptidoglycan from M . catarrhalis was particularly potent . The impressive capacity of M . catarrhalis peptidoglycan to induce in low concentration the secretion of tumour necrosis factor and nitrite and tumouricidal activity may, in addition to its lipopolysaccharide, contribute to the extraordinary potential of this organism to trigger the functional activities of macrophages.

J Bacteriol, 1992 Sep, 174(17), 5748 - 52
Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase; Marquardt JL et al.; The Escherichia coli gene murZ, encoding the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase, has been cloned and sequenced . Identified by screening an E . coli genomic library for clones that conferred phosphomycin resistance, murZ encoded a 419-amino-acid polypeptide and was mapped to 69.3 min on the E . coli chromosome . MurZ protein was purified to near homogeneity and found to have the expected UDP-N-acetylglucosamine enolpyruvyl transferase activity . Sequence analysis of the predicted product revealed 44% identity to OrfR from Bacillus subtilis (K . Trach, J.W . Chapman, P . Piggot, D . LeCoq, and J.A . Hoch, J . Bacteriol . 170:4194-4208, 1988), suggesting that orfR may also encode a UDP-N-acetylglucosamine enolpyruvyl transferase enzyme . MurZ is also homologous to the aromatic amino acid biosynthetic enzyme enolpyruvyl shikimate phosphate synthase, the other enzyme known to catalyze an enolpyruvyl transfer.

J Bacteriol, 1992 Sep, 174(17), 5593 - 6
Efficiency of homologous intermolecular recombination at different locations on the Bacillus subtilis chromosome; Biswas I et al.; The efficiencies of intermolecular recombination at 12 different locations on the Bacillus subtilis chromosome were determined by transforming competent cells with a nonreplicative plasmid . The efficiencies varied by only about threefold but were significantly different (P less than 0.05 by a chi-square test) for approximately 20% of the locations . The recA gene product is required for recombination, and the addA gene product appears to affect the variation in a site-specific way.

Gene, 1992 Sep 1, 118(1), 149 - 50
Nucleotide sequence of the URA1 gene of Saccharomyces cerevisiae; Roy A; The complete nucleotide sequence of the URA1 gene encoding dihydroorotic acid dehydrogenase (DHOdehase) is presented . This enzyme catalyses the conversion of DHO to orotic acid and plays a major role in the pyrimidine pathway, as DHO is the effector of the positive control of the transcription of at least four genes, URA1, URA3, URA4 and URA10 . Comparisons between the amino acid sequence of the yeast DHOdehase and sequences of DHOdehases previously isolated from Dictyostellum discoidum, Escherichia coli and Bacillus subtilis reveal no obvious homologies.

Mol Microbiol, 1992 Sep, 6(18), 2705 - 13
flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein; Carpenter PB et al.; We describe the sequence and characterization of the Bacillus subtilis flhF gene . flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif . The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP . flhF is located in a large operon consisting of chemotaxis and flagellar genes . Cells deficient in flhF are nonmotile . Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene . Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins . These data suggest that flagellar biosynthesis in B . subtilis requires GTP.

Protein Eng, 1992 Sep, 5(6), 543 - 50
Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease; Margarit I et al.; Oligonucleotide-directed mutagenesis has been used to replace glycine residues by alanine in neutral protease from Bacillus subtilis . One Gly to Ala substitution (G147A) was located in a helical region of the protein, while the other (G189A) was in a loop . The effects of mutational substitutions on the functional, conformational and stability properties of the enzyme have been investigated using enzymatic assays and spectroscopic measurements . Single substitutions of both Gly147 and Gly189 with Ala residues affect the enzyme kinetic properties using synthetic peptides as substrates . When Gly replacements were concurrently introduced at both positions, the kinetic characteristics of the double mutant were roughly intermediate between those of the two single mutants, and similar to those of the wild-type protease . Both mutants G147A and G189A were found to be more stable towards irreversible thermal inactivation/unfolding than the wild-type species . Moreover, the stabilizing effect of the Gly to Ala substitution was roughly additive in the double mutant G147A/G189A, which shows a 3.2 degrees C increase in Tm with respect to the wild-type protein . These findings indicate that the Gly to Ala substitution can be used as a strategy to stabilize globular proteins . The possible mechanisms of protein stabilization are also discussed.

Proteins, 1992 Sep, 14(1), 120 - 4
Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB; Schindelin H et al.; The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells . CspB was crystallized in two different forms using vapor diffusion methods . The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A . The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A . These crystals grow with polyethylene glycol 4000 as precipitant.

Mol Gen Genet, 1992 Sep, 234(3), 494 - 7
Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology; Khasanov FK et al.; To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome . In this system the recombination frequencies were measured between ts pE194 derivatives carrying segments of the chromosomal beta-gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature . Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for beta-gluconase activity . A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp . It was found that approximately 70 bp of homology is required for detectable homologous recombination . Homologous recombination was not detected when only 25 bp of homology between plasmid and chromosome were provided . The data indicate that homology requirements for recombination in B . subtilis differ from those in Escherichia coli.

Mol Gen Genet, 1992 Sep, 234(3), 429 - 32
Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation; Taura T et al.; The Escherichia coli gene ssyB was cloned and sequenced . The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24 . The correct map position of nusB is 9.5 min rather than 11 min as previously assigned . It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.

J Appl Bacteriol, 1992 Sep, 73(3), 251 - 6
Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis; Condon S et al.; The influence of different sporulation temperatures (30, 37, 44 and 52 degrees C) upon heat resistance of Bacillus subtilis was investigated . Heat resistance was greater after higher sporulation temperatures . Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested . Heat resistance increased about tenfold in the range of 30-44 degrees C . Sporulation at 52 degrees C did not show any further increase in heat resistance . This effect was constant over all the range of heating temperatures tested (100-120 degrees C) . z value remained constant (z = 9 degrees C) . Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature . Spores obtained from cells incubated at 32 or 52 degrees C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.

Gene, 1992 Sep 1, 118(1), 147 - 8
Sequence of the Bacillus subtilis homolog of the Escherichia coli cell-division gene murG; Miyao A et al.; The Bacillus subtilis homology of the Escherichia coli murG gene {encoding UDP-N-acetylglucosamine:N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase} was cloned in E . coli K-12 and sequenced . The murG homolog encodes a protein of M(r) 39,936 {363 amino acid (aa) residues} of which 108 aa residues (29.8%) are identical with the E . coli murG product.

Biosci Biotechnol Biochem, 1992 Sep, 56(9), 1455 - 60
Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp . 221; Takami H et al.; The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp . 221 was cloned in Escherichia coli and expressed in Bacillus subtilis . An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases . The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues . The alkaline protease from alkaliphilic Bacillus sp . 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%) . Also Bacillus sp . 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.

Mol Microbiol, 1992 Sep, 6(18), 2573 - 82
Analysis of the transcriptional activity of the hut promoter in Bacillus subtilis and identification of a cis-acting regulatory region associated with catabolite repression downstream from the site of transcription; Oda M et al.; Levels of transcripts initiated at a hut promoter in Bacillus subtilis were analysed . The addition of histidine to the culture medium increased the level of the transcript sixfold . In the presence of histidine and glucose together, the level of the transcript was reduced to the level in the absence of induction . Furthermore, addition of a mixture of 16 amino acids to cultures of induced cells and of catabolite-repressed cells decreased levels of the transcript 16-fold and 2.6-fold, respectively . Thus, it appears that at least three regulatory mechanisms associated with induction, catabolite repression, and amino acid repression, control the transcriptional activity of the hut promoter . Expression of the hut promoter-lacZ fusions that contained various regions of the hutP gene and deletion analysis of the hutP region revealed a cis-acting sequence associated with catabolite repression that was located between positions +204 and +231 or around position +203.

J Gen Microbiol, 1992 Sep, 138 ( Pt 9), 1949 - 61
Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier; Lazarevic V et al.; The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR . Proteins LytA, LytB and LytC are endowed with export signal peptides . Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein . LytB and LytC, the modifier and the amidase, are highly basic . After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively . These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases . The C-terminal moiety of LytB exhibits homology to the product of spoIID . LytR is a 35 kDa protein which acts as an attenuator of the expression of both lytABC and lytR operons . Transcription of the lytABC operon proceeds from two promoters: PD, identified as P28-7 (Gilman et al., 1984), and an upstream PA . The former only is subject to LytR attenuation . Translational initiation of lytB and lytC is directed by UUG start codons, suggesting that lytA, B and C undergo coupled translation . Transcription of lytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences . Both promoters are attenuated by LytR . It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e . that it belongs to the fla regulon and that its activity, or part of it, is co-regulated with flagellar motility . The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the lytABC operon is discussed.

J Clin Microbiol, 1992 Sep, 30(9), 2346 - 52
Usefulness of the ID32 staph system and a method based on rRNA gene restriction site polymorphism analysis for species and subspecies identification of staphylococcal clinical isolates; Chesneau O et al.; The usefulness of the ID32 Staph System and a method based on rRNA gene restriction site polymorphism was evaluated by the study of 42 staphylococcal clinical isolates phenotypically difficult to identify . The ID32 Staph micromethod and the genomic method are adapted for recognition of 27 and 31 staphylococcal taxa, respectively . The genomic method is based on a Dice analysis of the hybridization patterns obtained by cutting the cellular DNA either with EcoRI or with HindIII and by probing with pBA2, containing the Bacillus subtilis gene encoding 16S rRNA, labeled either with {alpha-32P}dCTP or with acetylaminofluorene . This study showed that the nonradioactive labeling provided a better resolution of the hybridizing bands than radioactive labeling . Of the 42 isolates selected, only 22 could be assigned to a staphylococcal species by the ID32 Staph System, whereas 35 could be identified by the genomic method . This latter method also enabled the screening of three unclassified isolates having hybridization patterns more closely related to each other than to any of the 31 staphylococcal taxa investigated . These three isolates could belong to a staphylococcal taxon not yet described.

Appl Environ Microbiol, 1992 Sep, 58(9), 3157 - 62
Metal-based formulations with high microbicidal activity; Sagripanti JL; Substances were evaluated for their relative potencies in inactivating Junin virus, Escherichia coli, and spores of Bacillus subtilis . Under the conditions of our test, glutaraldehyde was the most efficient agent among all substances currently recommended for disinfecting and sterilizing medical devices . Either copper or iron ions by themselves were able to inactivate virus with an efficiency similar to that of substances currently used for disinfection and sterilization . The microbicidal effect of metals, however, was enhanced further by the addition of peroxide . The mixtures of copper and peroxide described here were more efficient than glutaraldehyde in inactivating viruses and bacteria . The addition of a metal chelator to metal-peroxide mixtures further increased the microbicidal potency of the reagent . The formulations described in this study should be harmless to people but able to quickly and efficiently inactivate microorganisms, particularly viruses.

J Bacteriol, 1992 Sep, 174(17), 5633 - 8
New thermosensitive plasmid for gram-positive bacteria; Maguin E et al.; We isolated a replication-thermosensitive mutant of the broad-host-range replicon pWV01 . The mutant pVE6002 is fully thermosensitive above 35 degrees C in both gram-negative and gram-positive bacteria . Four clustered mutations were identified in the gene encoding the replication protein of pVE6002 . The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position . Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed . The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range . Using pVE6002 as the delivery vector for a transposon Tn10 derivative in Bacillus subtilis, we observed transposition frequencies of about 1%.

Gene, 1992 Sep 1, 118(1), 109 - 13
The DNA polymerase-encoding gene of Bacillus subtilis bacteriophage SPO1; Scarlato V et al.; The bacteriophage SPO1 DNA polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced . The aa sequence of SPO1 DNA polymerase shows a high degree of similarity with that of DNA polymerase I from Escherichia coli (Po1I) . Alignment with the sequences of Po1I, and the phi 29 and SPO1 DNA polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.

Biochim Biophys Acta, 1992 Aug 21, 1122(3), 278 - 82
Interaction of catalytic-site mutants of Bacillus subtilis alpha-amylase with substrates and acarbose; Takase K; The interactions of the three catalytic-site mutants of Bacillus subtilis alpha-amylase/(DN176 {Asp-176----Asn}, EQ208 {Glu-208----Gln} and DN269 {Asp-269----Asn}) with substrates and a pseudo-oligosaccharide inhibitor, acarbose, have been studied by means of difference absorption spectroscopy and affinity chromatography . The addition of maltopentaose or soluble starch to the inactive mutant enzymes mostly resulted in difference spectra characteristic of tryptophan perturbation, enabling determination of the dissociation constants . The results show that conversion of Glu-208 to Gln greatly enhanced substrate binding, implying that Glu-208 interacts unfavorably with the substrate's ground state, preventing its optimal fit to the active site . The affinity for acarbose was greatly reduced in DN269 and EQ208, but less so in DN176, suggesting that Asp-269 and Glu-208 are more important than Asp-176 in stabilizing the transition state . These results are consistent with Glu-208 and Asp-269 being the key catalytic residues, as proposed for Taka-amylase A.

J Mol Biol, 1992 Aug 20, 226(4), 1037 - 50
Sporulation regulatory protein GerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes; Zheng L et al.; The mother-cell line of gene expression during sporulation in Bacillus subtilis is a hierarchical cascade consisting of at least four temporally controlled gene sets, the first three of which each contain a regulatory gene for the next gene set in the pathway . gerE, a member of the penultimate gene set, is a regulatory gene whose products is required for the transcriptional activation of genes (coat protein genes cotB and cotC) in the last gene set . The gerE product also influences the expression of other members of the penultimate gene set (coat protein genes cotA and cotD appear to be repressed and activated, respectively) . We now report that the purified product of gerE (GerE) is a DNA-binding protein that adheres to the promoters for cotB and cotC . We also show that GerE stimulates cotB and cotC transcription in vitro by RNA polymerase containing the mother-cell sigma factor sigma K . These findings support the view that GerE is a positively acting, regulatory protein whose appearance at a late stage of development directly activates the transcription of genes in the last known temporal class of mother-cell-expressed genes . In addition, GerE stimulates cotD transcription and inhibits cotA transcription in vitro by sigma K RNA polymerase, as expected from in vivo studies, and, unexpectedly, profoundly inhibits in vitro transcription of the gene (sigK) that encodes sigma K . The effects of GerE on cotD and sigK transcription are just the opposite of the effects exerted by the earlier-appearing, mother-cell regulatory protein spoIIID, suggesting that the ordered appearance of first SpoIIID, then GerE, ensures proper flow of the regulatory cascade controlling gene expression in the mother cell.

J Mol Biol, 1992 Aug 20, 226(4), 931 - 3
Functional analysis of the intramolecular chaperone . Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule; Kobayashi T et al.; The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis . The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme . Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding . With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated . These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide . For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region . These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.

Biochemistry, 1992 Aug 18, 31(32), 7411 - 21
Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II); Hagerhall C et al.; Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide . The enzyme complex was overproduced 2-3-fold in membranes of B . subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent . The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified . Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation . Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide . Chemical analysis demonstrated two protoheme IX per complex II . One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K . The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K . Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes . Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B . subtilis cytochrome b558 isolated and in complex II . This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.

FEMS Microbiol Lett, 1992 Aug 15, 74(2-3), 241 - 6
Membrane-protein phosphorylation in the Bacillus subtilis cell cycle; Sandler N et al.; Sphingosine, an inhibitor of Ca(2+)-dependent protein kinases in eukaryotic cells, inhibited initiation of DNA replication in Bacillus subtilis at a concentration of 10 microM, without inhibiting elongation . The tumor promoter 12-tetradecanoyl 13-phorbol acetate, (TPA), an activator of protein kinase C in eukaryotic cells, partially counteracted the inhibition of initiation by sphingosine . Phosphorylation of polypeptides was observed in vivo at initiation of DNA replication in B . subtilis . Sphingosine, TPA, and vancomycin affected this protein phosphorylation.

Nucleic Acids Res, 1992 Aug 11, 20(15), 4015 - 20
Solid-phase synthesis of the nucleopeptide fragment H-Asp-Ser{pAAAGTAAGCC}-Glu-OH from the nucleoprotein of Bacillus subtilis phage phi 29; Dreef-Tromp CM et al.; The naturally occurring DNA-nucleopeptide H-Asp-Ser{5'-pAAAGTAAGCC-3'}-Glu-OH was prepared via a solid-phase phosphite triester approach using N-2-(tert-butyldiphenylsilyloxymethyl)benzoyl protected nucleosides . The oligonucleotide was linked via the extremely base-labile oxalyl ester anchor to the solid support.

Nucleic Acids Res, 1992 Aug 11, 20(15), 3939 - 44
Crosstalk between plasmid vegetative replication and conjugative transfer: repression of the trfA operon by trbA of broad host range plasmid RK2; Jagura-Burdzy G et al.; Previous deletion and complementation analysis has indicated that the region between trfA and kilBI (trbB) encodes trans-acting factor, designated trbA, required for conjugative transfer of broad host range plasmid RK2 . In analysing the nucleotide sequence of this region we have discovered a gene encoding a 12 kDa polypeptide . The predicted amino acid sequence of this protein shows similarity at its C-terminal to KorA from the central control operon of RK2 and at its N-terminal to immunity repressor protein from phage phi 105 of Bacillus subtilis as well as the Sin protein of B . subtilis which regulates alternate developmental processes including sporulation, motility and competence . We show that TrbA represses transcription of both trfA (vegetative replication) and kilBI (trbB) (required for conjugative transfer and whose product has similarity to ComG, required for competence of B . subtilis) and may help to coordinate expression of both sets of functions . This region has similarities to some temperate bacteriophage immunity regions in modulating divergent transcription required for alternative means of propagation.

FEBS Lett, 1992 Aug 10, 308(1), 18 - 21
Biosynthesis of bacillomycin D by Bacillus subtilis . Evidence for amino acid-activating enzymes by the use of affinity chromatography; Besson F et al.; Bacillomycin D is an antifungal lipopeptide produced by B . subtilis . The formation of the peptidyl bonds of bacillomycin D occurs non-ribosomally, as demonstrated by the use of chloramphenicol, an inhibitor of protein biosynthesis . Amino acid-activating enzymes were found in B . subtilis cell lysates purified by affinity chromatography on a gel containing L-Pro, an amino acid of bacillomycin D . Presence of ATP during this purification increases the binding of enzymatic proteins and their activity . An enzyme, with an apparent molecular weight of 230 kDa, catalyzed ATP-PPi exchange reactions, which were mediated by specific amino acids, corresponding to a partial sequence of bacillomycin D.

J Bacteriol, 1992 Aug, 174(16), 5482 - 4
Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus; Becker-Rudzik M et al.; We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus . Normalized alignment scores comparing the trpC gene of R . capsulatus with the trpC genes of other bacterial species are reported . An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found.

J Bacteriol, 1992 Aug, 174(16), 5475 - 8
The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis; Brantl S et al.; To prove the hypothesis that the amount of RepR protein is the rate-limiting factor for replication of plasmid pIP501 in Bacillus subtilis, the repR gene was placed under control of the inducible promoter pspac . The plasmid copy number of the pIP501 derivative pRS9 could be deliberately adjusted between approximately 1 and 50 to 100 molecules per cell by varying the concentration of the inducer isopropyl-beta-D-thiogalactopyranoside . Construction of a repR-lacZ fusion proved that the increase in copy number was due to a proportional increase in the amount of RepR protein.

J Bacteriol, 1992 Aug, 174(16), 5466 - 70
Physical distance between the site of type II DNA binding to the membrane and oriC on the Bacillus subtilis 168 chromosome; Itaya M et al.; The precise physical locations of the oriC region and the region for type II DNA binding to the membrane on the Bacillus subtilis 168 chromosome were determined . The DNA regions were physically mapped by creating new restriction sites (NotI and SfiI) within these regions . The physical distance between oriC and the type II DNA-binding region was verified with the creation of a novel sequence cleaved by endonuclease I-SceI in each of the above regions . Complete removal of the defined type II membrane-binding region produced no noticeable phenotype.

J Bacteriol, 1992 Aug, 174(16), 5430 - 5
Mutagenesis and mapping of the gene for a sporulation-specific penicillin-binding protein in Bacillus subtilis; Buchanan CE et al.; Penicillin-binding protein (PBP) 5* is produced by Bacillus subtilis only during sporulation and is believed to be required for synthesis of the peptidoglycan-like cortex layer of the spore . The structural gene (dacB) for PBP 5* was insertionally mutagenized by integration of a plasmid bearing an internal fragment of the gene, and the phenotype of the null mutant was characterized . The mutant had no apparent vegetative growth or germination defect, but it produced extremely heat-sensitive spores . This property is consistent with a defect in the amount or assembly of the cortex and supports the hypothesis that PBP 5* is required for synthesis of this structure . Analysis of the progeny after spontaneous excision of the integrated plasmid led to the conclusion that expression of the dacB gene was required only in the mother cell compartment during sporulation, which is also consistent with a role for PBP 5* in cortex synthesis and with its location in the outer forespore membrane . Genetic mapping located dacB midway between aroC (206 degrees) and lys (210 degrees) on the B . subtilis chromosome . This is a region where there are no other known spo, ger, or PBP genes . In related studies, we found that a null mutant of dacA, the structural gene for vegetative PBP 5, produced normal heat-resistant spores, which suggests that this PBP is not essential for cortex synthesis . In addition, a candidate for another sporulation-specific PBP was revealed on gels at approximately the same position as PBP 5* . The two PBPs could be distinguished by immunoassays.

J Bacteriol, 1992 Aug, 174(16), 5317 - 23
Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme; Green JM et al.; In Escherichia coli, p-aminobenzoate (PABA) is synthesized from chorismate and glutamine in two steps . Aminodeoxychorismate synthase components I and II, encoded by pabB and pabA, respectively, convert chorismate and glutamine to 4-amino-4-deoxychorismate (ADC) and glutamate, respectively . ADC lyase, encoded by pabC, converts ADC to PABA and pyruvate . We reported that pabC had been cloned and mapped to 25 min on the E . coli chromosome (J . M . Green and B . P . Nichols, J . Biol . Chem . 266:12971-12975, 1991) . Here we report the nucleotide sequence of pabC, including a portion of a sequence of a downstream open reading frame that may be cotranscribed with pabC . A disruption of pabC was constructed and transferred to the chromosome, and the pabC mutant strain required PABA for growth . The deduced amino acid sequence of ADC lyase is similar to those of Bacillus subtilis PabC and a number of amino acid transaminases . Aminodeoxychorismate lyase purified from a strain harboring an overproducing plasmid was shown to contain pyridoxal phosphate as a cofactor . This finding explains the similarity to the transaminases, which also contain pyridoxal phosphate . Expression studies revealed the size of the pabC gene product to be approximately 30 kDa, in agreement with that predicted by the nucleotide sequence data and approximately half the native molecular mass, suggesting that the native enzyme is dimeric.

Virology, 1992 Aug, 189(2), 640 - 6
Deoxyuridylate-hydroxymethylase of bacteriophage SPO1; Wilhelm K et al.; Phage SPO1 of Bacillus subtilis carries hydroxymethyl-deoxyuridylate in place of thymidylate in its DNA . The enzyme, responsible for the conversion of dUMP to HmdUMP, is a dUMP hydroxymethylase, encoded by the SPO1 gene 29 . Here we describe the cloning and sequencing of the gene and the overexpression of the gene product . DNA hybridization using the DNA of bacteriophage T4 dCMP-hydroxymethylase gene as a probe, allowed us to identify and map g29 on a 3.9-kb restriction fragment, EcoRI*11 . We determined the nucleotide sequence . One of the open reading frames detected, coding for a putative 44.6-kDa protein, showed significant amino acid homologies with all known thymidylate synthases . Gp29 was overexpressed in the pT7 system . Extracts prepared from induced cells show hydroxymethylase activity in a tritium release assay.

J Bacteriol, 1992 Aug, 174(15), 5123 - 6
Membrane ultrastructure of alkaliphilic Bacillus species studied by rapid-freeze electron microscopy; Khan S et al.; Cells of Bacillus firmus OF4 and Bacillus alcalophilus were examined by rapid-freeze freeze-fracture and freeze-substitution electron microscopy . No special vesicular structures linked to growth at alkaline pH were found, either within or associated with the cytoplasmic membrane . The cytoplasmic membranes of the alkaliphilic bacilli and the neutrophilic Bacillus subtilis BD99 were indistinguishable . Distinctive intramembrane particle rings, presumed to be flagellar structures on the basis of distribution and morphological characteristics, were found in all of these species . These observations indicate that the adaptations required to effect oxidative phosphorylation and flagellar rotation at extreme alkaline pH occur without gross morphological rearrangement.

J Bacteriol, 1992 Aug, 174(15), 5063 - 71
Sequence organization and regulation of the Bacillus subtilis menBE operon; Driscoll JR et al.; Menaquinone (MK) plays a central role in the respiratory chain of Bacillus subtilis . The biosynthesis of MK requires the formation of a naphthoquinone ring via a series of specific reactions branching from the shikimate pathway . "Early" MK-specific reactions catalyze the formation of o-succinylbenzoate (OSB) from isochorismate, and "late" reactions convert OSB to dihydroxynaphthoate, by utilizing an OSB-coenzyme A intermediate . We have cloned and sequenced the B . subtilis menE and menB genes encoding, respectively, OSB-coenzyme A synthase and dihydroxynaphthoate synthase . The MenB open reading frame encodes a potential polypeptide of 261 amino acid residues with a predicted size of 28.5 kDa, while the MenE open reading frame could encode a 24.4-kDa polypeptide of 220 amino acid residues . Probable promoter sequences were identified by high-resolution primer extension assays . Organization of these genes and regulatory regions was found to be menBp menB menEp menE . Expression of menE was dependent on both menEp and menBp, indicating an operonlike organization . A region of dyad symmetry capable of forming a stable RNA secondary structure was found between menB and menE . Culture cycle-dependent expression of menB and menE was measured by steady-state transcript accumulation . For both genes, maximal accumulation was found to occur within an hour after the end of exponential growth . The menBp and menEp promoters have sequences compatible with recognition by the major vegetative form of B . subtilis RNA polymerase, E sigma A . Both promoter regions also were found to contain homologies to a sequence motif previously identified in the menCDp region and in promoters for several B . subtilis tricarboxylic acid cycle genes.

J Bacteriol, 1992 Aug, 174(15), 4885 - 92
Characterization of a Bacillus subtilis sporulation operon that includes genes for an RNA polymerase sigma factor and for a putative DD-carboxypeptidase; Wu JJ et al.; At early stages of sporulation, the spoIIA locus is transcribed as a tricistronic (1.7-kb) operon, coding for sigma F and for two proteins that modulate the activity of sigma F . The locus is transcribed as a longer (2.9-kb) transcript at the late stages of sporulation . We show here that the longer transcript contains an additional open reading frame whose product has extensive sequence homology with DD-carboxypeptidases; the corresponding gene is designated dacF . Cotranscription of a morphogene, such as dacF, with the gene for a sigma factor suggests a way to couple transcription regulation with morphogenesis . The predicted N-terminal sequence of the DacF protein and the inhibition of sporulation by a translational dacF-lacZ fusion both suggest that the protein has a signal peptide for transport into or across a membrane . Expression of a dacF-lacZ transcriptional fusion was in the forespore . The 5' end of the 2.9-kb transcript was determined by primer extension analysis . The region 5' to the end showed no homology to promoters recognized by known sigma factors but was homologous to the corresponding region of the forespore-specific 0.3-kb gene of Bacillus subtilis.

Mol Gen Genet, 1992 Aug, 234(2), 285 - 96
Organization and regulation of the Bacillus subtilis odhAB operon, which encodes two of the subenzymes of the 2-oxoglutarate dehydrogenase complex; Resnekov O et al.; The primary structure of Bacillus subtilis 105 kDa 2-oxoglutarate dehydrogenase (E10) was deduced from the nucleotide sequence of the odhA gene and confirmed by N-terminal sequence analysis . The protein is highly homologous to E1o of Azotobacter vinelandii and Escherichia coli and of bakers' yeast cells . The 5' end of the odhAB mRNA was determined and the promoter region for the odhAB operon was localized to a 375 bp DNA fragment . The cellular concentration of the 4.5 kb odhAB transcript was found to be growth stage dependent; its concentration during growth in nutrient sporulation medium decreased abruptly at the end of the exponential growth phase and it was not detectable in early stationary phase . This decrease in the cellular concentration of the transcript is not the result of an increased rate of decay of the full-length odhAB mRNA, suggesting that transcription is down-regulated at the end of the exponential growth phase . The cellular concentration of the odhA and odhB gene products, E1o and dihydrolipoamide transsuccinylase (E2o), remains essentially constant throughout the growth curve in nutrient sporulation medium, indicating that both are rather stable proteins . In exponentially growing cells, glucose in nutrient sporulation medium repressed the cellular concentration of the odhAB mRNA, as well as that of E1o and E2o, about four-fold . This effect is most likely the result of a decreased rate of transcription from the odhAB promoter, since neither the stability nor the 5'-end of the transcript were affected by glucose in the medium . It is concluded that the cellular concentration of the 2-oxoglutarate dehydrogenase multienzyme complex (E1o and E2o) is regulated mainly at the transcriptional level.

Can J Microbiol, 1992 Aug, 38(8), 794 - 7
Pectin decomposition and associated nitrogen fixation by mixed cultures of Azospirillum and Bacillus species; Khammas KM et al.; Cocultures of different Azospirillum species with Bacillus polymyxa or Bacillus subtilis allow the efficient utilization of pectin as carbon and energy sources for nitrogen fixation . The nitrogenase activity obtained with cocultures was as high as 30-80 nmol C2H4 h-1 mL-1, a much higher value than that obtained with pure cultures of either Azospirillum (up to 13 nmol C2H4 h-1 mL-1) or B . polymyxa (up to 2 nmol C2H4 h-1 mL-1) alone . To establish to what extent each partner contributed to nitrogenase activity, acetylene reduction was assayed as a function of time and it was also measured on Azospirillum cultivated in the cultures filtrates of the Bacillus . The results suggested that the nitrogenase activity was mostly produced by Azospirillum . The nitrogenase activity occurred at the expense of the degradation and fermentation products of the pectin . The new pectinolytic species, Azospirillum irakense, utilized both degradation and fermentation products of pectin, whereas the nonpectinolytic strains (Azospirillum brasilense, Azospirillum lipoferum, Azospirillum amazonense) utilized only the fermentation products of pectin, including acetic and succinic acids . These cocultures can be considered as metabolic associations, where the Bacillus produces degradation and fermentation products of pectin, which can be used by Azospirillum species.

Wei Sheng Wu Xue Bao, 1992 Aug, 32(4), 296 - 8
{Expression and secretion of alpha-amylase gene from Bacillus subtilis in E . coli}; Zhu W et al.; The E . coli which carrying the alpha-amylase gene fragment cloned from B . subtilis secreted the gene products into the medium . The reason is the exogenous gene fragment act on the cell wall of E . coli by some way, gives rise to the change of its structure . It leads up to the alpha-amylase and some periplasm proteins passing through the cell wall into the medium . It also causes the change of host colonial morphology . The secrete process are non-specific.

Mol Microbiol, 1992 Aug, 6(15), 2085 - 94
Isolation of arginine auxotrophs, cloning by mutant complementation, and sequence analysis of the argC gene from the cyanobacterium Anabaena species PCC 7120; Floriano B et al.; Arginine auxotrophs of the dinitrogen-fixing cyanobacterium Anabaena species strain PCC 7120 were isolated after ultraviolet light mutagenesis and penicillin enrichment . Two of these auxotrophs were complemented by a cosmid gene library of the wild-type strain established in Escherichia coli that was transferred en masse to the mutants by conjugation . The gene complementing one of those mutants was found to complement an E . coli argC mutant . Sequencing analysis of the gene showed that it encodes a 322-residue polypeptide that is homologous to the ArgC protein of E . coli, Bacillus subtilis and Streptomyces clavuligerus and to the C-terminal moiety of the Saccharomyces cerevisiae ARG5,6 gene product, N-acetylglutamate semialdehyde dehydrogenase . A cysteine residue present in a highly conserved domain in the five proteins is probably located in the active site of the enzyme . Conserved among the ArgC proteins, sequences resembling the primary structure of nucleotide-binding domains are also found . Downstream of the Anabaena argC gene seven nearly perfect repeats of a heptanucleotide (consensus sequence:5'-CTAATGA-3') are found.

J Gen Microbiol, 1992 Aug, 138 ( Pt 8), 1717 - 27
The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae precursor rRNA; Kempsell KE et al.; Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon . The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region . The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427) . A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli . The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of precursor rRNA secondary structure . Sequences upstream from the 5'-end of Mycobacterium leprae 16S rRNA were also investigated . Homologous schemes of secondary structure were deduced for precursor rRNA of both M . tuberculosis and M . leprae; although the principal features are common to both species there are notable differences.

EMBO J, 1992 Aug, 11(8), 3117 - 27
Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence; Putzer H et al.; In Bacillus subtilis, two genes, thrS and thrZ, encode distinct threonyl-tRNA synthetase enzymes . Normally, only the thrS gene is expressed . Here we show that either gene, thrS or thrZ, is sufficient for normal cell growth and sporulation . Reducing the intracellular ThrS protein concentration induces thrZ expression in a dose-compensatory manner . Starvation for threonine simultaneously induces thrZ and stimulates thrS expression . The 5'-leader sequences of thrS and thrZ contain, respectively, one and three transcription terminators preceded by a conserved sequence . We show that this sequence is essential for the regulation of thrS via a transcriptional antitermination mechanism . We propose that both genes, thrS and thrZ, are regulated by the same mechanism such that the additional regulatory domains present before thrZ account for its non-expression . In contrast to Escherichia coli, structurally similar regulatory domains, i.e . the consensus sequence preceding a terminator structure, are found in the leader regions of most aminoacyl-tRNA synthetase genes of Gram-positive bacteria . This suggests that they are regulated by a common mechanism.

Mutat Res, 1992 Aug, 274(2), 79 - 84
Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion; Osburne MS et al.; The din23 fusion encodes a B . subtilis SOS-inducible regulatory region fused to the E . coli lacZ gene (Love et al., 1985) . A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon . The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents . The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.

Biosci Biotechnol Biochem, 1992 Aug, 56(8), 1275 - 8
Degradation of streptomyces metalloprotease inhibitor (SMPI) by neutral protease from Bacillus subtilis var . amylosacchariticus; Tsuru D et al.; The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var . amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI) . The degree of inhibition was, however, significantly less than that for thermolysin (TLN) . During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated . Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.

FEMS Microbiol Lett, 1992 Aug 1, 74(1), 109 - 13
Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases; Kuroda A et al.; The Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B . subtilis and B . licheniformis autolysins (CwlA and CwlM, respectively) in addition to that of the major B . subtilis autolysin (CwlB) . Even though the substrate for the enzyme reaction was changed from B . subtilis cell wall containing a teichoic acid to Micrococcus luteus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.

Mol Ecol, 1992 Aug, 1(2), 95 - 103
Sexuality in a natural population of bacteria--Bacillus subtilis challenges the clonal paradigm; Istock CA et al.; Reproduction by binary fission necessarily establishes a clonal genotypic structure in bacterial populations unless a high rate of genetic recombination opposes it . Several genetic properties were examined for a wild population of Bacillus subtilis in the Sonoran Desert of Arizona to assess the extent of recombination in a natural population . These properties included allozyme variation revealed by multilocus enzyme electrophoresis, phage and antibiotic resistance, and restriction fragment length polymorphism with Southern hybridization . Evidence of extensive genetic recombination was found along with evidence of modest clonal structure . Recombination must be frequent relative to binary fission in this population . This mixed population structure provides broader options for bacterial evolution than would a purely clonal structure.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3607 - 15
Expression of Escherichia coli dam gene in Bacillus subtilis provokes DNA damage response: N6-methyladenine is removed by two repair pathways; Guha S et al.; The dam gene of Escherichia coli encodes a DNA methyltransferase that methylates the N6 position of adenine in the sequence GATC . It was stably expressed from a shuttle vector in a repair- and recombination-proficient strain of Bacillus subtilis . In this strain the majority of plasmid DNA molecules was modified at dam sites whereas most chromosomal DNA remained unmethylated during exponential growth . During stationary phase the amount of unmethylated DNA increased, suggesting that methylated bases were being removed . An ultraviolet damage repair-deficient mutant (uvrB) contained highly methylated chromosomal and plasmid DNA . High levels of Dam methylation were detrimental to growth and viability of this mutant strain and some features of the SOS response were also induced . A mutant defective in the synthesis of adaptive DNA alkyltransferases and induction of the adaptive response (ada) also showed high methylation and properties similar to that of the dam gene expressing uvrB strain . When protein extracts from B . subtilis expressing the Dam methyltransferase or treated with N-methyl-N'-nitro-N-nitroso-guanidine were incubated with {3H}-labelled Dam methylated DNA, the methyl label was bound to two proteins of 14 and 9 kD . Some free N6-methyladenine was also detected in the supernatant of the incubation mixture . We propose that N6-methyladenine residues are excised by proteins involved in both excision (uvrB) and the adaptive response (ada) DNA repair pathways in B . subtilis.

Eur J Biochem, 1992 Jul 15, 207(2), 677 - 85
Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene; Groch N et al.; A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli . Yields of the purified protein are at least 20 mg/l culture medium . The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein . N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E . coli . Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy . According to gel-retardation experiments, the mutant protein {Phe47----Trp}HBsu shows identical DNA binding to wild-type HBsu protein . Analysis of fluorescence binding data reveals that {Phe47----Trp}HBsu binds double-stranded DNA with a dissociation constant in the micromolar range . Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely . A dimer of {Phe47----Trp}HBsu appears to contact three or four base pairs of DNA . These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.

J Biol Chem, 1992 Jul 15, 267(20), 14509 - 14
The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis; Dahl MK et al.; Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations) . We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B . subtilis . We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase . We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence . The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS . Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype . We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro . In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of degradative enzymes in vivo.

Biochim Biophys Acta, 1992 Jul 15, 1131(3), 253 - 60
Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168; Dartois V et al.; The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli . Enzyme activity measurements showed no fatty acid chain length preference . A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing . The nucleotide sequence was determined on two independent clones expressed in E . coli . In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon . The sequence of the wild-type lip gene from B . subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR) . When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis . However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B . subtilis may function as the catalytic site . Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species . The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.

J Mol Biol, 1992 Jul 5, 226(1), 85 - 99
Mutagenesis of the Bacillus subtilis "-12, -24" promoter of the levanase operon and evidence for the existence of an upstream activating sequence; Martin-Verstraete I et al.; The levanase operon of Bacillus subtilis is controlled by RNA polymerase associated with sigma 54 factor and by the LevR activator that is homologous to the NifA/NtrC family of regulators . A "-12, -24" promoter is present at the appropriate distance from the transcription start site . The drastic down effect of base substitutions in the TGGCAC, TTGCA consensus sequence on the expression of the levanase operon confirmed the involvement of the "-12, -24" region in promoter function . Deletion derivatives of the upstream sequence of the operon promoter were constructed using translational levD'-'lacZ fusions and were integrated as single copies at the amyE locus of the B . subtilis chromosome . A cis-acting DNA sequence that is required for activation of the operon promoter by LevR was identified . This regulatory sequence is about 50 base-pairs long and is centered 125 base-pairs upstream from the transcription start site in a region containing a 16 base-pair palindromic structure . This region of dyad symmetry functions as a regulatory element when placed up to at least 600 base-pairs upstream from the "-12, -24" promoter, although the efficacy of activation is lowered . Thus, in common with most sigma 54-dependent promoters, an upstream activating sequence (UAS) is involved in the control of expression of the levanase operon . The isolation and characterization of eight mutations in the UAS region confirmed the importance of the palindromic structure in promoter activation . Moreover, the expression of the levanase operon was inhibited by placing the UAS in trans on a multicopy plasmid, probably through titration of the LevR polypeptide . In conclusion, the levanase promoter region can be divided into two regulatory sequences: the "-12, -24" promoter recognized by the sigma 54 RNA polymerase holoenzyme and the UAS, an inverted repeat sequence that is probably the LevR binding site.

Biochem J, 1992 Jul 1, 285 ( Pt 1), 99 - 103
Instability of the protoplast membrane of facultative alkaliphilic Bacillus sp . C-125 at alkaline pH values below the pH optimum for growth; Aono R et al.; Cell walls of facultative alkaliphilic Bacillus sp . C-125 consist of three polymers (peptidoglycan, teichuronopeptide and teichuronic acid) . Protoplasts prepared from the strain with egg-white lysozyme regenerated cell walls at neutral pH, but not at pH above 8.5 . The protoplasts were susceptible to lysis at alkaline pH . The protoplasts exposed to alkaline pH rapidly burst and lost ability to regenerate their cell walls . The alkali-instability was similar to that of protoplasts from neutrophilic Bacillus subtilis 168 . The membrane vesicles were also labile at alkaline pH . The acidic wall components of strain C-125 may contribute to stabilization of the cytoplasmic membrane of cells growing at alkaline pH, probably by shielding the membrane from direct exposure to an alkaline environment.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5991 - 5
Binding of the Bacillus subtilis spoIVCA product to the recombination sites of the element interrupting the sigma K-encoding gene; Popham DL et al.; The gene encoding sigma K, a transcription factor controlling mother-cell-specific gene expression at a late stage of sporulation, is interrupted by the skin element in Bacillus subtilis . The skin element is excised from the mother cell chromosome by a DNA rearrangement that depends on the spoIVCA gene product . This protein has no other role in sporulation than promoting skin excision and exhibits sequence similarity to a family of bacterial site-specific recombinases . An expression library of B . subtilis DNA in lambda gt11 was screened for the presence of a gene encoding a protein able to bind in vitro to an oligonucleotide matching the inverted repeat sequences present at the ends of the skin element . Several bacteriophages were found to contain the spoIVCA gene . A cell extract containing the SpoIVCA protein protected the inverted repeats and their neighboring sequences from DNase I digestion and methylation . SpoIVCA decreased the electrophoretic mobility of a DNA fragment containing its binding sequence and simultaneously bent the DNA . A single molecule of SpoIVCA bound initially to the repeat sequence followed by binding of a second molecule to create a complex straddling the recombination site.

Radiat Res, 1992 Jul, 131(1), 72 - 80
Inactivation action spectra of Bacillus subtilis spores with monochromatic soft X rays (0.1-0.6 nm) of synchrotron radiation; Munakata N et al.; Five types of Bacillus subtilis spores differing in DNA repair and recombinational capacities were exposed in vacuum to monochromatic soft X rays from synchrotron radiation . The inactivation rate constants were obtained from exposure-survival curves upon irradiations at 12 wavelengths in the range of 0.1000 nm (12.40 keV) to 0.6000 nm (2.066 keV) . Spores of two repair-deficient strains, UVS (uvrA ssp) and UVP (uvrA ssp polA), exhibited almost equal sensitivities to those of wild-type UVR+, while those of two recombination-deficient strains, RCE (recE) and RCF (recF), exhibited higher sensitivities in the whole wavelength range . This suggested that the repair of DNA damage produced by soft X rays was dependent on the recombinational capabilities . Inactivation action spectra based on photon fluence showed that the effectiveness of the radiation increased as the wavelengths became longer . Abrupt changes in the effectiveness occurred around the wavelengths corresponding to the absorption edges of K-shell electrons of phosphorus and calcium . In both cases, the sensitivity was the highest at the wavelengths of the resonance absorption peak, the next highest at those of the higher energy, and the lowest at the lower energy . Mass energy absorption coefficients of spores were obtained from the transmission of a flake made of spores . They were used to derive inactivation action spectra based on absorbed doses . In these spectra, basal levels of the sensitivity seemed constant, and enhancements of the sensitivity were observed consistent with the absorption by calcium and phosphorus . Thus calcium and phosphorus atoms were the predominant targets for the absorption events leading to the inactivation of spores in the wavelength range examined.

J Bacteriol, 1992 Jul, 174(14), 4727 - 35
Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli; Craven MG et al.; A shotgun cloning of Bacillus subtilis DNA into pBR322 yielded a 2-kb fragment that suppresses the cold-sensitive defect of the nusA10(Cs) Escherichia coli mutant . The responsible gene encodes an open reading frame that is greater than 50% identical at the amino acid level to the E . coli rph gene, which was formerly called orfE . This B . subtilis gene is located at 251 degrees adjacent to the gerM gene on the B . subtilis genetic map . It has been named rph because, like its E . coli analog, it encodes a phosphate-dependent exoribonuclease activity, RNase PH, that removes the 3' nucleotides from precursor tRNAs . The cloned B . subtilis rph gene also suppresses the cold-sensitive phenotype of other unrelated cold-sensitive mutants of E . coli, but not the temperature-sensitive phenotype of three temperature-sensitive mutants, including the nusA11(Ts) mutant, that were tested.

J Bacteriol, 1992 Jul, 174(14), 4647 - 56
Regulation of transcription of the cell division gene ftsA during sporulation of Bacillus subtilis; Gholamhoseinian A et al.; Three distinct 5' ends of ftsA mRNA were identified by S1 mapping and by primer extension analysis . These are thought to represent three transcription start sites . The transcripts from the downstream and upstream sites were detected throughout growth . The transcript from the middle site was not detected during exponential growth but was detected within 30 min of the start of sporulation, when it was the predominant transcript . Insertion of a cat cassette in the middle promoter, ftsAp2 (p2), did not affect vegetative growth but prevented postexponential symmetrical division and spore formation . Transcription from p2 was dependent on RNA polymerase containing sigma H, and promoter p2 resembled the consensus sigma H promoter . Transcription from p2 did not require expression of the spo0A, spo0B, spo0E, spo0F, or spo0K loci . Northern (RNA) blot analysis indicated that ftsA is cotranscribed with the adjacent ftsZ gene . Multiple promoters provide a mechanism by which essential vegetative genes can be subjected to sporulation control independent of control during vegetative growth . In the case of ftsA,Z, the promoters provide a mechanism to permit septum formation in conditions of nutrient depletion that might be expected to shut down the vegetative division machinery.

J Bacteriol, 1992 Jul, 174(14), 4629 - 37
Mutational analysis of the precursor-specific region of Bacillus subtilis sigma E; Peters HK 3rd et al.; sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is formed from an inactive precursor protein (pro-sigma E) by the removal of 27 to 29 amino acids from the pro-sigma E amino terminus . By using oligonucleotide-directed mutagenesis, sequential deletions were constructed in the precursor-specific region of sigE and analyzed for their effect on the gene product's activity, ability to accumulate, and susceptibility to conversion into mature sigma E . The results demonstrated that the first 17 residues of the pro sequence contribute to silencing the sigma-like activity of pro-sigma E and that the amino acids between positions 12 and 17 are also important for its conversion into sigma E . Deletions that remove 21 or more codons from sigE reduce sigma E activity in cells which carry it, presumably by affecting pro-sigma E stability . A 26-codon deletion results in a gene whose product is not detectable in B . subtilis by either reporter gene activity or Western blot (immunoblot) assay . The primary structure as well as the size of the pro region of sigma E contributes to the protein's stability . The placement of additional amino acids into the pro region reduces the cell's ability to accumulate pro-sigma E . Additional sigE mutations revealed that the amino acids normally found at the putative processing site(s) of pro-sigma E are not essential to the processing reaction; however, a Glu residue upstream of these sites (position 25) was found to be important for processing . These last results suggest that the pro-sigma E processing apparatus does not recognize the actual site within pro-sigma E at which cleavage occurs but rater sequence elements that are upstream of this site.

J Bacteriol, 1992 Jul, 174(13), 4374 - 83
Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression; Mueller JP et al.; The Bacillus subtilis gsiA operon was induced rapidly, but transiently, as cells entered the stationary phase in nutrient broth medium . A mutation at the gsiC locus caused sporulation to be defective and expression of gsiA to be elevated and prolonged . The sporulation defect in this strain was apparently due to persistent expression of gsiA, since a gsiA null mutation restored sporulation to wild-type levels . Detailed mapping experiments revealed that the gsiC82 mutation lies within the kinA gene, which encodes the histidine protein kinase member of a two-component regulatory system . Since mutations in this gene caused a substantial blockage in expression of spoIIA, spoIIG, and spoIID genes, it seems that accumulation of a product of the gsiA operon interferes with sporulation by blocking the completion of stage II . It apparently does so by inhibiting or counteracting the activity of KinA.

J Bacteriol, 1992 Jul, 174(13), 4302 - 7
TmrB protein, responsible for tunicamycin resistance of Bacillus subtilis, is a novel ATP-binding membrane protein; Noda Y et al.; tmrB is the gene responsible for tunicamycin resistance in Bacillus subtilis . It is predicted that an increase in tmrB gene expression makes B . subtilis tunicamycin resistant . To examine the tmrB gene product, we produced the tmrB gene product in Escherichia coli by using the tac promoter . TmrB protein was found not only in the cytoplasm fraction but also in the membrane fraction . Although TmrB protein is entirely hydrophilic and has no hydrophobic stretch of amino acids sufficient to span the membrane, its C-terminal 18 amino acids could form an amphiphilic alpha-helix . Breaking this potential alpha-helix by introducing proline residues or a stop codon into this region caused the release of this membrane-bound protein into the cytoplasmic fraction, indicating that the C-terminal 18 residues were essential for membrane binding . On the other hand, TmrB protein has an ATP-binding consensus sequence in the N-terminal region . We have tested whether this sequence actually has the ability to bind ATP by photoaffinity cross-linking with azido-{alpha-32P}ATP . Wild-type protein bound azido-ATP well, but mutants with substitutions in the consensus amino acids were unable to bind azido-ATP . These C-terminal or N-terminal mutant genes were unable to confer tunicamycin resistance on B . subtilis in a multicopy state . It is concluded that TmrB protein is a novel ATP-binding protein which is anchored to the membrane with its C-terminal amphiphilic alpha-helix.

J Bacteriol, 1992 Jul, 174(13), 4218 - 22
Influence of attractants and repellents on methyl group turnover on methyl-accepting chemotaxis proteins of Bacillus subtilis and role of CheW; Hanlon DW et al.; The ability of attractants and repellents to affect the turnover of methyl groups on the methyl-accepting chemotaxis proteins (MCPs) was examined for Bacillus subtilis . Attractants were found to cause an increase in the turnover of methyl groups esterified to the MCPs, while repellents caused a decrease . These reactions do not require CheW . However, a cheW null mutant exhibits enhanced turnover in unstimulated cells . Assuming that the turnover of methyl groups on the MCPs reflects a change in the activity of CheA, these results suggest that the activation of CheA via chemoeffector binding at the receptor does not require CheW.

Infect Immun, 1992 Jul, 60(7), 2710 - 7
Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells; Portnoy DA et al.; The Listeria monocytogenes hemolysin listeriolysin O (LLO) plays a major role in mediating the escape of L . monocytogenes from a vacuolar compartment . In a previous report, it was shown that Bacillus subtilis expressing LLO could escape from a host vacuolar compartment and grow in the cytoplasm (J . Bielecki, P . Youngman, P . Connelly, and D . A . Portnoy, Nature {London} 345:175-176, 1990) . In the present study, two related thiol-activated hemolysins, streptolysin O (SLO) and perfringolysin O (PFO), were expressed in B . subtilis and their ability to mediate intracellular growth was monitored by visual inspection and by assaying for CFU . Like LLO, PFO was active within the vacuolar environment, whereas SLO showed negligible activity . However, expression of PFO seemed to damage the host cells . The pH of the vacuole probably had little to do with these results, since all three hemolysins showed full or enhanced activity at pH 5.5, although LLO showed greatly reduced activity at pH 7 . In addition, neutralization of the pH within host vacuoles by using weak bases had little effect on the lysis of the vacuole . The lack of SLO activity is probably caused by its lower specific activity; the purified protein had 10-fold less activity on a molar basis . These results suggest that LLO is not unique in its capacity to mediate intracellular growth of B . subtilis.

Protein Eng, 1992 Jul, 5(5), 433 - 9
Bipartite organization of the Bacillus subtilis endo-beta-1,4-glucanase revealed by C-terminal mutations; Hefford MA et al.; The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-beta-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host . The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate . CD spectra indicated that the bulk of the alpha-helical secondary structure in EG470 was contained within EG300 . However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and approximately 4- to 5-fold higher with carboxymethylcellulose (soluble cellulose) . These results along with data which show that EG470 binding capacity to microcrystalline cellulose is approximately 11 times more than that of EG300, demonstrate the importance of residues 330-499 for non-catalytic binding of cellulose . A construct of the cel gene carrying a deletion of codons 330-499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine-326 and serine-321, respectively . Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1365 - 70
Molecular cloning and characterization of an alkalophilic Bacillus sp . C125 gene homologous to Bacillus subtilis sec Y; Kang SK et al.; A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp . C125 has been cloned into plasmid pUC119 using the B . subtilis secY gene as a probe . The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs . The similarity of these ORFs to the sequences of the B . subtilis proteins indicated that they were the genes for ribosomal protein L15-SecY-adenylate kinase, in that order . The gene product of the alkalophilic Bacillus sp . C125 secY homologue was composed of 431 amino acids and its M(r) value has been calculated to be 47,100 . The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments . The total amino acid sequence of alkalophilic Bacillus sp . C125 secY homologue showed 69.7% homology with that of B . subtilis secY . Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).

Res Microbiol, 1992 Jul-Aug, 143(6), 559 - 67
A degU-containing SP beta prophage complements superactivator mutations affecting the Bacillus subtilis degSU operon; Podvin L et al.; The Bacillus subtilis degSU two-gene operon encodes a two-component regulatory system which positively controls expression of a variety of genes encoding degradative enzymes . Point superactivator mutations in either degS or degU increase the production of these enzymes . A specialized transducing SP beta phage which partially complements several of these mutations was isolated from a phage library of the B . subtilis chromosome . This phage was shown to contain the degU (wild-type) gene . This unexpected co-dominance is discussed.

Genetika, 1992 Jul, 28(7), 38 - 45
{Determination of the minimal length DNA homologous region required for plasmid integration into the Bacillus subtilis chromosome via homologous recombination}; Khasanov FK et al.; With a view to determine a minimal sequence length of homology necessary for RecE-dependent homologous recombination in Bacillus subtilis cells, we developed a system, based on interaction between plasmid replicon and bacterial chromosome . Recombination frequencies were measured between ts plasmid pE194 derivatives carrying chromosomal beta-glucuronidase gene (bglS) fragments of various length, and a bacterial chromosome . The homologous recombination events resulted in bglS gene disruption . Approx . 70 bp of homology were found to be necessary for detectable homologous recombination . Homologous recombination was not detected when homology was equal 25 bp . These data indicate that homology requirement for recombination in B . subtilis differs from that in Escherichia coli.

Biokhimiia, 1992 Jul, 57(7), 1021 - 30
{Purification and properties of GTP-cyclohydrolase from Bacillus subtilis}; Boretskii IuR et al.; Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography . The N-terminal amino acid sequence and amino acid composition of the protein were determined . According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa . The active enzyme has several isoforms separable by native electrophoresis . The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+ . The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents . The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2 . The pyrimidine product of the GTP-cyclohydrolase reaction . 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified . Another product of this reaction is pyrophosphate.

Biochimie, 1992 Jul-Aug, 74(7-8), 755 - 62
Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene; Cheo DL et al.; Expression of the Bacillus subtilis recA gene is induced following DNA damage as well as during the development of the competent state . DNA damage-induction of the recA gene occurs by a RecA-dependent mechanism, whereas competence-induction occurs by a RecA-independent mechanism . To examine the molecular mechanisms that control the expression of the recA gene, a deletion analysis of the recA promoter region was performed . A regulatory region that is required for repression of recA expression was identified upstream of the recA promoter . Deletion of this regulatory region derepressed expression and abolished damage-induction of the recA promoter . Within this region are sequences similar to the consensus sequence that has been identified within DNA damage-inducible promoter regions of other B subtilis genes . Another regulatory region was identified that is required for the RecA-independent, competence-specific induction of the recA gene . Deletion of these sequences significantly reduced competence-induction of the recA promoter.

Biochimie, 1992 Jul-Aug, 74(7-8), 749 - 54
Achievement of complete Bacillus subtilis microcycle sporulation by the addition of S-adenosylmethionine and phospholipids; Petridou S et al.; In an attempt to find factors that may be responsible for the initiation of sporulation, a system in which the germination and outgrowth phases were separate was applied to Bacillus subtilis . Outgrowth of the germinated spores to only the primary singlet cells was followed in chemically defined medium . Addition of specific metabolites induced the primary singlet cells to sporulate via microcycle sporulation . Experiments are described that led to complete sporulation by the addition of diaminopimelic acid, S-adenosyl-L-methionine and phosphatidylethanolamine.

Biochimie, 1992 Jul-Aug, 74(7-8), 735 - 48
A Bacillus subtilis morphogene cluster that includes spoVE is homologous to the mra region of Escherichia coli; Henriques AO et al.; It is known that there is a strong similarity in amino acid sequence between the products of the Escherichia coli morphogenes ftsW (mra region at 2 min) and rodA (mrd region at 14 min) and the Bacillus subtilis SpoVE protein which is required for spore cortex formation . We show here that the predicted amino acid sequences coded for by the genes flanking spoVE are homologous to the products of the E coli genes murD and murG, which flank ftsW, and are involved in peptidoglycan biosynthesis . During vegetative growth and early stationary phase spoVE is cotranscribed with murD and murG in the form of very long polycistronic messages originating upstream of murD . However, this transcriptional activity is shut-off soon (approximately 1 h) after the cells enter stationary phase, and spoVE is then transcribed at two times during sporulation from its own promoter(s) . Insertional in vitro mutagenesis of the region revealed that although murD and murG are essential for normal vegetative growth, spoVE is only required for sporulation: spoVE null mutants display a sporulation stage V phenotype indistinguishable by light microscopy from the phenotype conferred by the spoVE85 and spoVE153 alleles that originally defined the locus.

Biochimie, 1992 Jul-Aug, 74(7-8), 713 - 21
Evidence that recombination between reiterated sequences in the Bacillus subtilis chromosome does not occur via unequal crossing over; Stojanovic S et al.; An experimental system was designed to permit the detection of recombination events occurring via unequal crossing over between sister bacterial chromosomes in Bacillus subtilis . It exploits the fact that during spore development, genetic and metabolic cooperation occurs between two different cell types, only one of which survives . During the early stages of sporulation, the two chromosomes of the developing sporangiole lie in the same cell and recombination between them is possible, in principle . Internal duplications flanking a selectable antibiotic-resistance gene have been introduced into the spoIIIC, spoIVA and spoVJ genes, whose correct expression in the mother cell (non-surviving compartment) is necessary for completion of spore development . After incubation in a sporulation-inducing medium in the absence of selective pressure, these strains sporulate at a low frequency and up to 30% of the progeny are Spo- . They result from mosaic sporangioles, in which only the chromosome segregated into the mother cell compartment of the developing sporangiole contains a reconstituted spo gene . In mosaic sporangioles generated by unequal crossing over between sister bacterial chromosomes, the insertionally inactivated spo gene, segregated into the pre-spore compartment, would carry an extra copy of the duplication initially present . Analysis of the products of 124 independent recombination events giving rise to mosaic sporangioles provided no evidence for the occurrence of unequal crossing over.

Biochimie, 1992 Jul-Aug, 74(7-8), 705 - 11
Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene; Smith K et al.; The product of the spoIIM gene of Bacillus subtilis is required for complete septum migration and forespore engulfment during sporulation . To investigate whether expression of spoIIM is required in the forespore compartment of the sporangium, we have constructed a new integrational vector, pKSV7, which contains temperature-sensitive replication functions derived from pE194ts . The presence of the conditionally defective replication origin greatly stimulates plasmid excision when sporulation occurs at the permissive temperature . This facilitates the use of a genetic technique employed by Illing et al to distinguish genes whose expression must occur in the forespore from genes that may be expressed exclusively in the mother cell compartment . The results of the integration/excision experiments using pKSV7 support the conclusion that spoIIM must be expressed in the forespore . Biochemical analysis of forespore and mother cell fractions suggests that spoIIM is also expressed in the mother cell . The conditional integrational vector pKSV7 replicates at high copy number in E coli and allows the identification of inserts in the polylinker cluster by disruption of alpha-complementation and thus should be useful for other kinds of genetic manipulations in B subtilis.

Biochimie, 1992 Jul-Aug, 74(7-8), 689 - 94
The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis; Karmazyn-Campelli C et al.; The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus . This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step . The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library . Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene . After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine . The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature . The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation . Possible additional roles of FtsA during sporulation are discussed.

Biochimie, 1992 Jul-Aug, 74(7-8), 679 - 88
Suppressors of a spo0A missense mutation and their effects on sporulation in Bacillus subtilis; Grossman AD et al.; The spo0A gene product of Bacillus subtilis is a transcriptional regulator that is required for the initiation of sporulation . It has not been possible to isolate mutations that suppress the sporulation defect caused by spo0A null mutations . We describe the isolation and characterization of mutations that suppress the severe sporulation defect caused by a spo0A missense mutation (spo0A9V) . Two suppressor mutations, spa2 and spa4, have been characterized in combination with, and separated from, the spo0A9V mutation . Both were located in the carboxyl half of Spo0A, in the putative DNA binding, transcriptional activation region . spa2 was in codon 174, causing a leucine to arginine change (spo0A174LR), and spa4 was in codon 162 (of 267), causing a histidine to arginine change (spo0A162HR) . spa2 and spa4 significantly restored sporulation to the spo0A9V mutant, however, the appearance of heat resistant spores was delayed relative to wild-type . When separated from spo0A9V, that is, as single mutations in spo0A, spa4 caused a delay in sporulation, while spa2 allowed apparently normal sporulation . The spa mutations caused interesting phenotypes when combined with other early sporulation mutations . spa2 suppressed the sporulation defect caused by spo0E11 . This was most easily seen in spo0E11 abrB double mutants, which had a much more severe sporulation defect than the spo0E11 single mutant . That is, spo0E11 and abrB mutations caused a synthetic (synergistic) sporulation phenotype . Both the spa2 spo0A9V and the spa4 spo0A9V alleles greatly enhanced the sporulation defect caused by mutations in spoIIJ, spo0J and spo0K . The significance of these synthetic sporulation defects is discussed.

Biochimie, 1992 Jul-Aug, 74(7-8), 669 - 78
Early spo gene expression in Bacillus subtilis: the role of interrelated signal transduction systems; Smith I et al.; The early spo genes are subject to a number of different control mechanisms . We found that at least one histidine kinase, SpoIIJ, is important for the expression of early spo genes but that two others, ComP and DegS, also affect sporulation, especially when SpoIIJ is absent . This indicates the existence of a signal transduction network which may gather information from several sources to feed into the sporulation pathway . Early spo gene expression is inhibited by overproduction of two response regulators, SpoOF and ComA . This effect is eliminated by the elevated presence of their cognate histidine kinases, SpoIIJ and ComP, respectively . This suggests that the unphosphorylated response regulators cause the inhibition of sporulation.

Biochimie, 1992 Jul-Aug, 74(7-8), 661 - 7
Protein filaments may initiate the assembly of the Bacillus subtilis spore coat; Aronson AI et al.; The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat . These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects . Only a partial inner coat was present on the spores which were lysozyme-sensitive . The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores . Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides . These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain . The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V . Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells . CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins . Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments . Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments . Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochimie, 1992 Jul-Aug, 74(7-8), 651 - 60
Effect of mutant small, acid-soluble spore proteins containing cysteine or tryptophan on DNA properties in vivo and in vitro; Sanchez-Salas JL et al.; Two derivatives of the alpha/beta-type small acid-soluble spore protein (SASP) SspCwt have been constructed, each containing a residue potentially useful for physico-chemical analysis of protein-protein or protein-DNA interactions . In one mutant protein (SspCtrp) residue 27 (Met) was replaced by Trp; in the second (SspCcys) residue 48 (Asn) was replaced by Cys . Both mutant proteins were expressed in Bacillus subtilis spores at levels similar to those of SspCwt, and SspCcys and SspCtrp restored ultraviolet light (UV) resistance and plasmid negative supercoiling in spores lacking major alpha/beta-type SASP to levels similar to those restored by SspCwt . While the purified mutant proteins bound more weakly to DNA than SspCwt, all three had the same relative affinity for different DNAs, ie poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than pUC19, and purified SspCcys and SspCtrp gave the same pattern of DNase protected bands with pUC19 as SspCwt . Binding of SspCcys or SspCtrp to poly(dG).poly(dC) in vitro also prevented the formation of cyclobutane type cytosine dimers upon UV irradiation, as does binding of SspCwt . These data indicate that the two mutant proteins are extremely similar to SspCwt in their interaction with DNA, and thus may be useful in probing SASP-SASP and SASP-DNA interactions directly by physical or chemical techniques . Indeed, binding of SspCtrp to poly(dG).poly(dC) resulted in a 2.5-fold enhancement of the proteins Trp fluorescence.

Biochimie, 1992 Jul-Aug, 74(7-8), 635 - 40
Intergenic suppression of stage II sporulation defects by a mutation in the major vegetative sigma factor gene (rpoD) of Bacillus subtilis; Lee A et al.; The Bacillus subtilis intergenic suppressor mutations crsA and rvtA, previously shown to restore sporulation competence to a variety of strains containing stage 0 sporulation defects, also suppress lesions in the stage II sporulation genes spoIIF, spoIIN and spoIIJ . They do not rescue sporulation in other stage II through stage V sporulation mutations . Cells containing spoIIN, spoIIF96 and spoIIJ::Tn917 mutations fail to transcribe spoIID, a late stage II gene . Introduction of crsA47 into spoIINts279, spoIIF96, or spoIIJ::Tn917 mutant backgrounds circumvents the need for the spoIIF, IIN, and IIJ products, restoring both expression of spoIID, and sporulation competence.

Biochimie, 1992 Jul-Aug, 74(7-8), 627 - 34
The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis; Bird T et al.; The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis . We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spoIIA promoter . Core RNA polymerase or holoenzyme containing sigma A was reconstituted with sigma H protein and used to transcribe the spoIIA promoter . Formation of heparin resistant transcription initiation complexes required that the spoIIA template be supercoiled . Topoisomers of the spoIIA template were created and transcribed at various temperatures . Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spoIIA promoter.

Biochimie, 1992 Jul-Aug, 74(7-8), 601 - 12
The interaction between Bacillus subtilis sigma-A (sigma A) factor and RNA polymerase with promoters; Chang BY et al.; The P2 promoter from Bacillus subtilis sigma-A (sigma A) operon and the strong phi 29 phage G3b promoter were used to study their interactions with free sigma A and with RNA polymerase holoenzymes (E sigma A and E sigma 70) . No binding of free sigma A to the tested promoters was observed, suggesting that the B subtilis free sigma A does not bind promoter by itself for the initiation of RNA transcription . Different footprints of B subtilis RNA polymerase holoenzyme (E sigma A) on the P2 and G3b promoters were detected . The footprint on the P2 promoter is mainly in the -10 downstream region of the bottom strand (noncoding strand) DNA and limited on the top strand (coding strand), whereas the footprints on both strands of the G3b promoter are very clear . These results suggest that the footprint regions of RNA polymerase on a promoter and the strength of its binding to the promoter depend on the properties of the specific promoter DNA sequence . It also suggests that the -10 and its downstream regions are more important than the -35 region for the formation of the E sigma A-P2 promoter open complex . Footprints of B subtilis E sigma A and E coli E sigma 70 on the same G3b promoter are very similar on the top strand but different on the bottom strand, with the footprint being about 17 bases wider (-4 to +13) in the case of E coli E sigma 70 . Since this region contains most of the bases involved in promoter DNA melting, we suggest that E coli and B subtilis RNA polymerases have different efficiency in forming the open complex with heterologous promoter DNA during initiation of transcription.

Biochimie, 1992 Jul-Aug, 74(7-8), 619 - 26
Spo0A activates and represses its own synthesis by binding at its dual promoters; Strauch MA et al.; The Spo0A protein of Bacillus subtilis controls the onset of sporulation by regulating transcription of various genes in both positive and negative manners depending on the promoters affected . The expression of the spo0A gene occurs from two promoters (Pv,Ps), separated by 148 bp, and transcription switches from Pv to Ps early in the sporulation program . DNase I footprint analysis of the spo0A promoter region revealed three distinct sites of Spo0A binding: -4 to +19 relative to Pv, -17 to +1 relative to Ps, and a region between Pv and Ps . The Pv region and the region between the two promoters was sufficient for repression of Pv . Induction of Ps also required these regions which are upstream of -52 relative to Ps . Mutant Spo0A proteins containing asp----asn mutations at asp10 and asp56 were inactive in repression of the abrB promoter in vivo yet still retained DNA-binding activity . The results presented are consistent with a model in which the phosphorylated form of Spo0A acts directly at its promoters to achieve induction of Ps and repression of Pv . These effects at the spo0A promoter were independent of the presence of the major kinase, KinA.

J Bacteriol, 1992 Jul, 174(13), 4308 - 16
In vivo and in vitro characterization of the secA gene product of Bacillus subtilis; Takamatsu H et al.; The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y . Sadaie, H . Takamatsu, K . Nakamura, and K . Yamane, Gene 98:101-105, 1991) . The B . subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E . coli . The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence . The B . subtilis div+ gene was overexpressed in E . coli under the control of the tac promoter, and its product was purified to homogeneity . The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein . The antiserum against B . subtilis Div weakly cross-reacted with E . coli SecA . On the other hand, B . subtilis Div could not replace E . coli SecA in an E . coli in vitro protein translocation system . The temperature-sensitive growth of the E . coli secA mutant could not be restored by the introduction of B . subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa . The B . subtilis div+ gene is the B . subtilis counterpart of E . coli secA, and we propose that the div+ gene be referred to as B . subtilis secA, although Div did not function in the protein translocation system of E . coli.

Biochimie, 1992 Jul-Aug, 74(7-8), 641 - 50
The DNA-binding protein HBsu is essential for normal growth and development in Bacillus subtilis; Micka B et al.; The hbs operon of Bacillus subtilis comprises a single gene which is localized between the spoIVA and mtrA open reading frames, and is situated at 204 degrees of the standard map of B subtilis . Expression of hbs is initiated from two distinct promoters called P1 and P2 . The transcription initiation sites have been mapped by primer extension analysis . Sequences upstream from P1 show a -35 and -10 region which may be recognized by the vegetative form of RNA polymerase E sigma A, whereas sequences upstream from P2 may be recognized by either E sigma C or E sigma H minor forms of RNA polymerase . In vegetative cells, hbs is highly and equally transcribed from both promoters, P1 and P2 . In contrast, in sporulating cells, hbs is expressed predominantly from P2 . In order to study the physiological role of HBsu, we must overcome our failure to interrupt the hbs gene within the B subtilis chromosome by using a previously constructed strain, BM19, bearing hbs under the control of the IPTG-inducible spac-1 promoter . In this strain, growth was found to depend highly on hbs expression . In the absence of IPTG, growth was strongly affected culminating in a filamentous cell morphology . Although sporulation in IPTG-uninduced BM19 cells was poor, due to the limited cell growth, the outgrowth of those spores was delayed by 1 h . In contrast, in the presence of IPTG, a condition that induces hbs expression, normal outgrowth of spores was observed . The proposed essentiality of the hbs gene product for growth and development in B subtilis may be attributed to its interaction with replication and transcription as a consequence of its facility to wrap DNA and to condense the chromosome into nucleosomelike structures . A comparative sequence analysis of HBsu with 18 homologous histonelike proteins of diverse origin demonstrated their high conservation throughout evolution.

Biochimie, 1992 Jul-Aug, 74(7-8), 613 - 8
Activity of ribosomal and tRNA promoters of Bacillus subtilis during sporulation; Okamoto K et al.; The rrnB operon in Bacillus subtilis includes 21 tRNA genes downstream of the ribosomal genes . In addition to the dual promoters upstream of these ribosomal genes, a second promoter is found within the tRNA gene region . In this study, these two promoter regions were inserted before the lacZ gene and each was integrated as a single-copy into the B subtilis chromosome to examine their activity during sporulation . Both promoters exhibited similar strength and temporal downregulation from vegetative growth through t3 of sporulation . At t3, transcription from both promoters was approximately 20% of that in logarithmic growth . No obvious function of the second promoter is evident except to boost transcription of downstream genes . This function may be important because the ribosomal promoters of rrnB are weak relative to other ribosomal promoters . For instance, the rrnO promoters were much stronger than the rrnB ribosomal promoters and considerably more active at t3 . Thus, all ribosomal promoters are not of the same strength nor are they transcriptionally downregulated to the same extent during sporulation . However, both the rRNA and tRNA promoters in the rrnB operon are similar in strength and downregulation.

J Bacteriol, 1992 Jul, 174(13), 4361 - 73
Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system; Mueller JP et al.; The Bacillus subtilis glucose starvation-inducible transcription units, gsiA and gsiB, were characterized by DNA sequencing, transcriptional mapping, mutational analysis, and expression in response to changes in environmental conditions . The gsiA operon was shown to consist of two genes, gsiAA and gsiAB, predicted to encode 44.9- and 4.8-kDa polypeptides, respectively . The gsiB locus contains a single cistron which encodes a protein of unusual structure; most of its amino acids are arranged in five highly conserved, tandemly repeated units of 20 amino acids . The 5' ends of gsiA and gsiB mRNAs were located by primer extension analysis; their locations suggest that both are transcribed by RNA polymerase containing sigma A . Expression of both gsiA and gsiB was induced by starvation for glucose or phosphate or by addition of decoyinine, but only gsiA was induced by exhaustion of nutrient broth or by amino acid starvation . Regulation of gsiA expression was shown to be dependent upon the two-component signal transduction system ComP-ComA, which also controls expression of genetic competence genes . Mutations in mecA bypassed the dependency of gsiA expression on ComA . Disruption of gsiA relieved glucose repression of sporulation but did not otherwise interfere with sporulation, development of competence, motility, or glucose starvation survival . We propose that gsiA and gsiB are members of an adaptive pathway of genes whose products are involved in responses to nutrient deprivation other than sporulation.

Biosci Biotechnol Biochem, 1992 Jul, 56(7), 1166 - 8
Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action; Kato T et al.; A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto) . Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al . and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA . The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26 . The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.

Trends Biotechnol, 1992 Jul, 10(7), 247 - 56
Bacillus subtilis and its relatives: molecular biological and industrial workhorses; Harwood CR; The non-pathogenic bacterium Bacillus subtilis, since its first reported genetic transformation in 1959, has become a model system for the study of many aspects of the biochemistry, genetics and physiology of Gram-positive bacteria, and particularly of sporulation and associated metabolism . Extensive knowledge of the molecular biology of B . subtilis has led to the recent development of this bacterium as a host for the industrial production of heterologous proteins . Although difficulties have been encountered, these are being systematically addressed and overcome.

J Bacteriol, 1992 Jul, 174(14), 4606 - 13
Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production; Ludovice M et al.; The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33 . The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da . The argC gene is linked to argE, as shown by complementation of argE mutants of E . coli . Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S . lividans and in the argC mutant S . lividans 1674 . Formation of AGPR is repressed by addition of arginine to the culture medium . The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E . coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp . A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein . Transformation of S . clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production.

Gene, 1992 Jul 1, 116(1), 69 - 74
New IS10 transposition vectors based on a gram-positive replication origin; Mahillon J et al.; We describe below a set of plasmid-based vehicles which can be used for delivery of IS10-derived transposons into Gram- bacteria . These vehicles replicate via a Gram+ plasmid origin that is inactive in Escherichia coli; they are easily maintained in Bacillus subtilis . Transposons are introduced by electroporation or transformation with the plasmid, and as in previous delivery systems, transpositions are selected with the appropriate antibiotic . This system should be particularly useful in situations where the standard delivery vehicles, based on bacteriophage lambda, are inappropriate . The system described incorporates a number of useful features: a variety of antibiotic markers (Er, Cm, Km or Tc), a polylinker containing restriction sites for rare-cutting endonucleases to facilitate physical mapping of chromosomal insertions, a mutant transposase that confers a relaxation in insertion specificity and positioning of the transposase-encoding gene outside of the transposing segment to ensure the stability of insertions once isolated.

FEBS Lett, 1992 Jun 29, 305(2), 115 - 20
Synthesis and characterization of a 29-amino acid residue DNA-binding peptide derived from alpha/beta-type small, acid-soluble spore proteins (SASP) of bacteria; Rao H et al.; A 29-amino acid residue peptide (SASP-peptide) derived from the sequence of the putative DNA-contacting portion at the carboxyl terminus of an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis has been synthesized by automated solid-phase methods and tested for its ability to interact with DNA . Circular dichroism (CD) spectroscopy reveals an interaction between this SASP-peptide and DNA, both by an increase in alpha-helix content of the peptide (which alone has a mostly random conformation) and by enhancement of the 275-nm CD band of the DNA . In contrast to results with intact alpha/beta-type SASP, however, the peptide does not induce a B----A conformational transition in the DNA . The SASP-peptide also binds to poly(dG).poly(dC) and protects this polynucleotide against DNase I digestion and UV light-induced cytosine dimer formation, parallel to findings made previously with native alpha/beta-type SASP . The results confirm the hypothesis that the carboxyl-terminal region of the alpha/beta-type SASP directly contacts DNA and possesses some, but not all, of the functional characteristics of the intact molecule.

Biochim Biophys Acta, 1992 Jun 22, 1126(2), 119 - 24
Unsaturated and branched chain-fatty acids in temperature adaptation of Bacillus subtilis and Bacillus megaterium; Suutari M et al.; The effect of growth temperature on the cellular fatty acid profiles of Bacillus subtilis and Bacillus megaterium was studied over a temperature range from 40 to 10 degrees C . As the growth temperature of B . subtilis was reduced, the lower-melting point anteiso-acids increased, while the higher-melting point iso-acids decreased . Consequently the ratio of branched- to straight-chain acids was unaffected by temperature, although changes in the position of fatty acid branching and the degree of unsaturated branched-chain fatty acids occurred . In B . megaterium a more complicated, biphasic behaviour was observed . Saturated, straight-chain and iso-branched acids decreased only from 40 degrees C down to 20-26 degrees C, and anteiso-acids decreased only from 20-26 degrees C to 10 degrees C, while unsaturated acids increased over the whole temperature range studied . Thus, in B . megaterium total branched-chain acids decreased and straight-chain acids increased as temperature decreased . However, the overall cellular content of lower-melting point fatty acids increased with decreasing temperature in both bacilli, and unsaturated fatty acids appeared to be essential components in the adaptation of the microbes to changes in temperatures . Since changes in the relative amounts of branched- and straight-chain fatty acid biosynthesis are known to reflect differences in fatty acid primers, temperature seems to affect not only the activity of the fatty acid desaturases but also the formation or availability of these primers . The results indicate, however, that notable species-specific regulatory features exist in this genus of bacteria.

FEBS Lett, 1992 Jun 22, 305(1), 67 - 73
Characterization of the pcp gene encoding the pyrrolidone carboxyl peptidase of Bacillus subtilis; Awade A et al.; Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp) is an enzyme that catalyzes the removal of the N-terminal pyroglutamyl group from some peptides or proteins . Its value in protein chemistry and bacterial diagnosis makes this enzyme an interesting subject of study . The present paper reports for the first time the cloning and characterization of a pyrrolidone carboxyl peptidase gene (pcp) . This gene is present in a single copy in the genome of Bacillus subtilis as indicated by Southern blot hybridization analysis . The pcp transcripts were analyzed in Escherichia coli by Northern blot hybridization and S1 nuclease mapping . The deduced amino acid sequence predicts a protein of 215 amino acids with a calculated molecular weight of 23,777 Da . The pcp gene has been over-expressed in E . coli, allowing the identification and partial characterization of Pcp protein.

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