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<