Bacillus subtilis

J Bacteriol, 1996 Apr, 178(7), 1980 - 9
Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms; Mendelson NH et al.; Factors governing the morphogenesis of Bacillus subtilis colonies as well as the spatial-temporal pattern of expression of a reporter gene during colony development were examined by systematically varying the initial nutrient levels and agar concentrations (wetness), the relative humidity throughout incubation, and the genotype of the inoculum . A relationship between colony form and reporter gene expression pattern was found, indicating that cells respond to local signals during colony development as well as global conditions . The most complex colony forms were produced by motile strains grown under specific conditions such that cells could swim within the colony but not swarm outward uniformly from the colony periphery . The wetness of the growth environment was found to be a critical factor . Complex colonies consisted of structures produced by growth of finger-like projections that expanded outward a finite distance before giving rise to a successive round of fingers that behaved in a similar fashion . Finger tip expansion occurred when groups of cells penetrated the peripheral boundary . Although surfactin production was found to influence similar colony forms in other B . subtilis strains, the strains used here to study reporter gene expression do not produce it . The temporal expression of a reporter gene during morphogenesis of complex colonies by motile strains such as M18 was investigated . Expression arose first in cells located at the tips of fingers that were no longer expanding . The final expression pattern obtained reflects the developmental history of the colony.

J Bacteriol, 1996 Apr, 178(7), 1971 - 9
LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family; Schnetz K et al.; Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization . Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family . Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family . The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t . The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold . This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t . However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization.

Biochemistry, 1996 Mar 19, 35(11), 3636 - 40
Peptidyl-prolyl cis-trans isomerase of Bacillus subtilis: identification of residues involved in cyclosporin A affinity and catalytic efficiency; Gothel SF et al.; The 17-kDa peptidyl-prolyl cis-trans-isomerase from Bacillus subtilis (PPiB) is a member of the cyclophilin family and shows strong homology to PPIases of eukaryotic origin (40%) and less identify to PPIase sequences of Gram-negative bacteria (27-32%) . Although the majority of residues that form the PPIase active site are highly conserved, three residues, V52, H90, and H109 in the sequence of the B.subtilis PPIase, were found to differ from the sequences found in human (hCyP) and Escherichia coli (eCyP) . Also, the binding affinity of cyclosporin A (CsA) to the different PPIases varies in IC(50) values from 6 nM for human PPIase hCyPA and 84 nM for the human hCyPB to over 120 nM for B . subtilis and 3000 nM for E . coli . In addition, a variety of k(cat)/K(m) values, ranging from 1.1 mM(-1) s(-1) for the B . subtilis PPIase to over 10 mM(-1) s(-1) for human and 13 mM(-1) s(-1) for E . coli, were detected using the common substrate suc-Ala-Ala-Pro-Phe-pNA . Through site-specific mutagenesis we demonstrate that the differences in the three mentioned residues are mainly responsible for the variations in IC(50) and k(cat)/K(m) values . Replacement of H90 to N90, or H109 to W109, resembling the amino acid sequence of human hCyPA, resulted in more efficient CsA binding (IC(50) value for H90N, 60 nM, and for H109W, 95 nm), whereas replacement of H90 to R90, or H109 to F109, resembling the amino acid sequence of E . coli eCyP, resulted in less efficient CsA binding (IC(50) value for H90R, 2000nM, and for H109F, 5000 nM) . In addition to lower CsA affinity, mutant protein H109F shows a k(cat)/K(m) value of 10.5 mM(-1) s(-1), comparably high to that of the wild-type E . coli protein . In contrast, other mutants like C57F, H90N, H90R, and H109W do not differ significantly in k(cat)/K(m) values from wild-type PPiB . Replacement of V52 to M52, which is conserved in E . coli and all known eukaryotic PPIases, does not show any effect in CsA binding affinity (IC(50) value for V52M, 120 nM), but it raises the catalytic efficiency by 12-fold to k(cat)/K(m) of 14 mM(-1) s(-1) . In conclusion, our studies suggest that the unique histidine residues H90 and H109 in B . subtilis PPIase are, at least in part, responsible for its intermediate CsA affinity and that the v52 residue confers the low conversion rate.

FEMS Microbiol Lett, 1996 Mar 15, 137(1), 13 - 8
Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines; Ghim SY et al.; The pyrimidine nucleotide biosynthetic (pyr) operon in Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which PyrR, a bifunctional pyr RNA-binding attenuation protein/uracil phosphoribosyltransferase, plays a crucial role . A convenient procedure for isolation of pyrR mutants with defects in the regulation of pyr operon expression is described . The selection is based on the selection of spontaneous mutations that convert the pyrimidine-sensitive growth of cpa strain (lacking arginine-repressible carbamyl phosphate synthetase) to pyrimidine resistance . Twelve such mutants were isolated and sequenced . All resulted from point mutations in the pyrR gene.

Eur J Biochem, 1996 Mar 15, 236(3), 843 - 6
Aminopeptidase from Streptomyces griseus: primary structure and comparison with other zinc-containing aminopeptidases; Maras B et al.; The aminopeptidase from Streptomyces griseus is a calcium-activated metalloenzyme, which contains 2 mol tightly bound zinc/mol protein . This aminopeptidase rapidly hydrolyzes peptide bonds formed by N-terminal hydrophobic amino acids, such as leucine, methionine and phenylalanine . We have determined the complete primary structure of the protein, which contains 284 amino acid residues, yielding a molecular mass of 29723 Da . A search in the Swiss-Prot database for sequence similarities revealed a low degree of identity (26-34%) to Saccharomyces cerevisiae aminopeptidase Y, Aeromonas proteolytica aminopeptidase, and a hypothetical 49.5-kDa protein from Bacillus subtilis, which is supposed to belong to the aminopeptidase Y family . In all these proteins, the residues that are known to be involved in zinc coordination are conserved.

Biochim Biophys Acta, 1996 Mar 15, 1289(2), 217 - 20
A simple solid-state NMR method for determining peptidoglycan crosslinking in Bacillus subtilis; Shenouda NS et al.; The crosslink index of the peptidoglycan of intact cell walls of Bacillus subtilis grown in media containing L-{15N}aspartic acid has been determined by a combination of cross-polarization magic-angle spinning 15N NMR and gas chromatography/mass spectrometry . The crosslink index of 62% decreased to 52% when the bacteria were exposed to the antibiotic cephalothin.

Biochemistry, 1996 Mar 5, 35(9), 2926 - 33
A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates; Zapf JW et al.; Transient phosphorylation at an aspartate residue on the Spo0F protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli . The response regulator Spo0F-P is stable to hydrolysis (t1/2 > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated forms of Spo0F . Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate Spo0F, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors . Rapid hydrolysis of Spo0F-P generated with phosphoramidate by proteins downstream in the phosphorelay (Spo0B and Spo0A) is consistent with phosphorylation at the active site of Spo0F . The initial rate of Spo0F-P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as Spo0F, function as phosphoryl transferase enzymes (McCleary et al., 1993) . The results establish that Spo0F functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation.

Mikrobiol Z, 1996 Mar-Apr, 58(2), 46 - 55
{The effect of live cultures of Bacillus subtilis on body nonspecific resistance}; Kudriavtsev VA et al.; Alive cultures of Bacillus subtilis, antagonists of pyogenic microflora, have been studied for their effect on functional activity of macrophagal cells and induction of endogenic serum alpha-interferon in the female mice of the SBA line . It is established that even a single intravaginal and intraperitoneal introduction of bacilli in a dose of 1 milliard cells in 0.1 ml of cultural fluid stimulates migration, absorption and especially bactericidal activity of macrophages of peritoneal exudate . Introduction of alive cultures of aerobic bacilli essentially stimulates the in vivo production of the serum and alpha-interferon induced in vitro by the Newcastle disease virus . The highest immune-stimulating effect under a single introduction of B . subtilis is achieved 66 h later and has a tendency to gradual decrease.

Mol Microbiol, 1996 Mar, 19(5), 941 - 8
Three two-component signal-transduction systems interact for Pho regulation in Bacillus subtilis; Sun G et al.; The Pho regulon of Bacillus subtilis is controlled by three two-component signal-transduction systems: PhoP/PhoR, ResD/ResE, and the phosphorelay leading to the phosphorylation of SpoOA . Two of these systems act as positive regulators, while the third is involved in negative regulation of the Pho regulon . Under phosphate-starvation-induction conditions, the response regulator (RR) PhoP, and the histidine protein kinase (HK) PhoR, are involved in the induction of Pho-regulon genes including the phoPR operon and genes encoding the major vegetative alkaline phosphatases, phoA and phoB . ResD (the RR) and ResE (the HK) are positive regulators of both aerobic and anaerobic respiration in B . subtilis . Current data suggest that they are also positive regulators of the Pho regulon, as is the transition-state regulatory protein AbrB . Data presented reveal that ResDE and AbrB are involved in activation of the Pho regulon through separate regulatory pathways . SpoOA approximately P (RR) exerts a negative effect on the Pho regulon through its repression of AbrB, and possibly through repression of ResDE . Both pathways converge to regulate transcription of the phoPR operon.

Mol Microbiol, 1996 Mar, 19(5), 1047 - 60
Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly; Barak I et al.; Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor sigma F, which directs early forespore-specific gene expression . We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B . subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium . We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa . Each of five point-mutation alleles, which have traditionally defined the locus, and two transposon-generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression . The amino-terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane-spanning segments . The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous-phase exposed domains . An in-frame fusion joining the 3' end of the B . megaterium spoIIE coding sequence to the 5' end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation-specific fluorescent signal localized to the sites of sporulation septum assembly . We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development.

Mol Microbiol, 1996 Mar, 19(6), 1295 - 306
Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20; Meijer WJ et al.; We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions . Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B . subtilis chromosomal replication terminator . Here, we demonstrate that this segment is part of a functional replication terminator . This newly identified element, designated TerLS20, is the first replication terminator identified on a theta plasmid from a Gram-positive bacterium . TerLS20 is distinct from other known replication terminators in the sense that it is functional in both orientations . The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.

Mol Microbiol, 1996 Mar, 19(6), 1151 - 7
Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in Bacillus subtilis; Perego M et al.; Bacteria use two-component signal transduction systems to sense and respond to their environment . A sensor kinase and a response-regulator transcription factor work in concert by phosphorylation/dephosphorylation through kinase and phosphatase activities to maintain a level of phosphorylated response regulator commensurate with the level of signal input . Signal input can be accommodated through stimulation of the kinase activity or the phosphatase activity of the two-component system . With some notable exceptions, the sensor kinases recognize a single stimulatory ligand . A new dimension in the regulation of two-component signal transduction systems was discovered in the Rap phosphatases which dephosphorylate the SpoOF response-regulator of Bacillus subtilis independent of the sensor kinases . This family of phosphatases is encoded by at least six chromosomal genes . Although not all of the phosphatases of the family have activity on phosphorylated SpoOF, the two best-characterized members, RapA and RapB, prevent sporulation by dephosphorylating this response regulator component of the phosphorelay . Phosphatase activity of RapA is regulated by a gene, phrA, in the same transcriptional unit, that encodes a peptide secreted from the cell which may serve as a quorum sensor . Most of the Rap phosphatase operons have a gene coding for a protein with some similarity to PhrA in their transcription units, but it is uncertain whether all of these play a role in regulation . The Rap phosphatases are postulated to be a mechanism for allowing signals other than those that affect the sensor kinases to regulate the signal transduction pathway . They may have been recruited to help regulate sporulation because the multiple signals regulating this process may outstrip the recognition capacity of the kinases.

Phytochemistry, 1996 Mar, 41(4), 1191 - 5
Antibacterial hydroperoxysterols from Xanthosoma robustum; Kato T et al.; Three new hydroperoxysterols, 24-hydroperoxy-4 alpha, 14 alpha- dimethylcholesta-8,25-dien-3 beta-ol, 25-hydroperoxy-4 alpha, 14 alpha-dimethylcholesta-8,23-dien-3 beta-ol and 25-hydroperoxycycloart-23-en- 3 beta-ol, have been isolated from the aerial parts of Xanthosoma robustum, besides 24-hydroperoxycycloart-25-en-3 beta-ol, 4 alpha, 14 alpha-dimethylcholesta-8,24-dien-3 beta-ol and cycloartenol . Additionally, the two new diols, 4 alpha, 14 alpha-dimethylcholesta-8,25-dien-3 beta, 24-diol and 4 alpha, 14 alpha-dimethylcholesta-8,23-dien-3 beta, 25-diol were obtained from the first two hydroperoxysterols, respectively, by reduction with triphenylphosphine . The structures have been defined by chemical and spectroscopic studies . The four hydroperoxysterols exhibited antibacterial activities against Escherichia coli, Bacillus subtilis and Micrococcus luteus.

Phytochemistry, 1996 Mar, 41(4), 1041 - 6
Antimicrobial steroids from the fungus Fomitopsis pinicola; Keller AC et al.; Phytochemical examination of the dichloromethane extract of the European fungus Fomitopsis pinicola led to the isolation of a new lanostanoid derivative identified from spectral and chemical evidences as 3 alpha-(4-carboxymethyl-3-hydroxy-3-methylbutanoyloxy)-lanosta++ +-8,24-dien-21-oic acid . In addition, seven known triterpenes, polyporenic acid C, 3 alpha-acetyloxylanosta-8,24-dien-21-oic acid, ergosta-7,22-dien-3 beta-ol,21-hydroxylanosta-8,24-dien-3-one, pinicolic acid A, trametenolic acid B and pachymic acid, were also isolated . Antimicrobial activity against Bacillus subtilis in a TLC bioassay was observed for five of the isolated steroids.

Prikl Biokhim Mikrobiol, 1996 Mar-Apr, 32(2), 231 - 6
{Hydrolyzing ability of yeast proteases in relation to protein substrates}; Nekliudov AD et al.; The possibility of use of brewer's yeast as multienzymatic preparations for hydrolyzing of animal blood proteins was demonstrated . The kinetic characteristics of hydrolysis of blood proteins by activated brewer's yeast mass and neutral proteinase from Bacillus subtilis 102 and the activation energies were estimated . Brewer's yeast biomass may be used as a source of enzymes of broad substrate specificity and noncontiguous amino acids, in particular, isoleucine, to increase the biological efficiency of blood proteins by 15-30%.

Prikl Biokhim Mikrobiol, 1996 Mar-Apr, 32(2), 194 - 202
{Some patterns in formation of antibody-antigen-antibody complexes on a solid phase: experimental study and mathematical modeling}; Zherdev AV et al.; Equilibrium and kinetic constants were determined for interactions between alpha-amylase from Bacillus subtilis and polyclonal antibodies (with immobilization of either reagent) . The effects of desorption of immobilized molecules and intermolecular complexes on the immunochemical reaction were studied . Models of sandwich enzyme immunoassay, whereby complexes of immobilized antibody-determined antigen-labeled antibody were formed, were developed from these results . The results obtained from the model and experiments were compared . The desorption was shown to cause the hook-effect, that is, a decrease in the label binding at high concentrations of the antigen.

Vet Med (Praha), 1996 Mar, 41(3), 93 - 5
High-voltage electrophoretic identification of residual antibiotics in milk; Krcmar P et al.; Residual antibiotics in milk were identified by high-voltage electrophoresis in 1% agarose gel and bioautographic detection . The test strain Bacillus subtilis BGA was used for the detection, pH of the culture medium was 8.0 . An electrophoretic identification map of 13 selected antibiotics has been set up and the following minimal inhibition concentrations (MIC), expressed in microgram per 1 g, have been established: benzylpenicillin 0.06; ampicillin 0.25; streptomycin 0.5; dihydrostreptomycin 0.2; spectinomycin 40; gentamycin 0.06; neomycin 0.15; oxytetracycline 5; tetracycline 2.5; chlortetracycline 1; erythromycin 0.01; tylosin 1; chloramphenicol 20 . The established values of MIC were compared with the Maximum Residue Limits (MRL) as defined currently in the legislation of the European Union . The condensation of samples by freeze-drying increased the sensitivity of the method which was used for the identification of residual antibiotics detected by microbiological screening techniques in milk.

J Bacteriol, 1996 Mar, 178(5), 1473 - 5
The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain; Markham PN et al.; Rhodamine and tetraphenylphosphonium, the substrates of the Bacillus subtilis multidrug efflux transporter Bmr, induce the expression of Bmr through direct interaction with its transcriptional activator BmrR . Here we show that the C-terminal domain of BmrR, expressed individually, binds both these compounds and therefore can be used as a model for molecular analysis of the phenomenon of multidrug recognition.

J Bacteriol, 1996 Mar, 178(5), 1374 - 85
Regulators of aerobic and anaerobic respiration in Bacillus subtilis; Sun G et al.; Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration . The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD . They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially . ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex) . The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins . resD null mutations are more deleterious to the cell than those of resE . resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate . A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation . The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis.

J Mol Biol, 1996 Mar 1, 256(3), 436 - 48
The Bacillus subtilis response regulator Spo0A stimulates transcription of the spoIIG operon through modification of RNA polymerase promoter complexes; Bird TH et al.; Sporulation in Bacillus subtilis is dependent on the response regulator Spo0A, which both represses and activates transcription in vitro . The activity of Spo0A is increased by phosphorylation . We previously demonstrated that the phosphorylation increased the ability of Spo0A to stimulate in vivo transcription from the promoter for the spoIIG operon, one of the operons known to be regulated by Spo0A in vivo . In the work reported here we have examined the kinetics of transcription initiation at the spoIIG operon promoter using a single round transcription assay and the kinetics of formation of spoIIG promoter-RNA polymerase complexes using DNase I footprinting . Both the kinetic assays and the footprint assays indicated that the initial binding of the polymerase to the template was not dependent on the presence of Spo0A . The phosphorylated form of Spo0A stimulated the rate of initiation by affecting a step that occurred after the initial interaction of the polymerase with the template . Phosphorylation of Spo0A may stimulate transcription by modifying preinitiation complexes containing the polymerase and the promoter.

FEBS Lett, 1996 Feb 26, 381(1-2), 29 - 31
A functional Spo0A is required for maximal aprE expression in Bacillus subtilis; Olmos J et al.; The initiation of sporulation in Bacillus subtilis is under control of the transcriptional factor Spo0A . Most Spo0A mutants fail to initiate the sporulation process and all the sporulation initiated processes such as the synthesis of subtilisin . However, the product of spo0A9V, one of the several spo0A mutants characterized, distinguishes itself in the fact that, while it appears to effectively repress abrB, it fails to activate the spoIIA operon . The aim of this study was to examine the effect of the spo0A9V mutation on aprE expression and we found that in different genetic backgrounds, the spo0A9V mutation has a negative effect on aprE::lacZ expression.

J Mol Biol, 1996 Feb 23, 256(2), 301 - 18
The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination; Ayora S et al.; Inactivation of Bacillus subtilis orf1177 in an otherwise Rec+ strain reduced genetic exchange and DNA repair . When the mutation was transferred into a set of recombination-deficient and repair-deficient strains, the DNA repair and recombination ability of the double or triple mutant strains was drastically reduced . B . subtilis Orf1177 protein shares substantial homology with the Escherichia coli Mdf, RecG and UvrB proteins . In vivo analysis of UV-induced mutations suggests that Orf1177 is necessary for strand-specific DNA repair, as is the case for the E . coli MFD protein . Therefore, orf1177 and Orf1177 were termed mfd gene and Mfd protein, respectively . The purified Mfd protein has a native molecular mass of 140 kDa (expected molecular mass 133 kDa) . The Mfd protein is a sequence-independent DNA binding protein with weak ATPase activity . The Mfd protein was able to displace in vitro B . subtilis or E . coli RNA polymerase stalled at a lesion . Therefore, Mfd protein appears to target the transcribed strand for repair by recognizing a stalled RNA polymerase and dissociating it from the DNA . In addition, the strong recombination-deficient phenotype of mfd- rec- strains suggest that Mfd protein is involved in homologous DNA recombination.

Gene, 1996 Feb 22, 169(1), 17 - 23
Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region; Suh JW et al.; We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs) . The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17 . The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons . This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36 . Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16 . Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters . The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required . Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization . Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs.

Proc Natl Acad Sci U S A, 1996 Feb 20, 93(4), 1549 - 53
Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis; Perego M et al.; Rap phosphatases are a recently discovered family of protein aspartate phosphatases that dephosphorylate the Spo0F--P intermediate of the phosphorelay, thus preventing sporulation of Bacillus subtilis . They are regulators induced by physiological processes that are antithetical to sporulation . The RapA phosphatase is induced by the ComP-ComA two-component signal transduction system responsible for initiating competence . RapA phosphatase activity was found to be controlled by a small protein, PhrA, encoded on the same transcript as RapA . PhrA resembles secreted proteins and the evidence suggests that it is cleaved by signal peptidase I and a 19-residue C-terminal domain is secreted from the cell . The sporulation deficiency caused by the uncontrolled RapA activity of a phrA mutant can be complemented by synthetic peptides comprising the last six or more of the C-terminal residues of PhrA . Whether the peptide controls RapA activity directly or by regulating its synthesis remains to be determined . Complementation of the phrA mutant can also be obtained in mixed cultures with a wild-type strain, suggesting the peptide may serve as a means of communication between cells . Importation of the secreted peptide required the oligopeptide transport system . The sporulation deficiency of oligopeptide transport mutants can be suppressed by mutating the rapA and rapB genes or by introduction of a spo0F mutation Y13S that renders the protein insensitive to Rap phosphatases . The data indicate that the sporulation deficiency of oligopeptide transport mutants is due to their inability to import the peptides controlling Rap phosphatases.

FEMS Microbiol Lett, 1996 Feb 15, 136(2), 151 - 6
Permeability of dormant spores of Bacillus subtilis to gramicidin S; Tanimoto Y et al.; Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis, had a partial inhibitory effect on L-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth . The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly . Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores . An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region . These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B . subtilis dormant spores.

FEMS Microbiol Lett, 1996 Feb 15, 136(2), 117 - 22
Characterization of iturin synthetase in the wild-type Bacillus subtilis strain producing iturin and in an iturin deficient mutant; Feignier C et al.; The multi-enzyme system responsible for the biosynthesis of iturin, an antifungal lipopeptide of Bacillus subtilis, was partially purified by chromatography on different affigels . In the wild-type strain, two subunits of the iturin synthetase (ITs and ITagp) were characterized: ITs activated only L-Ser, one of the iturin amino acid components, and ITagp activated L-Asn, D-Asn, L-Gln and L-Pro, amino acids corresponding to a partial sequence of iturin . In an iturin deficient mutant, the activity of the ITagp subunit was modified.

Experientia, 1996 Feb 15, 52(2), 175 - 9
Unusual antimicrobial compounds from Aeollanthus buchnerianus; Dellar JE et al.; Using bioassay guided isolation, three novel 12 carbon polyoxygenated fatty acids and a novel abietane diterpene have been isolated from the chloroform extract of aerial parts of Aeollanthus buchnerianus (Lamiaceae) . Rigorous spectroscopic methods were used for compound identification . (Z,Z)-8zeta-acetoxy-5zeta-hydroxydodeca-2,6-dienoic acid and (Z,Z)-5zeta, 8zeta-dihydroxydodeca-2,6-dienoic acid inhibited the spore germination of Cladosporium cucumerinum (both with Minimum Inhibitory Dose (MID) values of 1 microgram) and Aspergillus niger (MID 5 and 25 microgram respectively) . Further, they also reduced the hyphal growth of Pythium ultimum . (Z)-5zeta-hydroxy-6zeta,7zeta,8zeta-triacetox ydodeca-2-dienoic acid exhibited short term inhibition of the growth of Cladosporium cucumerinum . The novel abietane diterpenoid, (rel)-14alpha-acetoxyabiet-7-en-18-oic acid inhibited the growth of the gram positive bacteria Bacillus subtilis, Staphylococcus aureus and Streptomyces scabies (MIC values 80, 20 and 20 micrograms ml(-1) respectively).

Biochem Biophys Res Commun, 1996 Feb 15, 219(2), 463 - 8
Secretion of active subtilisin YaB by a simultaneous expression of separate pre-pro and pre-mature polypeptides in Bacillus subtilis; Chang YC et al.; Alkaline elastase YaB, produced by alkalophilic Bacillus YaB, is an extracellular serine protease having 55% homology to subtilisin BPN' and thus could be called subtilisin YaB . It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease . To examine if the pro-peptide of subtilisin YaB functions in trans to guide the folding of secreted subtilisin YaB in vivo, we made genes encoding the pre-pro, pro and pre-mature portions and placed them under the control of the spac-1 promoter on a multi-copy plasmid . When simultaneous expression in Bacillus subtilis of both the pre-pro and pre-mature genes was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG), protease activity was detected in the medium . On the other hand, we could not detect protease activity when the expression of either the pre-mature gene alone or both the pro and pre-mature genes was induced . From these results, we concluded that the pro region functions in trans and outside the cells for the proper folding and activation of the enzyme.

Nucleic Acids Res, 1996 Feb 15, 24(4), 628 - 39
Sequence analysis of 56 kb from the genome of the bacterium Mycoplasma pneumoniae comprising the dnaA region, the atp operon and a cluster of ribosomal protein genes; Hilbert H et al.; To sequence the entire 800 kilobase pair genome of the bacterium Mycoplasma pneumoniae, a plasmid library was established with contained the majority of the EcoR1 fragments from M.pneumoniae . The EcoR1 fragments were subcloned from an ordered cosmid library comprising the complete M.pneumoniae genome . Individual plasmid clones were sequenced in an ordered fashion mainly by primer walking . We report here the initial results from the sequence analysis of -56 kb comprising the dnaA region as a potential origin of replication, the ATPase operon and a region coding for a cluster of ribosomal protein genes . The data were compared with the corresponding genes/operons from Bacillus subtilis, Escherichia coli, Mycoplasma capricolum and Mycoplasma gallisepticum.

Genes Dev, 1996 Feb 15, 10(4), 478 - 88
Transcription factor Spo0A switches the localization of the cell division protein FtsZ from a medial to a bipolar pattern in Bacillus subtilis; Levin PA et al.; Entry into sporulation by the Gram-positive bacterium Bacillus subtilis is governed by two transcription factors, Spo0A and sigma H, and involves a switch in the site of division from a medial to a polar location . We report that at the onset of sporulation, assembly of the cell division protein FtsZ shifts from midcell to potential division sites near both poles . The switch to a bipolar pattern of FtsZ localization is dependent on Spo0A . Additionally, synthesis of an activated form of Spo0A during growth artificially activates the switch in FtsZ localization and results in the formation of polar septa . The sigma H factor, on the other hand, is dispensable for the switch in the position of the FtsZ assembly site, although it is required for formation of the polar septum . Our results suggest that during the transition from growth to sporulation, Spo0A induces the expression of genes that suppress FtsZ assembly at the midcell site and activate sites at both poles, whereas sigma H induces genes required for a subsequent step in cytokinesis.

Mol Gen Genet, 1996 Feb 5, 250(2), 230 - 6
A T7 promoter-specific, inducible protein expression system for Bacillus subtilis; Conrad B et al.; The adaptation and application of the Escherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression in Bacillus subtilis is reported . The expression cassette used in Bacillus subtilis was tightly regulated and T7 RnA polymerase (T7 RNAP)appeared 30 minutes after induction . The efficiency of T7 promoter-specific gene expression in B.subtilis was studied using one secretory and two cytosolic proteins of heterologous origin . The accumulation of E . coli beta-galactosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter brockii in B . subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity . The alpha-amylase of Thermactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10-20 h after T7 RNAP induction, but was also deposited in cellular fractions . The addition of rifampicin inhibited chi-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation.

Gene, 1996 Feb 2, 168(1), 61 - 5
Cloning and sequencing of a new holin-encoding gene of Bacillus licheniformis; Kyogoku K et al.; A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec) . Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptides of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively) . Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1-G2, respectively, exhibited higher extra-cellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398 . The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin-encoding gene . Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of beta-galactosidase into the extracellular fraction.

Gene, 1996 Feb 2, 168(1), 55 - 60
Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase; Fischer C et al.; We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169 degrees on the chromosome . An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment . This fragment contained an incomplete and two complete open reading frames (ORFs) . The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25038 Da), was found to be responsible for the observed reduced mannitol fermentation . The 3' part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identify . However, the function of these two proteins remains unknown . The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72889 Da . PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases . Several of the essential aa in the loops known to form the active site of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA . After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.

Gene, 1996 Feb 2, 168(1), 123 - 4
Sequence of a gene encoding a putative primary sigma factor from Borrelia burgdorferi strain B31; Tsai CP et al.; Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced . Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa) . Comparison with Escherichia coli sigma 70 and Bacillus subtilis sigma 43 showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding.

J Biol Chem, 1996 Feb 2, 271(5), 2621 - 6
Analysis of abrB mutations, mutant proteins, and why abrB does not utilize a perfect consensus in the -35 region of its sigma A promoter; Xu K et al.; The Bacillus subtilis global regulator AbrB is a DNA-binding protein composed of six identical monomers of 96 amino acids that shows specificity to the promoter regions of its target genes including its own . We have sequenced thirteen previously uncharacterized abrB mutations . Four mutant AbrB proteins were purified, and their DNA-binding properties and multimeric structures were examined . AbrB23 (R25S) had no appreciable DNA binding activity but retained a hexameric structure, indicating that Arg25 is important in DNA interactions . Three other mutant proteins, AbrB1 (C56Y), AbrB19 (Gln83-->termination codon), and AbrB100 (L69P), showed decreased DNA binding and altered multimeric interactions . Analysis of the expression and AbrB binding affinities of mutant abrB promoters demonstrated that a consensus -35 region is incompatible with proper autoregulation of the abrB gene.

Mol Divers, 1996 Feb, 1(2), 121 - 4
Antibiotic activity of polyketide products derived from combinatorial biosynthesis: implications for directed evolution; Fu H et al.; A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria . A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified . Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized . When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively . We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways . If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation . Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules . They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties.

Biosci Biotechnol Biochem, 1996 Feb, 60(2), 271 - 6
Bacillus stearothermophilus cell shape determinant gene, mreC and mreD, and their stimulation of protease production in Bacillus subtilis; Kubo M et al.; Protease production stimulating genes were isolated from a soybean protein degrading bacterium, Bacillus stearothermophilus HA19 . The cloned fragment stimulated production of a 37-kDa protease in B . subtilis . The nucleotide sequence of the genes and their flanking regions were identical to the B . subtilis cell shape determinant genes mreC and mreD {J . Bacteriol., 176, 6729-6742 (1992); J . Bacteriol., 176, 6717-6728 (1992)} . The mreC and mreD genes in B . subtilis stimulate secretion of a neutral protease (37-kDa), and the protease activity in the culture medium reached 2500 U per ml (approximately 10 times higher than the host strain) after 24 h of cultivation in L broth, suggesting the mreCD genes regulate protease expression and the protease is related to the cell shape determination in Bacilli . The protease productions in B . subtilis carrying mreC or mreD deletion plasmids were not elevated, so the 37-kDa protease stimulation requires both mreC and mreD genes . The extracellular protease was purified, and the molecular mass of the enzyme was 37,000 Da by SDS-polyacrylamide gel electrophoresis and gel filtration . The optimum pH and temperature for the enzyme activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable at pH 7-10 . The enzyme was inactivated by EDTA, but not by phenylmethyl sulfonyl fluoride and diisopropyl fluorophosphate.

Orig Life Evol Biosph, 1996 Feb, 26(1), 47 - 59
Response of Bacillus subtilis spores to dehydration and UV irradiation at extremely low temperatures; Dose K et al.; Spores of Bacillus subtilis have been exposed to the conditions of extreme dehydration (argon/silica gel; simulated space vacuum) for up to 12 weeks at 298 K and 80 K in the dark . The inactivation has been correlated with the production of DNA-double strand-breaks . The temperature-dependence of the rate constants for inactivation or production of DNA-double strand-breaks is surprisingly low . Controls kept in the frozen state at 250 K for the same period of time showed no sign of deterioration . In another series of experiments the spores have been UV irradiated (253.7 nm) at 298 K, 200 K and 80 K after exposure to dehydrating conditions for 3 days . Fluence-effect relationships for inactivation, production of DNA-double strand-breaks and DNA-protein cross-links are presented . The corresponding F37-values for inactivation and production of DNA lesions are significantly increased only at 80 K (factor of 4 to 5) . The data indicate that the low temperatures that prevail in the outer parts of the Solar System or at the nightside of Mars or the Moon are not sufficiently low to crucially inhibit inactivation by dehydration . Our data place further constraints on the panspermia hypothesis.

Appl Microbiol Biotechnol, 1996 Feb, 44(6), 746 - 52
Purification and characterization of a truncated Bacillus subtilis alpha-amylase produced by Escherichia coli; Marco JL et al.; A Bacillus subtilis amylase gene was inserted into a plasmid which was transferred to Escherichia coli . During cloning, a 3' region encoding 171 carboxy-terminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein . The transformed cells produced substantial amylolytic activity . The active protein was purified to apparent homogeneity . Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of the B . subtilis complete alpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa) . This truncated enzyme form hydrolysed starch with a Km of 3.845 mg/ml . Activity was optimal at pH 6.5 and 50 degrees C, and the purified enzyme was stable at temperatures up to 50 degrees C . While Hg2+, Fe3+ and Al+3 were effective in inhibiting the truncated enzyme, Mn2+ and Co2+ considerably enhanced the activity.

Mol Microbiol, 1996 Feb, 19(3), 587 - 98
Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome; Morel-Deville F et al.; To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of approximately 4 kb . The replicon was derived from a broad-host-range plasmid, pAM beta 1, which replicates by a unidirectional theta mechanism and its thermosensitive . The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194 . Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats . The integrated replicon was active at the permissive temperature, and approximately 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin . Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats . We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.

Mol Microbiol, 1996 Feb, 19(4), 901 - 7
Establishing differential gene expression in sporulating Bacillus subtilis: phosphorylation of SpoIIAA (anti-anti-sigmaF) alters its conformation and prevents formation of a SpoIIAA/SpoIIAB/ADP complex; Magnin T et al.; Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Bacillus subtilis . Its activity is controlled by an anti-sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA . We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated . Upon gel filtration in the presence of ADP, SpoIIAA-P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was coeluted with SpoIIAB, indicating the formation of a protein/protein complex . The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer . Its dissociation constant was 13 mu M . Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA-P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA-P was not a phosphorylated dimer of SpoIIAA . Our tentative conclusion, that SpoIIAA and SpoIIAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.

Proteins, 1996 Feb, 24(2), 238 - 46
Overexpression of Bacillus subtilis phosphoribosylpyrophosphate synthetase and crystallization and preliminary X-ray characterization of the free enzyme and its substrate-effector complexes; Bentsen AK et al.; Bacillus subtilis phosphoribosylpyrophosphate (PRPP) synthetase has been expressed to high levels in an Escherichia coli host strain devoid of endogenous PRPP synthetase . A rapid and efficient purification protocol has been developed allowing production of enzyme preparations with purity conforming to the stringent criteria required for crystallization . Crystallization experiments, in combination with dynamic light scattering studies, have led to the production of three crystal forms of the enzyme . These forms include the free enzyme, the enzyme in a binary complex with the substrate adenosine triphosphate (ATP), and the enzyme in a quaternary complex with the substrate analog alpha, beta-methylene adenosine triphosphate (mATP), the substrate ribose-5-phosphate (Rib-5-P), and the allosteric inhibitor adenosine diphosphate (ADP) . Diffraction data showed that all three crystal forms are suitable for structure determination . They crystallize in the same hexagonal space group, P6(3), with virtually identical unit cell dimensions of a = b = 115.6 angstroms, c = 107.8 angstrom, and with two molecules in the asymmetric unit . The self-rotation function showed the existence of a non-crystallographic twofold axis perpendicular to the c axis . The availability of the different complexes should allow questions regarding the molecular mechanisms of catalysis and allostery in PRPP synthetase to be addressed.

Planta Med, 1996 Feb, 62(1), 67 - 9
Antifungal and antibacterial chalcones from Myrica serrata; Gafner S et al.; The dichloromethane extract of the leaves of Myrica serrata inhibits growth of Cladosporium cucumerinum, Bacillus subtilis, and Escherichia coli on TLC plates . Activity-guided fractionation led the isolation of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (1), 2',4'-dihydroxy-6'-methoxy-5'-methylchalcone (aurentiacin A) (2), 2',6'-dihydroxy-4'-methoxy-3',5'-dimethyldihydrochalcone (3), 2'-hydroxy-4',6'-dimethoxy-3'-methyldihydrochalcone (4), and 2', 6'-dihydroxy-4'-methoxy-3'-methyldihydrochalcone (5) . In addition, the flavanones demethoxymatteucinol (6) and cryptostrobin (7) were also identified.

Curr Opin Struct Biol, 1996 Feb, 6(1), 53 - 61
RNA-protein complexes; Nagai K; The three commonly found RNA-binding domains, the ribonucleoprotein (RNP) domain, the double stranded RNA binding domain (dsRBD) and the K homology (KH) domain, have now been shown to have an alpha/beta fold similar to that found in many ribosomal proteins . Crystal structures of two hairpin RNA-protein complexes have been determined recently: the U1A spliceosomal protein bound to hairpin II of U1 small nuclear RNA, and MS2 bacteriophage capsid protein bound to a hairpin present at the ribosomal binding site of MS2 replicase mRNA . The crystal structure of the tryptophan operon RNA binding attenuation protein from Bacillus subtilis shows a novel structure with 11 monomers arranged in a doughnut-shaped ring that binds 11 copies of (U/G)AG triplets presented in the leader sequence of the tryptophan operon polycistronic message.

Curr Biol, 1996 Feb 1, 6(2), 111 - 4
Bacterial differentiation: sizing up sporulation; Jenal U et al.; New results on Bacillus subtilis sporulation suggest that size differences between the post-septation compartments trigger differential gene expression, which is then coordinated by communication between the nascent mother cell and forespore compartments.

Exp Cell Res, 1996 Feb 1, 222(2), 345 - 59
Affinity purification and comparative analysis of two distinct human uracil-DNA glycosylases; Caradonna S et al.; Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells . We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells . This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by the Bacillus subtilis bacteriophage, PBS2 . Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS-PAGE and silver staining . In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations . Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell . Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1 . With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment . Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes . Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently post-translationally processed into a lower molecular weight form . This is substantiated by mitochondrial import studies which reveal that in vitro expressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria . Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria . Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000 . SDS-PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell . Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus . Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA . These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme.

Appl Environ Microbiol, 1996 Feb, 62(2), 545 - 51
Comparative sporicidal effects of liquid chemical agents; Sagripanti JL et al.; We compared the effectiveness of glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate (plus a sublethal amount of hydrogen peroxide), sodium hypochlorite, and phenol to inactivate Bacillus subtilis spores under various conditions . Each chemical agent was distinctly affected by pH, storage time after activation, dilution, and temperature . Only three of the preparations (hypochlorite, peracetic acid, and cupric ascorbate) studied here inactivated more than 99.9% of the spore load after a 30-min incubation at 20 degrees C at concentrations generally used to decontaminate medical devices . Under similar conditions, glutaraldehyde inactivated approximately 90%, and hydrogen peroxide, formaldehyde, and phenol produced little killing of spores in suspension . By kinetic analysis at different temperatures, we calculated the rate of spore inactivation (k) and the activation energy of spore killing (delta E) for each chemical agent . Rates of spore inactivation had a similar delta E value of approximately 20 kcal/mol (ca.83.68 kJ/mol) for every substance tested . The variation among k values allowed a quantitative comparison of liquid germicidal agents.

J Bacteriol, 1996 Feb, 178(4), 1197 - 9
Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria; Pelicic V et al.; Expression in mycobacteria of the structural gene sacB, which encodes the Bacillus subtilis levansucrase, was investigated . sacB expression is lethal to Mycobacterium smegmatis and Mycobacterium bovis BCG in the presence of 10% sucrose . sacB could thus be used as a counterselectable marker in mycobacteria.

J Bacteriol, 1996 Feb, 178(4), 1178 - 86
Identification of a membrane protein involved in activation of the KinB pathway to sporulation in Bacillus subtilis; Dartois V et al.; The initiation of sporulation in Bacillus subtilis is dependent on the phosphorylation of the Spo0A transcription factor mediated by the phosphorelay and by two major kinases, KinA and KinB . Temporal expression of these kinases was analyzed, and an assessment of their respective contributions to the production of Spo0A-P was undertaken . The results show that KinB is expressed and activated prior to KinA; i.e., the two kinases are solicited sequentially in the sporulation process and are thought to be activated by different signaling pathways . A strategy was developed to isolate mutations specifically affecting the KinB pathway, using the newly improved mini-Tn10 delivery vector pIC333 . Several mutants were obtained, one of which carried a transposon in a gene coding for a small integral membrane protein, named KbaA . Inactivation of the kbaA gene appeared to affect KinB activity but not transcription of kinB . A Spo+ suppressor (kinB45) of the kbaA null mutation was isolated in the promoter region of kinB . An eightfold increase of kinB expression levels over wild-type levels was observed in the kinB45 mutant . Thus, overexpression of the kinB-kapB operon was sufficient to overcome the sporulation defect caused by inactivation of kbaA in a KinA- strain . Transcription of kinB was found to be repressed by SinR, while the kinB45 mutant was no longer sensitive to SinR regulation . Implications of these observations on the transcriptional regulation of kinB and the role of KbaA in KinB activation are discussed.

J Bacteriol, 1996 Feb, 178(4), 1088 - 93
hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes; Schulz A et al.; Whereas in Escherichia coli only one heat shock regulon is transiently induced by mild heat stress, for Bacillus subtilis three classes of heat shock genes regulated by different mechanisms have been described . Regulation of class I heat shock genes (dnaK and groE operons) involves an inverted repeat (CIRCE element) which most probably serves as an operator for a repressor . Here, we report on the analyses of an hrcA null mutant (delta hrcA), in which hrcA, the first gene of the dnaK operon, was deleted from the B . subtilis chromosome . This strain was perfectly viable at low and high temperatures . Transcriptional analysis of the deletion mutant revealed a high level of constitutive expression of both the dnaK and groE operons even at a low temperature . A further increase in the amount of groE transcript was observed after temperature upshift, suggesting a second induction mechanism for this operon . Overproduction of HrcA protein from a second copy of hrcA derived from a plasmid (phrcA+) in B . subtilis wild-type and delta hrcA strains prevented heat shock induction of the dnaK and groE operons at the level of transcription almost completely and strongly reduced the amounts of mRNA at a low temperature as well . Whereas the wild-type strain needed 4 h to resume growth after temperature upshift, the delta hrcA strain stopped growth only for about 1 h . Overproduction of HrcA protein prior to a heat shock almost completely prevented growth at a high temperature . These data clearly demonstrate that the hrcA product serves as a negative regulator of class I heat shock genes.

J Bacteriol, 1996 Feb, 178(3), 854 - 61
Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3-dihydroxybenzoate; Rowland BM et al.; Bacillus subtilis has duplicate isochorismate synthase genes, menF and dhbC . Isochorismate synthase is involved in the biosynthesis of both the respiratory chain component menaquinone (MK) and the siderophore 2,3-dihydroxybenzoate (DHB) . Several menF and dhbC deletion mutants were constructed to identify the contribution made by each gene product to MK and DHB biosynthesis . menF deletion mutants were able to produce wild-type levels of MK and DHB, suggesting that the dhbC gene product is able to compensate for the lack of MenF . However, a dhbC deletion mutant produced wild-type levels of MK but was DHB deficient, indicating that MenF is unable to compensate for the lack of DhbC . A menF dhbC double-deletion mutant was both MK and DHB deficient . Transcription analysis showed that expression of dhbC, but not of menF, is regulated by iron concentration . A dhbA'::lacZ fusion strain was constructed to examine the effects of mutations to the iron box sequence within the dhb promoter region . These mutations abolished the iron-regulated transcription of the dhb genes, suggesting that a Fur-like repressor protein exists in B . subtilis.

J Bacteriol, 1996 Feb, 178(3), 846 - 53
Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis; Nygaard P et al.; The isolation of mutants defective in adenine metabolism in Bacillus subtilis has provided a tool that has made it possible to investigate the role of adenine deaminase in adenine metabolism in growing cells . Adenine deaminase is the only enzyme that can deaminate adenine compounds in B . subtilis, a reaction which is important for adenine utilization as a purine and also as a nitrogen source . The uptake of adenine is strictly coupled to its further metabolism . Salvaging of adenine is inhibited by the stringent response to amino acid starvation, while the deamination of adenine is not . The level of adenine deaminase was reduced when exogenous guanosine served as the purine source and when glutamine served as the nitrogen source . The enzyme level was essentially the same whether ammonia or purines served as the nitrogen source . Reduced levels were seen on poor carbon sources . The ade gene was cloned, and the nucleotide sequence and mRNA analyses revealed a single-gene operon encoding a 65-kDa protein . By transductional crosses, we have located the ade gene to 130 degrees on the chromosomal map.

J Bacteriol, 1996 Feb, 178(3), 768 - 73
The permeability of the wall fabric of Escherichia coli and Bacillus subtilis; Demchick P et al.; To study the overall structure of the peptidoglycan fabric of the sacculi of gram-negative and gram-positive walls, actively growing cultures of Escherichia coli and Bacillus subtilis were treated with boiling sodium dodecyl sulfate solutions . The sacculi were then treated with enzymes to eliminate proteins and nucleic acids . These intact saccoli were probed with fluorescein-labeled dextrans with a range of known molecular weights . The penetration of the probes could be monitored by the negative-staining appearance in the fluorescence microscope . At several chosen times, the molecular weight fraction that allowed barely observable entry of the fluorescein-labeled probe and the molecular weight fraction that penetrated to achieve almost, but not quite, the concentration of probe in the solution external to the sacculi were determined . From three pairs of times and molecular weights that met one or the other of these two criteria, the effective pore size could be calculated . The minimum size of protein molecule that could diffuse through the pores was also calculated . Two mathematical models, which gave essentially the same results, were used to interpret the experimental data: one for the permeation of random coils through a surface containing holes and the other for rigid spheres diffusing through water-filled cylindrical pores . The mean estimate of the effective hole radius in walls from E . coli is 2.06 nm, and that of the effective hole size in walls from B . subtilis is 2.12 nm . These results are supported by experiments in which the loss of preloaded cells was monitored . Various fluorescein-labeled dextran samples were mixed with samples of intact cell walls, held for a long time, and then diluted . The efflux of the dextrans was monitored . Neither large nor small dextrans stained under these conditions . Only with dextran samples of a sufficiently small size were the sacculi filled during the preincubation period, and only with the largest of these could the probe not escape quickly . From the pore (or mesh) size, it can be concluded that the wall fabric of both organisms has few imperfections and that the major passageway is through the smallest possible pore, or "tessera," formed by the maximal cross-linking of the peptides from glycan chain to glycan chain compatible with the degree of rotational flexibility of the chains of repeating disaccharides of N-acetyl muramic acid and N-acetyl glucosamine . A tessera is composed of two chains of eight saccharides cross-linked by two octapeptides . The size of a globular hydrophilic molecule, if it did not bind to wall components, that could pass freely through the meshwork of an unstretched sacculus of either organism is roughly 25 kDa . We stress that this is only a rough estimate, and it may be possible for proteins of less than 50 kDa to pass through the native wall during normal growth conditions.

J Bacteriol, 1996 Feb, 178(3), 705 - 13
Transcriptional regulation of the Bacillus subtilis menp1 promoter; Qin X et al.; The Bacillus subtilis men genes encode biosynthetic enzymes for formation of the respiratory chain component menaquinone . The menp1 promoter previously was shown to be the primary cis element for menFD gene expression . In the present work, it was found that either supplementation with nonfermentable carbon sources or reutilization of glycolytic end products increased menp1 activity in the late postexponential phase . The effect on menp1 activity by a particular end product (such as acetoin or acetate) was prevented by blocking the corresponding pathway for end product utilization . Alteration of a TGAAA motif within the promoter region resulted in unregulated menp1 activity throughout the culture cycle, irrespective of the carbon source added.

Nucleic Acids Res, 1996 Jan 15, 24(2), 282 - 8
Distamycin-induced inhibition of formation of a nucleoprotein complex between the terminase small subunit G1P and the non-encapsidated end (pacL site) of Bacillus subtilis bacteriophage SPP1; Chai S et al.; The small subunit of the Bacillus subtilis bacteriophage SPP1 terminase (G1P) forms a sequence-specific nucleoprotein complex with the SPP1 non-encapsidated end (pacL site) during initiation of DNA encapsidation . Gel mobility shift assay was used to study the G1P-pacL interaction . Distamycin, a minor groove binder that induces local distortion of the DNA, inhibits G1P-pacL complex formation . The competition of G1P with distamycin for DNA binding at the pacL site is independent of the order of addition of the reactants . Other minor groove binders, such as spermine or Hoechst 33258, which do not distort DNA, failed to compete with G1P for pacL DNA binding . Cationic metals, which generate a repertoire of DNA structures different from that caused by the minor groove binders, can partially reverse the distamycin-induced inhibition of G1P binding to pacL DNA . The major groove binder methyl green, which does not distort sequence-directed bending of pacL DNA, competes with G1P for binding at the pacL site . Our data suggest that the natural sequence-directed bend that exists within the pacL site is the architectural element that facilitates assembly of a nucleoprotein complex and hence initiation of DNA encapsidation by bacteriophage SPP1.

Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 347 - 51
Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity; Seok YJ et al.; The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr) . The isolated amino-terminal domain (EIN) of E . coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr . An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein . A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield . Intact E . coli EI is effective in phosphotransfer from PEP to HPr from E . coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum . Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E . coli or M . capricolum requires the intermediacy of HPr . The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E . coli, B . subtilis, and M . capricolum as well as to EIAglc from E . coli . These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E . coli HPr.

FEBS Lett, 1996 Jan 2, 378(1), 98 - 100
Crystallisation of the Bacillus subtilis sporulation inhibitor SinR, complexed with its antagonist, SinI; Lewis RJ et al.; The transcription factor SinR, a pleiotropic regulator of late growth processes in Bacillus subtilis, has been crystallised as a complex with its antagonist SinI, in a form suitable for structural analysis . The SinI:SinR crystals diffract X-rays generated from a rotating copper anode source to 2.3 A spacing and a complete native dataset has been collected to this resolution limit . The space group of the crystals is P3(1)21 (or its enantiomorph P3(2)21) with cell dimensions a = b = 60.76 A, c = 87.79 A . Assuming that there is a single SinI:SinR heterodimer in the asymmetric unit, the crystals have a Vm of 2.53 A3.Da-1.

Biochimie, 1996, 78(11-12), 1017 - 24
Some novel transcription attenuation mechanisms used by bacteria; Yanofsky C et al.; A variety of transcription attenuation mechanisms are used by bacteria to regulate gene and operon expression . This review summarizes previous and current studies designed to elucidate the features of the specific attenuation mechanisms that regulate expression of the tryptophanase (tna) operon of Escherichia coli and the tryptophan (trp) operon of Bacillus subtilis . Initiation of transcription in the tna operon is regulated by catabolite repression . Once initiated, transcription is regulated by tryptophan-induced inhibition of Rho-mediated transcription termination in the leader region of the operon . An operon-encoded leader peptide, TnaC, containing a crucial tryptophan residue, plays an essential role in induction . This peptide appears to act in cis on the ribosome translating tnaC to inhibit its release at the tnaC stop codon . The stalled ribosome would block Rho's access to the tna transcript, thereby preventing termination . Transcription of the trp operon of B subtilis is regulated by an attenuation mechanism that responds to a tryptophan-activated eleven subunit RNA-binding regulatory protein, called TRAP . Activated TRAP binds to repeated GAG sequences in the leader segment of the trp operon transcript, disrupting an RNA antiterminator and promoting formation of a terminator . Activated TRAP also regulates translation of trpG in the folate operon by binding to repeat GAG sequences surrounding the trpG ribosome binding site . A temperature sensitive tryptophanyl-tRNA synthetase (trpS) mutant was previously observed to overexpress the trp operon and trpG, when grown at elevated temperatures in the presence of tryptophan . We have found that the trpS defect increases trp operon and trpG expression by interfering with TRAP's ability to act . We suggest that either accumulation of uncharged tRNA(Trp) or overproduction of a TRAP-binding transcript reduces the level of functional TRAP in the trpS mutant.

Acta Microbiol Immunol Hung, 1996, 43(4), 289 - 99
The development of a Bacillus subtilis 168 culture condition for enhanced and accelerated beta-mannanase production; el-Helow ER et al.; A shaken flask cultivation condition for enhanced and accelerated beta-mannanase formation by Bacillus subtilis 168 was achieved . Among five examined fermentation media a formula that supported enzyme generation and retarded biomass yield and sporulation was selected . The deficiency of biomass production in this medium was mastered by choosing a seed culture medium that accelerated growth and initiation of beta-mannanase synthesis . With respect to enzyme production, the optimum pH and temperature were 7 and 37 degrees C, respectively . The biosynthesis of the enzyme was extremely influenced by the cell growth state as a modulator, glucose as a catabolite repressor, and galactomannan as an inducer . A galactomannan concentration of 4 g l-1 induced a beta-mannanase activity level of 17.5 U ml-1 after 24 h of incubation at the experimental condition . Higher inducer concentrations supported growth rather than enzyme production . The influence of inoculum size was so remarkable that, at optimum, a crude filtrate with an enzyme activity of 33U ml-1 was yielded within 4 hours . It appears that this is among the highest rates reported for beta-mannanase production . We have also demonstrated that blocking of the sporulation process at stage II do not affect enzyme production significantly . This would allow an extended enzyme production phase especially in a continuous culture.

Rocz Panstw Zakl Hig, 1996, 47(4), 439 - 44
{The effect of growth media on recovery of test microorganisms after exposure to saturated steam under pressure}; Krzywicka H et al.; The aim of this study was to find out which growth media give the best condition for the development of test bacteria after exposure to saturated steam under pressure . The test organisms were strains of Bacillus subtilis NCTC 3610 and Bacillus stearothermophilus NCTC 8923 . The test prepared from spore suspension were exposed to saturated steam under pressure 0.2 atn-B.subtilis, and 0.7 atn-B . stearothermophilus with various length of exposure /sublethal conditions/ . After the exposure the tests were placed in growth media . The obtained results show that the compositions of the medium in which spore-forming bacteria are grown after the exposure under sublethal conditions to saturated steam under pressure affects the recovery of the test organism . The media with glucose, tryptose and L-alanine provided the best conditions for growth.

Protein Eng, 1996 Jan, 9(1), 77 - 83
Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide; You L et al.; Sequential rounds of error-prone PCR to introduce random mutations and screening of the resultant mutant libraries have been used to enhance the total catalytic activity of subtilisin E significantly in a non-natural environment, aqueous dimethylformamide (DMF) . Seven DNA substitutions coding for three new amino acid substitutions were identified in a mutant isolated after two additional generations of directed evolution carried out on 10M subtilisin E, previously "evolved' to increase its specific activity in DMF . A Bacillus subtilis-Escherichia coli shuttle vector was developed in order to increase the size of the mutant library that could be established in B.subtilis and the stringency of the screening process was increased to reflect total as well as specific activity . This directed evolution approach has been extremely effective for improving enzyme activity in a non-natural environment: the resulting-evolved 13M subtilisin exhibits specific catalytic efficiency towards the hydrolysis of a peptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in 60% DMF solution that is three times that of the parent 10M and 471 times that of wild type subtilisin E . The total activity of the 13M culture supernatant is enhanced 16-fold over that of the parent 10M.

Annu Rev Genet, 1996, 30, 297 - 41
Molecular genetics of sporulation in Bacillus subtilis; Stragier P et al.; The process of sporulation in the bacterium Bacillus subtilis proceeds through a well-defined series of morphological stages that involve the conversion of a growing cell into a two-cell-chamber sporangium within which a spore is produced . Over 125 genes are involved in this process, the transcription of which is temporally and spatially controlled by four DNA-binding proteins and five RNA polymerase sigma factors . Through a combination of genetic, biochemical, and cell biological approaches, regulatory networks have been elucidated that explicitly link the activation of these sigma factors to landmark events in the course of morphogenesis and to each other through pathways of intercellular communication . Signals targeting proteins to specific subcellular localizations and governing the assembly of macromolecular structures have been uncovered but their nature remains to be determined.

Biochimie, 1996, 78(6), 381 - 9
Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis; Condon C et al.; In this review, we summarize progress on the regulation of the aminoacyl-tRNA synthetase genes in Bacillus subtilis . Most of the genes encoding this set of enzymes in B subtilis are members of a large family of Gram-positive genes and operons controlled by a novel antitermination mechanism that uses their cognate uncharged tRNA as the effector . A subset of these genes is, in addition, likely to be controlled at the level of mRNA processing and degradation . We describe the key experiments leading to these conclusions.

Biotechnol Prog, 1996 Jan-Feb, 12(1), 31 - 9
Intracellular expression of Vitreoscilla hemoglobin (VHb) enhances total protein secretion and improves the production of alpha-amylase and neutral protease in Bacillus subtilis; Kallio PT et al.; In an attempt to alleviate oxygen limitation during batch cultivations, a heterologous bacterial hemoglobin gene (vhb) of Vitreoscilla was introduced into Bacillus subtilis . Biochemically active VHb, as demonstrated by immunoblot analysis and carbon monoxide binding assay, was intracellularly expressed in B . subtilis from an inducible promoter-repressor (spac-lacI) . Expression of VHb in oxygen-limited B . subtilis batch bioreactor cultivations enhanced cell growth, decreased accumulation of acetate, and increased the total protein secreted into the culture medium by approximately 1.5-fold . In addition, VHb-expressing B . subtilis cultures exhibited increases of approximately 30% and 5-17% in neutral protease activity and alpha-amylase activity, respectively, relative to the parental, VHb-free strain.

Mol Microbiol, 1996 Jan, 19(2), 343 - 56
Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis; Luttinger A et al.; comR (pnpA) is a newly identified gene in Bacillus subtilis that is necessary for the expression of late competence genes . Transformability of a comR (pnpA) mutant is 1-5% of that seen in comR+ strains . Cloning and sequencing identified ComR as polynucleotide phosphorylase (PNPase) . The PNPase amino acid sequence has 50% identity and 67% similarity with the Escherichia coli enzyme . Enzymatic assays show that this is the only PNPase activity in B . subtilis . comR (pnpA) is necessary for comG-lacZ and comK-lacZ expression, but this requirement is bypassed by a mecA disruption . In B . subtilis, the loss of PNPase has little effect on expression from a fusion of the srfA promoter directly to lacZ, but is necessary for normal expression from certain srfA-lacZ fusions that include portions of the normal srfA transcript . When a srfA-lacZ translational fusion is tested in isogenic pnpA+ and pnpA derivatives of E . coli, lower expression is seen in the pnpA mutant . Since expression from lacZ fusions to comA, sinR, and mecA appeared similar in the B . subtilis pnpA and pnpA+ strains, the loss of PNPase does not have a strong general effect on gene expression . These results suggest that PNPase may be necessary for modification of the srfA transcript in order to activate translation or stabilize the transcript, and that this may be necessary for competence development . This is the first evidence of post-transcriptional effects on the development of competence in B . subtilis.

Mol Microbiol, 1996 Jan, 19(2), 329 - 42
In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase; Mitra S et al.; A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose . Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B . subtilis PNPase gene (Luttinger et al., 1996--accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase) . Processive 3'-to-5' exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site . Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure . A similar stall site was observed for an RNA that comprised the intergenic region between the B . subtilis rpsO and pnpA genes . The ability to initiate degradation of a substrate that had a stem structure at its 3' end differed for the B . subtilis and Escherichia coli PNPase enzymes.

Mol Microbiol, 1996 Jan, 19(2), 319 - 28
A dual role for the Bacillus subtilis glpD leader and the GlpP protein in the regulated expression of glpD: antitermination and control of mRNA stability; Glatz E et al.; The Bacillus subtilis glpD gene encodes glycerol-3-phosphate dehydrogenase . This gene is preceded by a leader region containing an inverted repeat which acts as a transcription terminator . Expression of glpD is controlled by antitermination of transcription at the inverted repeat . Antitermination is effected by the glpP gene product in conjunction with glycerol-3-phosphate and, consequently, GlpP mutants fail to grow on glycerol as a sole carbon and energy source . We have isolated a number of glycerol-positive revertants of GlpP mutants . Most of these revertants have mutations in the inverted repeat of the glpD leader and produce glycerol-3-phosphate dehydrogenase constitutively . Unlike wild-type bacteria, they are not sensitive to glucose repression of glpD . A few of the revertants are temperature sensitive, i.e . they grow on glycerol at 32 degrees C but not at 45 degrees C and produce glycerol-3-phosphate dehydrogenase only at 32 degrees C . Northern blot analyses demonstrated that the temperature-sensitive expression of glpD is due to destabilization of glpD mRNA . Furthermore, introduction of the wild-type glpP gene into the revertants stabilized the glpD mRNA . This is probably a result of a direct interaction between the GlpP protein and the leader of glpD mRNA . Besides its function in antitermination of transcription of glpD, it is suggested that GlpP is also involved in controlling glpD mRNA stability . Introduction of the glpP gene into the revertants also restored glucose repression, indicating that this repression is mediated by the GlpP protein.

Biosci Biotechnol Biochem, 1996 Jan, 60(1), 111 - 6
A regulatory mechanism for the balanced synthesis of membrane phospholipid species in Escherichia coli; Saha SK et al.; The mechanism that assures the balanced synthesis of zwitterionic (phosphatidylethanolamine) and acidic phospholipids (phosphatidylglycerol and cardiolipin) in Escherichia coli has been examined by genetically manipulating the two enzymes at the biosynthetic branch point, i.e., phosphatidylglycerophosphate synthase, encoded by pgsA, and phosphatidylserine synthase, encoded by pssA . A mutant in which the most part of the pssA gene was replaced with a drug resistance gene lacked phosphatidylserine synthase and phosphatidylethanolamine and required divalent metal ions for growth, as did a previously reported insertion-inactivated pssA mutant . When this mutant harbored a plasmid containing a Bacillus subtilis gene that encodes membrane-bound phosphatidylserine synthase, the phosphatidylethanolamine content was dependent on its activity, in contrast to that with the soluble E . coli counterpart . A defective mutation, pgsA3, caused reductions not only in acidic-phospholipid synthesis but also in phosphatidylethanolamine synthesis, despite the normal level of phosphatidylserine synthase activity . These results, together with previous observations, indicate that phosphatidylserine synthesis requires the membrane-associated form of phosphatidylserine synthase, which is related to the membrane-levels of acidic phospholipids, thus yielding balanced compositions of zwitterionic and acidic phospholipids.

Mol Microbiol, 1996 Jan, 19(1), 145 - 58
In vitro selection of optimal AbrB-binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures; Xu K et al.; The AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters . Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination . Using in vitro selection techniques, we have isolated optimal AbrB-binding sites from oligonucleotides containing 22 or 44 random base pairs . The best of these sites have an apparent in vitro Kd which is fivefold lower than a similar-sized DNA fragment containing the sequence corresponding to the AbrB-binding site on the spo0E gene . We tested one of the sites in vivo and found that it confers AbrB-mediated control upon a promoter not normally regulated by AbrB . In each of four separate trials, the selected sites possess motifs that converge to a simple consensus . It is argued that the nature and spacing of these motifs produce a type of three-dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Jan-Feb, (1), 75 - 7
{The efficacy of the new bacterial preparation biosporin in treating acute intestinal infections}; Gracheva NM et al.; The clinico-laboratory study of the new probiotic Biosporin revealed its effectiveness in the treatment of patients with acute enteric infections . The pronounced curative action of the preparation, manifested by the rapid normalization of stool, the disappearance of abdominal pains and the decrease of dysbiosis in the intestine, was demonstrated . The best results were registered after the administration of Biosporin containing 2 x 10(9) microbial cells (Bacillus subtilis and Bacillus licheniformis) . Biosporin was well tolerated by the patients, no side effects were observed . Biosporin is recommended for use in medical practice for the treatment of patients with acute enteric infections.

Crit Rev Microbiol, 1996, 22(2), 101 - 38
Alkaline-fermented foods: a review with emphasis on pidan fermentation; Wang J et al.; Alkaline-fermented foods constitute a group of less-known food products that are widely consumed in Southeast Asia and African countries . They can be made from different raw ingredients . For instance, Japanese natto, Thai thua-nao, and kinema are made from cooked soybeans, dawadawa from African locust beans, ogiri from melon seeds, ugba from African oil beans, kawal from fresh legale leaves, owoh from cotton seeds, and pidan from fresh poultry eggs . In alkaline-fermented foods, the protein of the raw materials is broken down into amino acids and peptides; ammonia is released during the fermentation, raising the pH of the final products and giving the food a strong ammoniacal smell . Most alkaline fermentations are achieved spontaneously by mixed bacteria cultures, principally dominated by Bacillus subtilis . In other cases, pure cultures can be used . For example, Japanese natto is inoculated with a pure culture of B . subtilis var natto . Pidan is a special example of alkaline fermentation . Instead of using microorganisms, pidan is made using an alkali-treated fermentation . Sodium hydroxide (NaOH) is produced from the reaction of sodium carbonate (Na2CO3), water (H2O), and calcium oxide (CaO) of pickle or coating mud . NaOH penetrates into the eggs, causing the physicochemical changes, color changes, and gelation . The appearance of pidan differs from fresh eggs in that the white becomes a semitransparent tea-brown color, and the yolk is solid or semisolid with a dark-green color . The nutritional value of pidan is slightly decreased compared with fresh eggs, but pidan has an extremely long shelf life and a pleasant, fragrant taste that is preferred by most people in Southeast Asian countries . In a small-scale laboratory study conducted by the authors, B . subtilis was not found in pidan . Four Staphylococcus spp . (S . cohnii, S . epidermidis, S . haemolyticus, and S . warneri) and two strains of Bacillus spp . (B . cereus and B . macerans) were isolated from pidan . Staphylococcus spp . did not contribute to the fermentation and were considered contaminants.

Adv Exp Med Biol, 1996, 379, 171 - 82
Random mutagenesis of the weak calcium binding site in subtilisin Carlsberg and screening for thermostability by temperature-gradient gel electrophoresis; Sattler A et al.; A random mutagenesis approach was directed to the weak calcium binding site of subtilisin Carlsberg in order to enhance the thermal stability of the enzyme by changing its calcium affinity . The structural motif of the binding site was altered by two strategies, the ligand strategy, which was directed to the amino acid ligands of the calcium ion and the conformation strategy, by which a part of the calcium cave was redesigned . Subtilisin mutants were expressed in Bacillus subtilis and screened for enhanced thermostability by a filter assay and by temperature-gradient gel electrophoresis (TGGE) . Characterization of selected mutants and application of TGGE to investigate the thermal stability of proteases and protease-inhibitor complexes in general is described.

Biosens Bioelectron, 1996, 11(4), 443 - 8
Covalent enzyme immobilization on paramagnetic polyacrolein beads; Varlan AR et al.; An optimized procedure of covalent glucose oxidase, urease, Bacillus subtilis alpha-amylase and Bacillus licheniformis alpha-amylase immobilization on paramagnetic, non-porous, polyacrolein beads is presented . The resulting insolubilized enzymes can be employed for extended periods of time without loss of activity . The conditions were optimized for maximizing the activity of the linked enzyme . Coated beads bearing up to 15 micrograms active enzyme/mg(beads) were obtained on reproducible basis . The paramagnetic feature of the particles facilitates the enzyme handling . In the magnetic field, the enzyme separation is fast and complete . Thus, the paramagnetic beads represent an excellent carrier for immobilized enzymes.

Trends Genet, 1996 Jan, 12(1), 31 - 4
Determination of cell fate in Bacillus subtilis; Errington J; On starvation, the soil bacterium Bacillus subtilis stops dividing and initiates sporulation, a simple developmental process involving the differentiation of two cell types . Sporulation begins with a reorganization of the cell cycle, to produce cells with the size and chromosome content appropriate for the developmental process . The central division that would normally occur, to produce a pair of identical daughter cells, is blocked and the cell divides asymmetrically to produce a small, polar prespore cell and a much larger mother cell . The developmental fates of the two cells are dictated by the localized activation of cell-specific transcription factors, which are controlled by mechanisms that respond to the cellular asymmetry.

J Biomed Mater Res, 1996 Summer, 33(2), 101 - 5
Glutaraldehyde retains its disinfectant properties in presence of hydroxypropylmethyl cellulose (HPMC) gel; Matchette LS et al.; Explanted medical devices are routinely sent to laboratories at the Center for Devices and Radiological Health for analysis . The shipping of these devices presents potential hazards to personnel as well as an opportunity for damage to the devices . In an effort to address these concerns, a viscous disinfecting shipping medium that would limit splashing and cushion a suspended device was proposed . Consequently, we investigated the disinfectant properties of adding a gelling agent, hydroxypropylmethyl cellulose (HPMC) to common disinfectants . We found that the germicidal effectiveness of 2.5% glutaraldehyde in 0.05 M borax when tested against Bacillus subtilis spores was not changed by the addition of 2% HPMC . In addition, HPMC appears to be compatible with 70% ethanol and at least one commercial disinfectant containing a quaternary ammonium compound . Preliminary experiments indicate that an HPMC-disinfectant gel is a potentially useful packaging agent for minimizing the hazards to personnel and materials during shipping of explanted medical devices . The use of such a medium would be subject to guidelines within the context of a program for handling biologically contaminated materials.

Mikrobiologiia, 1996 Jan-Feb, 65(1), 74 - 8
{Physiological effects of sunlight on Bacillus subtilis strains with varying capacity to repair photoproducts}; Lotareva OV; Nonlethal doses of sunlight were established to cause a growth delay in Bacillus subtilis strain 1201 possessing an unimpaired repair system . This effect was not observed with the 1201-10 uvr1 mutant deficient in DNA excision repair; however, addition of an extract of uvr1+ cells restorated the phenomenon of growth delay by sunlight in this mutant . Experiments with glass light filters showed that a weakened growth delay in strain 1201 could also be produced by rays of sunlight with wavelengths greater than 380 nm . Maximum growth delay was observed after irradiation with the complete spectrum of sunlight . Experiments with the protein synthesis inhibitor chloramphenicol indicated that sunlight promotes synthesis of proteins preventing the dying of cells . The presence of Casamino acids is favorable for the synthesis of protective proteins.

Radiats Biol Radioecol, 1996 Jan-Feb, 36(1), 63 - 7
{Effects of single and fractionated irradiation on Bacillus subtilis spores under different incubation conditions}; Mal'tsev VN et al.; Effect of 60Co gamma-rays on inactivation of Bac . subtilis spores was studied . In the course of investigation, the dose of gamma-rays was divided in to two parts with an interval of 4 or 24 hours between irradiation . Fractional irradiation was found to be more effective for decontamination of Bac . subtilis spores than single irradiation.

Plasmid, 1996 Jan, 35(1), 14 - 30
A positive selection vector for the analysis of structural plasmid instability in Bacillus subtilis; Meima R et al.; A system for the positive selection of structural plasmid rearrangements in Bacillus subtilis was developed . Random deletions removing a transcription terminator structure in the assay plasmid, designated pGP100, resulted in expression of the cat-86 gene, under control of a constitutive bacteriophage promoter . The resulting chlorampenicol-resistant colonies were analyzed for plasmid contents and were shown, by restriction analysis, to contain initially both the intact parental plasmid and a deletion variant . Sequence analysis of deletion derivatives revealed a consensus target site (5'-A-T-T-A-A/T-3') at or near deletion termini, which resembles topoisomerase I target sites . Endpoints on one side of the deletions were found to be clustered in the promoter region of the tetracycline resistance gene present on pGP100, the gene product of which is an integral membrane protein . Furthermore, deletion of the genes encoding the ATP-dependent exonuclease, AddAB, severely reduced the structural stability of pGP100 . The data indicate that similar mechanisms underlie deletion formation in pGP100, and a different plasmid-based system, pGP1, which we have analyzed previously.

Plasmid, 1996 Jan, 35(1), 1 - 13
Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria; Metcalf WW et al.; We describe several new cloning vectors for mutagenesis and allele replacement experiments . These plasmids have the R6K gamma DNA replication origin (oriR(R6K gamma) so they replicate only in bacteria supplying the pi replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of pi . They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria . Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA . Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker . One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants . Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones . Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker . Plasmid-free segregants with an allele replacement can be subsequently selected as TetS or SucR recombinants . A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 9 - 15
The role of CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis; Henkin TM; The CcpA protein has been identified as a key regulator of carbon metabolism in Bacillus subtilis . CcpA is a DNA binding protein in the LacI/GalR transcriptional repressor family, and genes which respond to CcpA contain common cis-acting target sequences (Ccp boxes) . A number of pathways involved in carbon source utilization are repressed by CcpA, while at least one gene which is involved in excretion of excess carbon is activated by CcpA . Genes repressed by CcpA generally contain Ccp boxes within or downstream of the promoter, while ackA, which is activated by CcpA, contains Ccp boxes upstream of the promoter . It therefore appears that CcpA acts globally to direct carbon flow in B . subtilis.

Nucleic Acids Res, 1996 Jan 1, 24(1), 41 - 5
NRSub: a non-redundant database for Bacillus subtilis; Perriere G et al.; In the context of the international project aimed at sequencing the whole genome of Bacillus subtilis we have developed a non-redundant, fully annotated database of sequences from this organism . Starting from the B.subtilis sequences available in the EMBL, GenBank and DDBJ collections we have removed all encountered duplications and then added extra annotations to the sequences (e.g . accession numbers for the genes, locations on the genetic map, codon usage, etc.) We have also added cross-references to the EMBL, MEDLINE, SWISS-PROT and ENZYME data banks . The present system results from merging of the NRSub and SubtiList databases and the sequence contigs used in the two systems are identical . NRSub is distributed as a flatfile in EMBL format (which is supported by most sequence analysis software packages) and as an ACNUC database, while SubtiList is distributed as a relational database under 4th Dimension . It is possible to access the data through two dedicated World Wide Web servers located in France and Japan.

Microbiology, 1996 Jan, 142 ( Pt 1), 71 - 7
Molecular analysis of an operon in Bacillus subtilis encoding a novel ABC transporter with a role in exoprotein production, sporulation and competence; Leskela S et al.; The levels of exoamylase and other exoenzymes of Bacillus subtilis are pleiotropically decreased by the ecs-26 (prs-26) and ecs-13 (prs-13) mutations . These mutations also cause a competence- and sporulation-deficient phenotype . In the present work, the ecs locus, which has been defined by the ecs-26 and ecs-13 mutations, was cloned and sequenced . Sequence analysis revealed a putative operon of three ORFs (ecsA, ecsB and ecsC) . ecsA can encode a putative polypeptide of 248 amino acid residues containing an ATP-binding site . The polypeptide shows about 30% sequence similarity with the ATP-binding components of numerous membrane transporters of the ABC-type (ATP-binding cassette transporters or traffic ATPases) . The ecs-26 mutation was found to result from a transition of one base pair chaning the glycine164 of EcsA to a glutamic acid residue in the vicinity of the putative ATP-binding pocket . ecsB was predicted to encode a hydrophobic protein with six membrane-spanning helices in a pattern found in other hydrophobic components of ABC transporters . The properties deduced for the ecsA and ecsB gene products are consistent with the interpretation that ecs encodes a novel ABC-type membrane transporter of B . subtilis . The third ORF, ecsC, can encode a putative polypeptide of 237 amino acid residues . The polypeptide does not resemble components of ABC transporters.

Photochem Photobiol, 1996 Jan, 63(1), 74 - 8
Experimental correspondence between spore dosimetry and spectral photometry of solar ultraviolet radiation; Munakata N et al.; The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores . Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer . To obtain the effective spectrum, the irradiance for each 1 nm wavelenght interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples . Integration of the effective spectrum provided the estimate for ID/min . The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993 . The average ratio (+/- SD) between them was 1.24 (+/- 0.16) for 14 data points showing high inactivation rates (> 0.05 ID/min) . Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.

J Bacteriol, 1996 Jan, 178(2), 560 - 3
A Bacillus subtilis malate dehydrogenase gene; Jin S et al.; A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase . Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation . In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.

J Bacteriol, 1996 Jan, 178(2), 546 - 9
Exchange of precursor-specific elements between Pro-sigma E and Pro-sigma K of Bacillus subtilis; Carlson HC et al.; sigma E and sigma K are sporulation-specific sigma factors of Bacillus subtilis that are synthesized as inactive proproteins . Pro-sigma E and pro-sigma K are activated by the removal of 27 and 20 amino acids, respectively, from their amino termini . To explore the properties of the precursor-specific sequences, we exchanged the coding elements for these domains in the sigma E and sigma K structural genes and determined the properties of the resulting chimeric proteins in B . subtilis . The pro-sigma E-sigma K chimera accumulated and was cleaved into active sigma K, while the pro-sigma K-sigma E fusion protein failed to accumulate and is likely unstable in B . subtilis . A fusion of the sigE "pro" sequence to an unrelated protein (bovine rhodanese) also formed a protein that was cleaved by the pro-sigma E processing apparatus . The data suggest that the sigma E pro sequence contains sufficient information for pro-sigma E processing as well as a unique quality needed for sigma E accumulation.

J Bacteriol, 1996 Jan, 178(2), 424 - 34
Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein; Saxild HH et al.; The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined . Sequence analysis showed that the genes were localized immediately downstream of the hut operon . Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp . A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure . In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine . By the use of lacZ fusions, the regulation was found to be at the level of transcription . The operon expression was subject to glucose repression . Upstream of the dra gene an open reading frame of 313 amino acids was identified . Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides . The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).

J Bacteriol, 1996 Jan, 178(1), 61 - 7
The gntP gene of Escherichia coli involved in gluconate uptake; Klemm P et al.; The gntP gene, located between the fim and uxu loci in Escherichia coli K-12, has been cloned and characterized . Nucleotide sequencing of a region encompassing the gntP gene revealed an open reading frame of 447 codons with significant homology to the Bacillus subtilis gluconate permease . Northern (RNA) blotting indicated that the gntP gene was monocistronic and was transcribed as an mRNA with an apparent molecular size of 1.54 kb . The transcriptional start point was determined by primer extension analysis . The gntP gene was found to be under catabolite repression and was not induced by gluconate . Also, expression seemed to be stringently controlled . Several observations indicated that the GntP protein is an inner membrane protein; it contains characteristic membrane-spanning regions and was isolated predominantly from the inner-membrane fraction of fractionated host cells . A topology analysis predicted a protein with 14 membrane-spanning segments . The inability of a mutant strain to grow on gluconate minimal medium could be relieved by introduction of a plasmid encoding the gntP gene . Finally, the kinetics of GntP-mediated gluconate uptake were investigated, indicating an apparent Km for gluconate of 25 microM.

J Bacteriol, 1996 Jan, 178(1), 35 - 45
mgpS, a complex regulatory locus involved in the transcriptional control of the puc and puf operons in Rhodobacter sphaeroides 2.4.1; Sabaty M et al.; A new method has been developed in order to select mutants showing decreased puc operon transcription in Rhodobacter sphaeroides 2.4.1 . A transcriptional fusion of a promoterless fragment derived from the sacB gene, encoding the levansucrase from Bacillus subtilis, to the upstream regulatory region of the puc operon has been constructed . With appropriate levels of exogenous sucrose, survivors of a sucrose killing challenge have been isolated . Subsequent analysis revealed the presence of both cis- and trans-acting "down" mutations in relation to puc operon expression . One of the trans-acting regulatory mutations was chosen for further study . The original mutation showed less than 2% of the level of puc operon transcription compared with the wild type under aerobic conditions and an 86% reduction under dark dimethyl sulfoxide conditions . This mutation can be complemented by a 3.9-kb BamHI DNA fragment derived from a cosmid contained within a genomic cosmid bank . DNA sequence analysis of this fragment revealed the presence of a 2.8-kb open reading frame, designated mgpS, which would encode a 930-amino-acid protein . The N-terminal portion of the putative protein product presents homologies to proteins of the RNA helicase family . Disruption of the chromosomal mgpS resulted in decreased transcription of both puc and puf, while the presence of mgpS in multicopy in the wild type, 2.4.1., increased puc expression by a factor of 2 under aerobic conditions . Structural analysis of the mgpS locus revealed that expression of mgpS was likely to be complex . A smaller protein containing the 472 C-terminal amino acids of MgpS is able to act by itself as an activator of puc transcription and is expressed independently of the large open reading frame in which it is contained.

J Bacteriol, 1996 Jan, 178(1), 216 - 22
Transcription of Bacillus subtilis degR is sigma D dependent and suppressed by multicopy proB through sigma D; Ogura M et al.; Production of Bacillus subtilis exoproteases is positively regulated by the DegS-DegU two-component regulatory system and other regulatory factors including DegR and ProB . It was shown that the expression of degR was virtually abolished in a sigD mutant and that the transcriptional initiation site in vivo is preceded by a sequence very similar to the consensus sequence of sigma D-recognized promoters . Alteration of the -10 sequence of the putative promoter greatly reduced the expression of degR . These results show that degR expression is driven by the alternative sigma factor, sigma D . It was found that degR expression was suppressed by multiple copies of proB on plasmid pLC1 and that this suppression was exerted at the transcriptional level through a target in the vicinity of the degR promoter . Furthermore, it was shown that the expression of another sigma D-directed gene, hag, was suppressed by pLC1 . Suppression by pLC1 diminished when the sequence of the -10 element of the degR promoter was changed to a sigma A-like promoter sequence . pLC1, however, did not suppress sigD expression . On the basis of these results, we conclude that multicopy proB on pLC1 inhibits transcription from sigma D-driven promoters by affecting some posttranscriptional process of sigma D.

J Bacteriol, 1996 Jan, 178(1), 191 - 8
Homology among nearly all plasmids infecting three Bacillus species; Zawadzki P et al.; We have surveyed naturally occurring plasmids in strains of Bacillus subtilis and the closely related species B . mojavensis and B . licheniformis . Previous studies have failed to find host-benefitting functions for plasmids of these species, suggesting that these plasmids are nonmutualistic . Only one type of plasmid was found in each plasmid-bearing strain, suggesting that most of the plasmids infecting these Bacillus species are in the same incompatibility group . A sample of 18 plasmids from these species ranged in size from 6.9 to 16 kb, with all but 6 plasmids falling into three size groups . These groups differed in the sizes of their host ranges and geographical ranges . All but 1 of the 18 plasmids from these three host species are homologous with one another . The cryptic plasmids from these three species are far less diverse than are plasmids (from other species) that are known to benefit their bacterial hosts . The low-level diversity among these cryptic plasmids is consistent with the hypothesis that host-benefitting adaptations play an important role in fostering the coexistence of plasmid populations, but other explanations for the low-level plasmid diversity are possible . Comparison of the phylogenies of the plasmids with those of their hosts suggests that Bacillus plasmids are horizontally transferred in nature at a low rate similar to that found for the colicin plasmids of Escherichia coli.

DNA Res, 1995 Dec 31, 2(6), 295 - 301
Cloning and sequencing of a 23-kb region of the Bacillus subtilis genome between the iol and hut operons; Yoshida K et al.; Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 23-kb chromosomal segment, which covers the region between the iol and hut operons, has been cloned and sequenced, creating a 99-kb contig from the gnt operon to the wapA locus . This region (23351 bp) contains 25 complete open reading frames (ORFs; genes) including deoR, dra, nupC and pdp and two partial ones . The region (5140 bp) containing these four genes, being also sequenced by H . H . Saxild et al., was sequenced by subjecting a long polymerase chain reaction product to random sequencing using phage M13mp19 . However, we could detect no conflict, between two independently determined sequences, which could be attributed to our sequencing method . A homology search for the 24 newly identified gene products revealed significant homology to known proteins in 14 of them . It was notable that three proteins, encoded by the successive genes (yxeMNO), exhibited meaningful homology to the E . coli GlnHPQ products constituting a periplasmic ATP-dependent transport system for glutamine.

Gene, 1995 Dec 29, 167(1-2), 335 - 6
Antibiotic-resistance cassettes for Bacillus subtilis; Guerout-Fleury AM et al.; The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors . These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.

Gene, 1995 Dec 29, 167(1-2), 105 - 9
Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes; Rowland B et al.; Menaquinone (MK) is a non-protein component of the Bacillus subtilis (Bs) electron transport chain synthesized from chorismate through a series of MK-specific reactions . The genes encoding biosynthesis of the naphthoquinone ring of MK are clustered at 273 degrees on the Bs chromosome . A 3.9-kb region capable of rescuing men mutants blocked in the early stages of MK biosynthesis was sequenced and found to contain three major open reading frames (ORFs) . The first ORF (menF) has a predicted size of 51.8 kDa and 34% amino-acid identity with the isochorismate synthases of Escherichia coli (EntC) and Aeromonas hydrophila (AmoA), ORF2 (menD) a predicted size of 60.2 kDa and 21% identity with MenD of E . coli . ORF3 has a predicted size of 21.4 kDa and 29% identity to triacylglycerol lipase of Psychrobacter immobilis . No sequence corresponding to menC was identified . Plasmid integrational studies of the men gene cluster had suggested the presence of promoters secondary to the previously identified p1 men promoter . Sequence analysis revealed a putative promoter region upstream from ORF3.

J Chromatogr A, 1995 Dec 22, 718(2), 291 - 7
Determination of beta-(1-3),(1-4)-D-glucans in barley by reversed-phase high-performance liquid chromatography; Perez-Vendrell AM et al.; An HPLC method for the determination of beta-glucan in barley was developed . The beta-glucan was hydrolysed with lichenase {endo-beta-(1-3),(1-4)-D-glucan-4-glucanhydrolase from Bacillus subtilis} to oligosaccharides, which were analysed by reversed-phase HPLC using water as the mobile phase at a flow-rate of 0.7 ml/min . The separation of the oligosaccharides was performed in a C18 stainless-steel column (Spherisorb ODS-2) with 5-microns particles in less than 10 min, with a refractive index detection.

J Ethnopharmacol, 1995 Dec 15, 49(3), 147 - 56
Biological screening of traditional medicinal plants from Papua New Guinea; Nick A et al.; Based on ethnopharmacological literature, 17 species of medicinal plants used in the traditional medicine in Papua New Guinea were collected . Extracts of different polarities were tested in a preliminary biological screening for their antimicrobial (Escherichia coli, Bacillus subtilis, Micrococcus luteus and Penicillium oxalicum) and molluscicidal activity against Biomphalaria glabrata as well as for their toxicity to brine shrimp . The pretreated plant extracts were also investigated for their ability to inhibit protein kinase C and tyrosine-specific protein kinase of epidermal growth factor receptor . Furthermore, all plants were screened for the presence of alkaloids.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 129 - 35
Changes in DNA supertwist as a response of Bacillus subtilis towards different kinds of stress; Krispin O et al.; We have investigated the change in DNA supercoiling in Bacillus subtilis after exposure to different kinds of stress . No or only minor effects are induced by solvents . Anaerobiosis and heat shock result in plasmids which are less negatively supercoiled . Cold shock and salt stress result in an increase in negative supercoiling . The reduction of linking number starts immediately after addition of NaCl, and reached a maximal value after 2 min . Primer extension experiments revealed that the mRNA level of the gyrA gene is not specifically changed by stress . The response in B . subtilis is inverse to the response in vitro . We discuss the possible effects of these inverse in vivo and in vitro responses for B . subtilis.

J Biol Chem, 1995 Dec 8, 270(49), 29138 - 44
The contrahelicase activities of the replication terminator proteins of Escherichia coli and Bacillus subtilis are helicase-specific and impede both helicase translocation and authentic DNA unwinding; Sahoo T et al.; Replication forks are arrested at sequence-specific replication termini primarily, perhaps exclusively, by polar arrest of helicase-catalyzed DNA unwinding by the terminator protein . The mechanism of this arrest is of considerable interest . This paper presents experimental evidence in support of four major points pertaining to termination of DNA replication . First, the replication terminator proteins of both Escherichia coli and Bacillus subtilis are helicase-specific contrahelicases, i.e . the proteins specifically impede the activities of helicases that are involved in symmetric DNA replication but not of those involved in conjugative DNA transfer and rolling circle replication . Second, the terminator protein (Ter) of E . coli blocks not only helicase translocation but also authentic DNA unwinding . Third, the replication terminator protein of Gram-positive B . subtilis is a polar contrahelicase of the primosomal helicase PriA of Gram-negative E . coli . Finally, the blockage of PriA-catalyzed DNA unwinding was abrogated by the passage of an RNA transcript through the replication terminator protein-terminus complex . These results are significant because of their relevance to the mechanistic aspects of replication termination.

Biochim Biophys Acta, 1995 Dec 6, 1253(2), 224 - 8
Purification and characterization of a new serine proteinase from Bacillus subtilis with specificity for amino acids at P1 and P2 positions; Yamagata A et al.; A proteinase was purified 230-fold to apparent homogeneity from culture filtrates of Bacillus subtilis by a series of column chromatographies on DE52, DEAE-Toyopearl, Cellulofine GC200M, and Mono-Q, using Boc-Ala-Ala-Pro-Ser-pNA as a substrate . The molecular weight of the proteinase was estimated to be 42,000 by SDS-PAGE in the presence of 2-mercaptoethanol . Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that this proteinase preferentially hydrolyzed the peptide bond on the carboxyl-terminal side of either serine or alanine residues at the P1 position and hydrophobic bulky amino acids at P2 . It was most active at pH 9.5 for the hydrolysis of Boc-Ala-Ala-Pro-Ser-pNA . The enzyme was inactivated by diisopropyl fluorophosphate (DFP), but not by tosyl-L-phenylalanine chloromethylketone (TPCK) or by EDTA . Based on the reactivity toward substrates and inhibitors, this enzyme differs from elastase- or subtilisin-like proteinase, hence it is a new type of proteinase with specificity for amino acids at P1 and P2 positions.

Biochemistry, 1995 Dec 5, 34(48), 15838 - 44
Methylation of minimalist 23S rRNA sequences in vitro by ErmSF (TlrA) N-methyltransferase; Kovalic D et al.; ermSF (synonym tlrA) from Streptomyces fradiae NRRL 2702 confers resistance to the macrolide-lincosamide- streptogramin type B (MLS) superfamily of antibiotics . ErmSF specifically methylates Bacillus subtilis 23S rRNA in vitro at A2085 (B . subtilis coordinate, which is equivalent to the Escherichia coli coordinate A2058) . In the present studies, partial B . subtilis 23S rRNA sequences containing portions of the peptidyltransferase circle which include A2085 were constructed in order to identify structural requirements needed for RNA to function as substrate of ErmSF . A model methylase substrate based on the 41-nucleotide construct DK111, ggCCUAUCCGUCGCGGGUUCGCCCGCGACAGGACGGA*AAGA, had methyl-acceptor activity . This sequence contains 23S rRNA stem 73 {Stade, K., et al . (1994) Nucleic Acids Res . 22, 1394-1399} underlined, flanking a tetraloop-like (UUCG), and the impaired sequence AAAGA, at the 3' end containing A2085 (A*) . A set of systematic alterations introduced into the sequence suggested that the four unpaired nucleotides in stem 73 are necessary for methyl-acceptor activity, whereas inversion of 11 out 13 paired bases in stem 73 conferred no significant reduction in methyl-acceptor activity.

EMBO J, 1995 Dec 1, 14(23), 5984 - 94
Anaerobic transcription activation in Bacillus subtilis: identification of distinct FNR-dependent and -independent regulatory mechanisms; Cruz Ramos H et al.; Bacillus subtilis is able to grow anaerobically using alternative electron acceptors, including nitrate or fumarate . We characterized an operon encoding the dissimilatory nitrate reductase subunits homologous to the Escherichia coli narGHJI operon and the narK gene encoding a protein with nitrite extrusion activity . Downstream from narK and co-transcribed with it a gene (fnr) encoding a protein homologous to E.coli FNR was found . Disruption of fnr abolished both nitrate and fumarate utilization as electron acceptors and anaerobic induction of narK . Four putative FNR binding sites were found in B.subtilis sequences . The consensus sequence, centred at position -41.5, is identical to the consensus for the DNA site for E.coli CAP . Bs-FNR contained a four cysteine residue cluster at its C-terminal end . This is in contrast to Ec-FNR, where a similar cluster is present at the N-terminal end . It is possible that oxygen modulates the activity of both activators by a similar mechanism involving iron . Unlike in E.coli, where fnr expression is weakly repressed by anaerobiosis, fnr gene expression in B.subtilis is strongly activated by anaerobiosis . We have identified in the narK-fnr intergenic region a promotor activated by anaerobiosis independently of FNR . Thus induction of genes involved in anaerobic respiration requires in B.subtilis at least two levels of regulation: activation of fnr transcription and activation of FNR to induce transcription of FNR-dependent promoters.

Poult Sci, 1995 Dec, 74(12), 2019 - 28
Stunting syndrome in broilers: effect of age and exogenous amylase and protease on performance, development of the digestive tract, digestive enzyme activity, and apparent digestibility; Shapiro F et al.; Day-old male, meat-type chicks raised in brooder batteries were infected by orally administering an inoculum prepared from intestines of broiler chicks infected with stunting syndrome (SS) . Naive controls were kept in a parallel room . The chicks were fed a commercial starter diet supplemented with two levels of enzyme preparations to 14 d of age . The experiment was continued to the age of 6 wk in order to estimate compensatory feed intake and growth . In a parallel study, digestibility of the feed was determined from 1 to 3 wk of age with control or inoculated chicks . The enzymes amylase and proteases were produced by Bacillus subtilis and Penicillium emersonii . Enzyme supplementation had no effect on feed intake, growth, or feed utilization, or on digestibility of fat, starch, protein, or energy . Because enzyme supplementation did not consistently affect performance of chicks and no interactions were observed between enzyme supplementation and infection status, data are presented for effects of infection only . Inoculation of SS-infective material reduced performance to 4 wk . Compensatory growth and feed intake were observed from the age of 4 wk onward . At the age of 6 wk the slight retardation of the inoculated chicks was not significant . On Week 1, retention of fat, starch, protein, and energy was significantly depressed in the inoculated chicks . At the age of 2 wk, retention of starch was not depressed, and at the age of 3 wk, the only consistent depression was that observed for fat . The proventriculus weight and content were consistently higher in inoculated chicks, as were the small intestine and intestinal content . The pH of the gizzard content was higher, and that of the small intestine content was lower, in the inoculated birds than in their control counterparts . Stunting syndrome infection was accompanied by a significant depression of trypsin activity in the pancreas at the age of 1 and 2 wk . At these periods, amylase and chymotrypsin were not affected . At 6 wk of age, the activities of amylase, trypsin, and chymotrypsin in the pancreas were higher in the inoculated than in the control birds . In the intestinal chime, amylase, trypsin, and chymotrypsin activities were lower in the inoculated birds on Week 1 and 2 (NS for amylase on Week 1) . On Week 6, the activity of all enzymes assayed was higher in the inoculated birds (NS for amylase) . It is suggested that the main factors depressing feed intake and growth in SS-infected birds are most probably beyond those of digestion.

Planta Med, 1995 Dec, 61(6), 562 - 3
Larvicidal constituents of Melantheria albinervia; Slimestad R et al.; Bioactivity-guided fractionation has led to the isolation of two larvicidal diterpenes, active against the yellow fever-transmitting mosquito Aedes aegypti, from a dichloromethane root extract of Melantheria albinervia (Asteraceae), a plant from Zimbabwe . These diterpenes were identified as ent-kaur-16-en-19-oic acid and 9(11),16-kauradien-19-oic acid . The diterpenes were also weakly active against Bacillus subtilis.

Proteins, 1995 Dec, 23(4), 607 - 9
Purification, crystallization, and preliminary X-ray analysis of Bacillus subtilis ferrochelatase; Hansson M et al.; Bacillus subtilis ferrochelatase (EC 4.99.1.1), the final enzyme in protoheme IX biosynthesis, was produced with an inducible T7 RNA polymerase expression system in Escherichia coli and purified from the soluble cell fraction . It was crystallized from polyethylene glycol solution using the microseeding technique . The crystals diffract to a minimum Bragg spacing of 2.1 A . The space group is P4(2) with unit cell dimensions a = b = 50.2 A, c = 120.1 A.

Genetics, 1995 Dec, 141(4), 1231 - 43
The size and continuity of DNA segments integrated in Bacillus transformation; Zawadzki P et al.; We investigated the size and continuity of DNA segments integrated in Bacillus subtilis transformation . We transformed B . subtilis strain 1A2 toward rifampicin resistance (coded by rpoB) with genomic DNA and with a PCR-amplified 3.4-kb segment of the rpoB gene from several donors . Restriction analysis showed that smaller lengths of donor DNA integrated into the chromosome with transformation by PCR-amplified DNA than by genomic DNA . Nevertheless, integration of very short segments (< 2 kb) from large, genomic donor molecules was not a rare event . With PCR-amplified segments as donor DNA, smaller fragments were integrated when there was greater sequence divergence between donor and recipient . There was a large stochastic component to the pattern of recombination . We detected discontinuity in the integration of donor segments within the rpoB gene, probably due to multiple integration events involving a single donor molecule . The transfer of adaptations across Bacillus species may be facilitated by the small sizes of DNA segments integrated in transformation.

Protein Sci, 1995 Dec, 4(12), 2478 - 86
Phosphorylation of serine-46 in HPr, a key regulatory protein in bacteria, results in stabilization of its solution structure; Pullen K et al.; The serine-phosphorylated form of histidine-containing protein (HPr), a component of the phosphoenolpyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy and solvent denaturation studies . The results indicate that phosphorylation of Ser 46, the N-cap of alpha-helix-B, does not cause a conformational change but rather stabilizes the helix . Amide proton exchange rates in helix-B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaturation, with a delta delta G of 0.7-0.8 kcal mol-1 . A mutant in which Ser 46 is replaced by aspartic acid shows a similar stabilization, indicating that an electrostatic interaction between the negatively charged groups and the helix macrodipole contributes significantly to the stabilization.

Appl Microbiol Biotechnol, 1995 Dec, 44(1-2), 112 - 7
Purification and characterization of an arabinofuranosidase from Bacillus polymyxa expressed in Bacillus subtilis; Morales P et al.; Two polypeptides showing alpha-L-arabinofuranosidase activity have been purified to homogeneity from culture supernatants of a Bacillus subtilis clone harbouring the xynD gene {Gosalbes et al . (1991) J Bacteriol 173: 7705-7710} from Bacillus polymyxa . Both polypeptides, with determined molecular masses of 64 kDa and 53 kDa, share the same sequence at their N termini, which also coincides with the sequence deduced for the mature protein from the previously determined sequence of nucleotides (Gosalbes et al . 1991) . The two polypeptides have been biochemically characterized . Arabinose is the unique product released from arabinose-containing xylans which are substrates for both enzyme forms . Other natural arabinose-containing polysaccharides, such as arabinogalactans, are not attacked by them but some artificial arabinose derivatives are good substrates for both polypeptides . Their arabinose-releasing activity on arabinoxylans facilitates the hydrolysis of the xylan backbone by some endoxylanases from Bacillus polymyxa.

DNA Cell Biol, 1995 Dec, 14(12), 1049 - 55
Origin of replication of the Bacillus subtilis chromosome: in vitro approach to the isolation of early replicating segments; Sasvari-Szekely M et al.; We have developed a permeable cell system for the study of the molecular mechanisms involved in the control and initiation of DNA replication at the origin of the Bacillus subtilis chromosome . Our system take advantage of the synchronous initiation of DNA replication that occurs in outgrowing B . subtilis spores and the curtailment of DNA elongation by novobiocin . Early replicating DNA sequences were identified by the use of 5-mercury-dCTP as substrate, which allows the isolation of nascent DNA chains by affinity chromatography on thiol agarose . The average size of the isolated nascent DNA was 1,000 bp, and more than 80% of the nascent DNA chains had RNA primers at their 5' end . The study of the temporal order of chromosome replication near the origin using this experimental system showed that a segment containing recF and gyrB replicated earlier than a segment containing gyrA and part of the rRNA operon (rrnO) . This observation is in agreement with previous in vivo data on the replication of origin region and supports the conclusion that the major activity in our in vitro system was the faithful replication of the ori region.

Appl Environ Microbiol, 1995 Dec, 61(12), 4244 - 50
A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination; Kiel JA et al.; We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance) . The procedure employs the high level of transformability of B . subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B . subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive . First, a thymine-auxotrophic B . subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus . In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected . Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene . Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene . The method was used to construct B . subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.

Curr Microbiol, 1995 Dec, 31(6), 340 - 4
Molecular cloning and nucleotide sequence of the 90k serine protease gene, hspK, from Bacillus subtilis (natto) No . 16; Yamagata Y et al.; We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No . 16 {Kato et al . 1992 Biosci Biotechnol Biochem 56:1166} . The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins . The structural gene, hspK, for the 90k serine protease was cloned and sequenced . The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues . The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence . The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k . It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein . The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases . The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.

J Bacteriol, 1995 Dec, 177(24), 7280 - 4
Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis; Hilden I et al.; The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon . The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as beta-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent on the promoter in front of the gcaD gene . Analysis of cDNA molecules prepared with gcaD-prs-ctc-specified mRNA as the template revealed an RNA transcript that encompassed all three cistrons.

J Bacteriol, 1995 Dec, 177(24), 7231 - 7
SecA proteins of Bacillus subtilis and Escherichia coli possess homologous amino-terminal ATP-binding domains regulating integration into the plasma membrane; McNicholas P et al.; The Bacillus subtilis secA homolog, div, was cloned and expressed at a variety of different levels in wild-type and secA mutant strains of Escherichia coli . Analysis of Div function showed that it could not substitute for SecA despite being present at a wide range of concentrations at or above the physiological level . Location of regions of functional similarity between the two proteins using div-secA chimeras revealed that only the amino-terminal ATP-binding domain of Div could functionally substitute for the corresponding region of SecA . The role of this domain was revealed by subcellular localization experiments that demonstrated that in both B . subtilis and E . coli Div had cytoplasmic, peripheral, and integral membrane distributions similar to those of its SecA homolog and that an intact ATP-binding domain was essential for regulating integration of this protein into the plasma membrane . These results suggest strongly that the previously observed cycle of membrane binding, insertion, and deinsertion of SecA protein (A . Economou and W . Wickner, Cell 78:835-843, 1994) is common to these two bacteria, and they demonstrate the importance of the conserved ATP-binding domain in promoting this cycle.

J Bacteriol, 1995 Dec, 177(24), 7060 - 9
Mechanics of bacterial macrofiber initiation; Mendelson NH et al.; The twisting and writhing during growth of single-cell filaments of Bacillus subtilis which lead to macrofiber formation was studied in both left- and right-handed forms of strains FJ7 and RHX . Filament bending, touching, and loop formation (folding), followed by winding up into a double-strand fiber, were documented . Subsequent folds that produced multistrandedness were also examined . The rate of loop rotation during winding up was measured for 26 loops from 16 clones . In most cases, the first loop formed turned at a lower rate than those produced by the following cycles of folding . The sequence of folding topologies differed in FJ7 and RHX strains and in left- versus right-handed structures . Right-handed FJ7 routinely gave rise to four-stranded helices at the second fold, whereas left-handed FJ7 and both left-handed and right-handed forms of RHX made structures with predominantly two double-stranded helical regions . Left-handed RHX structures frequently produced second folds within the initial loop itself, resulting in T- or Y-shaped fibers . Sixteen cases in which the initial touch of a filament to itself produced a loop that snapped open before it could wind up into a double-strand fiber were found . The snap motions were used to obtain estimates of the forces generated by helical growth of single filaments and to investigate theoretical models involving the material properties of cell filaments . In general, the mechanical behavior of growing single-cell filaments and fibers consisting of two-, three-, or four-strand helices was similar to that described for larger, mature, multifilament macrofibers . The behavior of multicellular macrofibers can be understood, therefore, in terms of individual cell growth.

J Bacteriol, 1995 Dec, 177(23), 7003 - 6
The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis; Marini P et al.; The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis . DNA sequencing and RNA blot hybridization studies indicated that the B . subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.

J Bacteriol, 1995 Dec, 177(23), 6999 - 7002
Delineation of AbrB-binding sites on the Bacillus subtilis spo0H, kinB, ftsAZ, and pbpE promoters and use of a derived homology to identify a previously unsuspected binding site in the bsuB1 methylase promote; Strauch MA; DNase I footprinting experiments showed that AbrB binds to the regulatory regions of the spo0H, kinB, ftsAZ, and pbpE genes . A conserved motif was found in these and other AbrB-binding sites . A search for Bacillus subtilis DNA sequences containing this motif led to the prediction that AbrB would bind to the promoter controlling the bsuB1 methylase gene . DNase I footprinting experiments confirmed this prediction.

J Bacteriol, 1995 Dec, 177(23), 6928 - 36
The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon; Stulke J et al.; The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis . The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L . One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family . It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity . We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS . This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr . In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr . The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity . This domain appears to be duplicated in the LevR protein . The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.

J Bacteriol, 1995 Dec, 177(23), 6919 - 27
Two different mechanisms mediate catabolite repression of the Bacillus subtilis levanase operon; Martin-Verstraete I et al.; There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression . The LevR activator interacts with its target, the upstream activating sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter . Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala . In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon . In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis . The CRE sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter . However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation . Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.

J Bacteriol, 1995 Dec, 177(23), 6751 - 60
The cytochrome bc complex (menaquinone:cytochrome c reductase) in Bacillus subtilis has a nontraditional subunit organization; Yu J et al.; We have identified an operon in Bacillus subtilis, designated qcr, that is thought to encode a quinone: cytochrome c reductase . Northern (RNA blot) analysis suggests a tricistronic operon . The operon is located at about 200 degrees on the B . subtilis map . Disruption of the operon leads to loss of a 22-kDa cytochrome c from membrane preparations . The structure of the putative protein products of the qcr operon suggests a protein complex that is closely related to but distinct from known cytochrome bc1 and b6f complexes, which catalyze electron transfer from a quinol to a c-type cytochrome or to plastocyanin . QcrA is similar to Rieske-type iron-sulfur proteins; QcrB is similar in size and sequence to b-type cytochromes from b6f complexes; and QcrC has a novel structure that resembles a fusion of a subunit IV (found in b6f complexes) to a cytochrome c . Transcription of the operon is induced at the end of exponential growth from a sigma A-like promoter . This transition state induction appears to be dependent on the downregulation of abrB expression, which is mediated by Spo0A activation . As bacteria move from the transition state into sporulation, transcription of the operon is reduced in a sigma F-dependent manner.

J Bacteriol, 1995 Dec, 177(23), 6727 - 31
AbrB modulates expression and catabolite repression of a Bacillus subtilis ribose transport operon; Strauch MA; A Bacillus subtilis ribose transport operon (rbs) was shown to be subject to AbrB-mediated control through direct AbrB-DNA binding interactions in the vicinity of the promoter . Overproduction of AbrB was shown to relieve catabolite repression of rbs during growth in the presence of poorer carbon sources such as arabinose but had much less effect when cells were grown in the presence of glucose, a rapidly metabolizable carbon source . A ccpA mutation relieved catabolite repression of rbs under all conditions tested . One of the AbrB-binding sites on the rbs promoter contains the putative site of action for the B . subtilis catabolite repressor protein CcpA, suggesting that competition for binding to this site could be at least partly responsible for modulating rbs expression during carbon-limited growth.

Biochim Biophys Acta, 1995 Nov 21, 1232(1-2), 67 - 74
Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement; Villani G et al.; Using the high efficiency of homologous gene recombination in Bacillus subtilis, a strategy for mutational analysis of the proton pumping aa3-600 quinol oxidase of this organism has been developed . The qox operon with the qoxA, qoxB, qoxC and qoxD genes, coding for the four subunits of this oxidase, was deleted and then replaced with mutated copies in which qoxC (subunit III) or qoxD (subunit IV) genes were deleted . The complete deletion of the qox operon caused disappearance of heme aa3-600 and a slight depression of the overall respiratory activity, compensated by alternative oxidase with no proton pumping activity . Deletion of qoxC probably resulted in a defective assembly of the aa3-600 quinol oxidase . The strain with deletion of qoxD gene expressed normal content of heme aa3-600 but exhibited a reduced respiratory activity and a significantly depressed proton pumping activity . These results show that subunit IV is critical for the activity of the proton pumping aa3-600 quinol oxidase.

Gene, 1995 Nov 7, 165(1), 45 - 50
The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure; Barnes MH et al.; The primary structure of the 3'-5' exonuclease (Exo) site of the Gram+ bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III) . It was found to differ significantly from the conventional three-motif substructure established for the Exo site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases . Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues . However, the conventional downstream motif, motif III, was replaced by motif III epsilon, a novel 55-amino-acid (aa) segment incorporating three essential aa (His565, Asp533 and Asp570) which are strictly conserved in three Gram+ Pol III and in the Ec Exo epsilon (epsilon) . Despite its unique substructure, the Gram+ Pol III-specific Exo site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization . The entire Exo site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs . Conversely, Pol and all other sequences downstream of the Exo site could be deleted with little apparent effect on Exo activity . Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model Exo site of EcPol I, remains to be established.

Gene, 1995 Nov 7, 165(1), GC37 - 51
Analysis of a Bacillus subtilis genome fragment using a co-operative computer system prototype; Medigue C et al.; Analysis of the huge volume of data generated by large scale sequencing projects requires the construction of new, sophisticated computer systems . These systems should be able to manage the biological data as well as the results of their analysis . They should also help the user to choose the most appropriate methods, and to string them together in order to solve a global analysis task . In this paper we present the prototype of a software system providing an environment for the analysis of large-scale sequence data . As a first step toward this end, this environment has been put to the test within the Bacillus subtilis genome sequencing project . This system integrates both the descriptive knowledge of the entities involved (genes, regulatory signals and the like) and the methodological knowledge comprising an extensible set of analytical methods . A knowledge representation based on two existing object-oriented models is used to implement this integrated system . In addition, the present prototype provides a suitable user interface both for displaying simultaneously the results generated by several methods and for interacting with the objects . We present in this paper the analysis of a B . subtilis genome fragment, present in data libraries but not annotated . Annotation of the genes present in the fragment allowed us to combine the results of several methods used for predicting coding sequences, and to characterize it as comprising a cryptic phage, the skin element . Comparison between the annotation of the skin element and a standard region of the chromosome indicated that local features of the nucleotide sequence could discriminate between phage and non-phage DNA sequence.

FEBS Lett, 1995 Nov 6, 374(3), 363 - 6
Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate; Kamal M et al.; A serine protease has been isolated and characterized from Bacillus subtilis, strain RT-5 (a thermostable soil isolate from the Tharparkar desert of Pakistan) able to grow at 55 degrees C . The primary structure was established by a combination of protein and DNA-sequence analyses . The amino-acid sequence, inhibition pattern and solubility properties identify the enzyme as a subtilisin . It has 43 amino-acid replacements toward subtilisin BPN' and as much as 83 replacements toward another subtilisin, confirming that strain variabilities are extensive between different subtilisin forms . However, the structure is identical to one of unknown functional properties deduced from DNA and is closely related to mesentericopeptidase but that homologue is not thermostable . From comparisons with that form and with subtilisin BPN', it is concluded that replacements of Ala --> Ser at positions 85 and 89, Ser --> Ala at position 88 and Asp or Ser --> Asn at position 259 may promote thermostability.

J Chromatogr A, 1995 Nov 3, 715(2), 247 - 58
Unusual chromatographic behaviour and one-step purification of a novel membrane proteinase from Bacillus cereus; Fricke B et al.; Cell envelopes of Bacillus cereus contain a casein-cleaving membrane proteinase (CCMP) and an insulin-cleaving membrane proteinase (ICMP), which differ in their substrate and inhibitor specificity from all Bacillus proteinases described previously . They remained localized in the cytoplasmic membrane after treatment with lysozyme and mutanolysin and they are strongly attached to the membrane compared with other known membrane proteinases . Only high a concentration of the Zwitterionic detergent sulfobetain SB-12 enabled an effective solubilization of both membrane proteinases . The usual conventional purification methods, such as chromatofocusing, ion-exchange chromatography and hydrophobic interaction chromatography in the presence of detergent concentrations beyond their critical micelle concentration, could not be applied to the purification, because the solubilized membrane proteinases bound strongly and irreversibly to the chromatographic matrix . In the search for other purification methods, we used a tentacle ion-exchanger (EMD trimethylaminoethyl-Fractogel) to reduce the hydrophobic interactions between the proteinases and the matrix . All contaminating proteins could be removed by a first gradient of sodium chloride without elution of CCMP; a second gradient with isopropanol and a decreasing salt concentration resulted in an efficiently purified CCMP . The ICMP was irreversibly denaturated . Purified CCMP is a member of the metalloproteinase family with a pH optimum in the neutral range and a temperature optimum of 40 degrees C, whose properties differ from the serine-type membrane proteinase of Bacillus subtilis described by Shimizu et al . {Agric . Biol . Chem., 47 (1983) 1775} . It consists of two subunits in sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Mr 53,000 and 65,000); however, the molecular mass of the purified enzyme could not be determined by size exclusion or SDS-PAGE, because the purified enzyme aggregated at the top of the gel matrix . CCMP solubilized before the purification process, could be eluted in the presence of 0.1% octylphenol-poly(ethyleneglycol ether)9-10 (Triton X-100) in two peaks of Mr 56,000 and 128,000, respectively . We discuss this special chromatographic behaviour of the CCMP from Bacillus cereus, with regard to the strong hydrophobic interactions of the enzyme with the chromatographic matrix and additional self-aggregation, which could only be dissolved by solvents such as isopropanol.

Mol Microbiol, 1995 Nov, 18(4), 755 - 67
Inactivation of mecA prevents recovery from the competent state and interferes with cell division and the partitioning of nucleoids in Bacillus subtilis; Hahn J et al.; The development of genetic competence in Bacillus subtilis requires the synthesis of ComK, a transcription factor, which is normally produced as a culture enters the stationary phase . This synthesis is known to be regulated in part by the protein MecA . Loss-of-function mutations in mecA result in overexpression of ComK and its appearance early during exponential growth . We show here that mecA inactivation also causes a loss of colony-forming ability, especially during stationary phase . This loss is accompanied by the appearance of cells in which normal nucleoid separation has failed to occur . Renografin gradient fractionation of mecA cultures grown to competence reveals that nearly 100% of the cells band at the low buoyant density characteristic of competent cells, and that this low density is competence-related . The loss of viability, the low buoyant density and the nucleoid separation defect, are all comK-dependent . The loss of viability can be reversed by even the transient introduction of mecA+ . It is proposed that these effects of ComK overexpression are related to the DNA replication arrest normally exhibited by the competent cell fraction and that MecA is needed to reverse this arrest and to permit escape from the competent state . The shift of nearly 100% of the cells to light buoyant density in a mecA mutant culture strongly suggests that the MecA protein is a regulator of the cell-type-specific expression of competence.

Mol Microbiol, 1995 Nov, 18(3), 471 - 8
The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites; Alonso JC et al.; The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia {correction of Eschrichia} coli HU} is provided . By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex . Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction . We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination . These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures . Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site . Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

Mol Microbiol, 1995 Nov, 18(3), 459 - 70
Visualization of the subcellular location of sporulation proteins in Bacillus subtilis using immunofluorescence microscopy; Pogliano K et al.; We describe the application of immunofluorescence microscopy to visualization of the subcellular localization of proteins involved in coat morphogenesis and chromosome packaging during the process of sporulation in Bacillus subtilis . In confirmation and extension of previous findings, we show that SpolVA, which is responsible for guiding coat formation to the surface of the outer membrane that surrounds the developing spore, assembles into a shell that is located close to or on the surface of this enveloping membrane . CotE, which is responsible for the formation of the outer layer of the coat, assembles into a second shell of apparently larger diameter . Assembly of SpolVA could be detected as early as the morphological stage of polar septation and closely followed the enveloping membrane of the mother cell during the stage of engulfment, thereby providing a sensitive and diagnostic marker for this phagocytic-like process . Surprisingly, the chromosome of the developing spore and the small, acid-soluble proteins, known as alpha/beta-type SASPs, that are known to coat the spore chromosome, were found to co-localize to a doughnut-like ring of approximately 1 micrometer in diameter . The use of a double mutant lacking the alpha/beta-type SASP demonstrated that these high abundance, DNA-binding proteins are responsible for packaging the chromosome of the developing spore into this unusual structure . We conclude that sporulation in B . subtilis is a fertile system for addressing cell biological problems in a bacterium and that immunofluorescence microscopy provides a sensitive method for visualizing protein subcellular localization at high resolution.

Appl Microbiol Biotechnol, 1995 Nov, 43(6), 1067 - 76
Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express alpha-amylase and glucoamylase separately or as bifunctional fusion proteins; de Moraes LM et al.; Eight constructions involving the Bacillus subtilis alpha-amylase gene (amyE), a mouse pancreatic alpha-amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one alpha-amylase and the glucoamylase, two double-cassette plasmids (expressing one or other alpha-amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences . Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B . subtilis alpha-amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic alpha-amylase/glucoamylase fusion (MAAase/GAase) . When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B . subtilis alpha-amylase and the glucoamylase exhibited both enzymic activities . Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch . The MAAse/GAase fusion protein was found to exhibit only alpha-amylase activity . Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the strains bearing plasmids coding for the fusion proteins and the separate enzymes . The strain carrying the double-cassette BsAAase + GAase, which produced one of the smallest hydrolysis haloes in the place test, showed the best performance, not only in digesting soluble and corn starch but also in using all of the hydrolysis products for growth . The transformant bearing the BsAAase/GAase fusion was able to grow on soluble starch, but not on corn starch.

PDA J Pharm Sci Technol, 1995 Nov-Dec, 49(6), 294 - 9
Performance of blow/fill/seal equipment under controlled airborne microbial challenges; Sinclair CS et al.; Fundamental investigations have been carried out into the effectiveness of aseptic fill using Blow/Fill/Seal machinery . Techniques have been developed to generate and to maintain over prolonged periods, controlled microbial challenges of spores of Bacillus subtilis var . niger dispersed within the air of a containment room housing a Blow/Fill/Seal machine set-up to undertake medium fill studies . The range of spore concentrations that have been generated extends from as low as 3 x 10(2) to as high as 10(7) spores m-3 . Responses to controlled microbial challenges have defined the relationship between product contamination and microbiological quality of the machine environment . The relationship is regular and amenable to extrapolation, so that it is now practicable to specify the microbiological quality of the machine environment, together with machine operating conditions, needed to attain a Sterility Assurance Level comparable to that targeted for terminal sterilization (i.e . 10(-6)) . The impacts of mould configuration, air shower operation and location of point of fill on product contamination have also been examined.

J Struct Biol, 1995 Nov-Dec, 115(3), 258 - 66
Electron microscopy of Bacillus subtilis GroESL chaperonin and interaction with the bacteriophage phi 29 head-tail connector; Tsuprun V et al.; The Bacillus subtilis GroESL chaperonin was isolated by sucrose density gradient centrifugation and the constituent GroES and GroEL moieties were purified by electrophoresis in agarose . Electron microscopic images of negatively stained GroEL and GroES oligomers and GroESL complexes were averaged using a reference-free alignment method . The GroEL and GroES particles had the sevenfold symmetry characteristic of their Escherichia coli counterparts . GroESL complexes, reconstituted efficiently in vitro from GroEL and GroES in the absence of added ADP or ATP, had the characteristic bullet- and football-like shapes in side view . Purified bacteriophage phi 29 head-tail connectors having a mass in excess of 0.4 MDa were shown to bind to GroESL at the end opposite to the GroES . The same GroESL-connector complexes were isolated from phage-infected cells in which capsid assembly was blocked, and thus the complex may have functional significance in phi 29 morphogenesis.

J Photochem Photobiol B, 1995 Nov, 31(1-2), 35 - 41
Biological dosimetry and action spectra; Tyrrell RM; In view of the current renewed interest in biological dosimeters, data obtained in the mid-1970s with the UV-sensitive Bacillus subtilis spore UVSSP both in natural sunlight in Rio de Janeiro and with defined monochromatic radiation is reproduced and reconsidered . The crucial issue of the correspondence of the wavelength dependence of the dosimeter response and the biological effect of interest is discussed with reference to end points such as cytotoxicity and melanoma and non-melanoma skin cancer induction . The necessity of taking into account UVA as well as UVB effects in the overall biological effectiveness of sunlight is evaluated and discussed . Gene activation is proposed as a basis for developing sensitive biological markers that can differentiate between UVA and UVB effects and also be used to determine UV penetration into target tissue.

J Antibiot (Tokyo), 1995 Nov, 48(11), 1240 - 7
Bacillomycin Lc, a new antibiotic of the iturin group: isolations, structures, and antifungal activities of the congeners; Eshita SM et al.; Bacillomycin Lc, a new antifungal antibiotic of the iturin class, was isolated from a strain of Bacillus subtilis as a set of five congeners . The structure as determined by chemical and spectrometric analyses has been shown to differ from that of bacillomycin L by sequence changes from aspartate-1 to asparagine-1 and from glutamine-5 to glutamate-5 . The five congeners differ from each other only in the structure of the aliphatic side chain of the constituent beta-amino acid . The hydrophobicity of the beta-amino acid affects the antifungal activity of the congener, as activity increased in the order of increased congener retention on a reversed-phase HPLC column.

Biosci Biotechnol Biochem, 1995 Nov, 59(11), 2172 - 5
Application of the upstream region of a Bacillus endoglucanase gene to high-level expression of foreign genes in Bacillus subtilis; Sumitomo N et al.; A 0.4-kb ScaI-HpaI fragment, 199 bp upstream of the structural gene for alkaline endoglucanase, from the alkalophilic Bacillus sp . KSM-64, was found to be essential for the extracellular production of the enzyme by recombinant Bacillus subtilis cells . We constructed a new vector, pHSP64 (5.5 kb), using pHY300PLK and part of the 5' region of the endoglucanase that contained a possible promoter region . Using recombinant B . subtilis cells that carried this vector, very high production of two endoglucanases and of chloramphenicol acetyltransferase was done.

Biosci Biotechnol Biochem, 1995 Nov, 59(11), 2149 - 50
Bacillus subtilis strains carry highly homologous direct repeat sequences on their chromosomes; Amano H et al.; Highly homologous 170-bp sequences were found to be carried in the same orientation by the chromosomes of Bacillus subtilis GSY908 (a 168 derivative), B . subtilis R, and B . subtilis var . natto . These sequences were in 5'- and 3'-flanking regions of a tetracycline-resistance determinant in B . subtilis GSY908 and B . subtilis R.

Biosci Biotechnol Biochem, 1995 Nov, 59(11), 2132 - 3
Substrate specificity of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6 toward arabinofurano-oligosaccharides; Kaneko S et al.; The substrate specificity of alpha-L-arabinofuranosidase (EC 3.2.1.55) from Bacillus subtilis 3-6 was explored using methyl 2-O-, methyl 3-O-, and methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranosides, and methyl 3,5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside . The enzyme hydrolyzed the methyl alpha-L-arabinofuranobiosides to arabinose and methyl alpha-L-arabinofuranoside in the order of (1-->2)- > (1-->3)- > (1-->5)-linkages . The enzyme hydrolyzed the (1-->3)-linkage more than the (1-->5)-linkage of the methyl alpha-L-arabinofuranotrioside.

Microbiology, 1995 Nov, 141 ( Pt 11), 2921 - 7
A ribonucleic antiterminator sequence (RAT) and a distant palindrome are both involved in sucrose induction of the Bacillus subtilis sacXY regulatory operon; Tortosa P et al.; The Bacillus subtilis sacXY regulatory operon is involved in sucrose induction of the levansucrase sacB gene by an antitermination mechanism . In the presence of sucrose, the activated SacY antiterminator protein stabilizes the secondary structure of a ribonucleic antiterminator sequence (RAT) located in the leader region of the sacB transcript, and overlapping a rho-independent transcription terminator . Formation of the SacY-RAT complex prevents alternative formation of the terminator, allowing transcription of the downstream sequences . In the absence of sucrose, inhibition of SacY activity by SacX leads to termination of transcription . Expression of sacXY is also sucrose-inducible . This induction was previously shown to be mediated by SacY itself and/or SacT, another antiterminator involved in induction of genes belonging to a distinct sucrose pathway . These antiterminators are not activated at the same concentration of sucrose . We show here that sacXY induction occurs through activation of either SacY or SacT antiterminators, at their respective sucrose activation concentration . This result demonstrates a link between SacY- and SacT-mediated metabolic pathways . In addition, the sacXY leader region carries a RAT-like sequence, which however does not appear to overlap any apparent rho-independent transcription terminator . Site-directed mutagenesis experiments on this RAT-like sequence demonstrated its involvement in sucrose induction . Deletions generated in the sacXY leader region showed that a palindrome, located 100 nt downstream from the RAT-like sequence, also acts as a cis-acting element . Computer analysis of the leader RNA suggested that formation of the secondary structure of the RAT-like sequence and the palindrome could be mutually exclusive.

Phytochemistry, 1995 Nov, 40(5), 1447 - 52
Antibacterial phloroglucinols and flavonoids from Hypericum brasiliense; Rocha L et al.; Three known phloroglucinols (japonicine A, uliginosin A and isouliginosin B) and a new phloroglucinol (hyperbrasiol A) have been isolated from a petrol extract of the leaves and flowers of Hypericum brasiliense . Their structures were established by spectroscopic methods (UV, DCI-MS, 1H and 13CNMR, including SINEPT, HMBC, HSQC, DQFCOSY experiments) . The substitution pattern of hyperbrasilol A was confirmed by X-ray crystallography . All four phloroglucinols were antibacterial against Bacillus subtilis in a TLC bioautographic assay . The flavonoids, kaempferol, luteolin, quercetin, quercitrin, isoquercitrin, hyperoside and guaijaverin, were isolated from a methanol extract of the same organs.

J Bacteriol, 1995 Nov, 177(22), 6679 - 83
Expression of kinA and accumulation of sigma H at the onset of sporulation in Bacillus subtilis; Asai K et al.; Induction of the Bacillus subtilis kinA gene, which codes for a major kinase of the phosphorelay pathway, required the spo0H gene, coding for the sigma H protein, but not the genes spo0A, spo0B, and spo0F at the onset of sporulation . Also, the levels of sigma H in spo0A, spo0B, and spo0F mutants were increased at the onset of sporulation, though induction of spo0H transcription in all of these mutants was appreciably inhibited . In addition, kinA expression was almost completely eliminated in a medium supplemented with excess glucose and glutamine, even though the usual stationary-phase-associated increase in sigma H was observed under these conditions.

J Bacteriol, 1995 Nov, 177(22), 6506 - 9
sigma E changed to sigma B specificity by amino acid substitutions in its -10 binding region; Tatti KM et al.; The association of a sigma factor (sigma) with RNA polymerase in bacteria determines its specificity of promoter utilization . To identify amino acid residues in sigma E from Bacillus subtilis that determine the specificity of its interaction with the nucleotides at the -10 region of its cognate promoters, we tested whether base pair substitutions in the -10 region of a sigma B-dependent promoter could signal its utilization by sigma E-RNA polymerase . We found that a combination of base pair substitutions at positions -15 and -14 of the sigma B-dependent ctc promoter resulted in its utilization by sigma E-RNA polymerase in vivo . We also found that the combination of two amino acid substitutions at positions 119 and 120 in sigma E changed its specificity for promoter utilization, resulting in a sigma factor that directed transcription from the sigma B-dependent ctc promoter, but not from sigma E-dependent promoters . These results suggest that amino acid residues at positions 119 and 120 determine, at least in part, the specificity of interactions between sigma E and the nucleotides in the -10 region of its cognate promoters.

J Bacteriol, 1995 Nov, 177(22), 6469 - 76
Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator; Sheehan B et al.; PrfA is a pleiotropic activator of virulence gene expression in the pathogenic bacterium Listeria monocytogenes . Several lines of evidence have suggested that a hierarchy of virulence gene activation by PrfA exists . This hypothesis was investigated by assessing the ability of PrfA to activate the expression of virulence gene fusions to lacZ in Bacillus subtilis . Expression of PrfA in this heterologous host was sufficient for activation of transcription at the hly, plcA, mpl, and actA promoters . Activation was most efficient at the divergently transcribed hly and plcA promoters . The putative PrfA binding site shared by these promoters is perfectly symmetrical and appears to represent the optimum sequence for target gene activation by PrfA . The activation of actA and mpl expression was considerably weaker and occurred more slowly than that observed at the hly and plcA promoters, suggesting that greater quantities of PrfA are required for productive interaction at these promoters . Interestingly, expression of an inlA-lacZ transcriptional fusion was very poorly activated by PrfA in B . subtilis, suggesting that other Listeria factors, in addition to PrfA, are required for PrfA-mediated activation at this promoter . Further support for the involvement of such factors was obtained by constructing and analyzing a prfA deletion mutant of L . monocytogenes . We observed that, in contrast to that of the other genes of the PrfA regulon, expression of inlA is only partially dependent on PrfA.

J Bacteriol, 1995 Nov, 177(22), 6462 - 8
Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK; Yuan G et al.; An inverted repeat sequence known as CIRCE (controlling inverted repeat of chaperone expression) in the Bacillus subtilis groE operon has been suggested to function as an operator . To identify the regulatory gene directly or indirectly involved in CIRCE-mediated heat-inducible groE expression, B . subtilis WBG2, carrying an integrated groE-bgaB transcription fusion in the amyE locus, was mutagenized . Dark blue colonies formed at 37 degrees C represent mutants which constitutively produce BgaB (a thermostable beta-galactosidase) at high levels . Seven mutants (WBG101 to WBG107) were selected for further characterization . They all overproduced BgaB, GroEL, and DnaK simultaneously at 37 degrees C . These mutants could be restored to normal by introducing a plasmid carrying a functional copy of orf39, the first gene in the B . subtilis dnaK operon . Genomic sequencing of these mutants demonstrated that they all carried a single mutation in orf39 . These mutations can be divided into three groups: (i) Gly-307 to Asp, (ii) Ser-122 to Phe, and (iii) Gly-63 to Glu . By using a binary vector system in E . coli, production of ORF39 was found to negatively regulate the expression of groE-bgaB in a CIRCE-specific manner . Under the heat shock condition, the negative regulation mediated by ORF39 was abolished . Mobility shift of the CIRCE-containing probe was also observed with the crude extract prepared from the E . coli strain that overproduced ORF39 . Therefore, ORF39 is the negative regulatory factor which regulates both groE and dnaK expression in B . subtilis . It is likely to function as a CIRCE-specific repressor.

J Bacteriol, 1995 Nov, 177(22), 6362 - 70
trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon; Merino E et al.; Expression of the Bacillus subtilis trpEDCFBA operon has been shown to be regulated by transcription attenuation in response to the availability of L-tryptophan . Regulation is mediated by the tryptophan-activated trp RNA-binding attenuation protein, TRAP, the product of mtrB . Formation of mutually exclusive RNA anti-terminator and terminator structures within trp leader RNA determines whether transcription will terminate in the leader region of the operon . Previous studies suggested that transcripts that escape termination are subject to translational regulation via the formation of a secondary structure that blocks ribosome access to the trpE ribosome-binding site . To assess the relative importance of these postulated events in trp operon regulation, we used site-directed mutagenesis to alter the putative elements involved in transcriptional and translational control . Using a trpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB+ and mtrB strains, during growth in the presence and absence of excess tryptophan . To verify that the altered regulatory regions behaved as expected, we carried out in vitro transcription assays with the wild-type and altered leader region templates and performed oligonucleotide competition assays with an oligonucleotide complementary to a segment of the transcription terminator . Our results establish that binding of TRAP to leader RNA regulates of transcription termination in the trp operon over about an 88-fold range and regulates translation of trpE over about a 13-fold range . The roles played by different trp leader RNA segments in mediating transcriptional and translational regulation are documented by our findings.

J Bacteriol, 1995 Nov, 177(21), 6294 - 6
Immunoelectron microscopic localization of one of the spore germination proteins, GerAB, in Bacillus subtilis spores; Sakae Y et al.; Ultrastructural localization of GerAB, one of the proteins of Bacillus subtilis spores related to L-alanine-initiated germination, was investigated by immunoelectron microscopy with antipeptide (residues 61 to 80 of GerAB) antiserum and a colloidal gold-immunoglobulin G complex . Immunogold particles were visualized in the boundary region between the cortex and coat of dormant spores, and they were broadly dispersed into the cortex region after germination.

J Bacteriol, 1995 Nov, 177(21), 6263 - 75
Adjacent and divergently oriented operons under the control of the sporulation regulatory protein GerE in Bacillus subtilis; Roels S et al.; The DNA-binding protein GerE is the latest-acting regulatory protein in the mother cell line of gene expression during sporulation in Bacillus subtilis . GerE directs the transcription of several genes that encode structural components of the protein coat that encases the mature spore . We report on the identification and characterization of a cluster of additional genes whose transcription is dependent on GerE . These genes, which are located in the replication terminus region of the chromosome (181 degrees on the genetic map), are arranged in adjacent and divergently oriented operons called cgeAB and cgeCDE, which consist of two and at least three genes, respectively . CgeD, the product of the second member of the cgeCDE operon, is strikingly similar to the product of a B . subtilis gene (ipa-63d) of unknown function and is similar at its amino terminus to certain glycosyl transferases involved in polysaccharide biosynthesis . Strains with mutations in the cgeAB and cgeCDE operons produce spores with altered surface properties, on which basis we propose that proteins encoded by these operons influence maturation of the outermost layer of the spore, perhaps by glycosylation of coat proteins at the spore surface.

J Bacteriol, 1995 Nov, 177(21), 6176 - 83
A periplasm in Bacillus subtilis; Merchante R et al.; The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively . Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation . The contents of the protoplast supernatant fraction represent an operational definition of the periplasm . In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts . The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B . subtilis 168 protein considered periplasmic . Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm . We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli . We discuss evidence that the maintenance of the components of this surface compartment in B . subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.

FEBS Lett, 1995 Oct 30, 374(2), 253 - 6
Point mutation of a conserved arginine (104) to lysine introduces hypersensitivity to inhibition by glyphosate in the 5-enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis; Selvapandiyan A et al.; The role of a conserved arginine (R104) in the putative phosphoenol pyruvate binding region of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis has been investigated . Employing site directed mutagenesis arginine was substituted by lysine or glutamine . Native and mutant proteins were expressed and purified to near homogeneity . Estimation of Michaelis and inhibitor constants of the native and mutant proteins exhibited altered substrate-inhibitor binding mode and constants . Mutation R104K hypersensitized the enzyme reaction to inhibition by glyphosate . The role of R104 in discriminating between glyphosate and phosphoenol pyruvate is discussed.

J Biol Chem, 1995 Oct 27, 270(43), 26012 - 9
Characterization of a bifunctional cellulase and its structural gene . The cell gene of Bacillus sp . D04 has exo- and endoglucanase activity; Han SJ et al.; Bacillus sp . D04 secreted a bifunctional cellulase that had a molecular weight of 35,000 . This cellulase degraded Cm-cellulose, cellotetraose, cellopentaose, p-nitrophenyl-beta-D-cellobioside, and avicel PH101 . Based on the high performance liquid chromatography analysis of the degradation products, this cellulase randomly cleaved internal beta-1, 4-glycosidic bonds in cellotetraose and cellopentaose as an endoglucanase . It also hydrolyzed the aglycosidic bond in p-nitrophenyl-beta-D-cellobioside and cleaved avicel to cellobiose as an exoglucanase . Cellobiose competitively inhibited the p-nitrophenyl-beta-D-cellobioside degrading activity but not Cm-cellulose degrading activity . Ten mM p-chloromercuribenzoate inhibited p-nitrophenyl-beta-D-cellobioside degrading activity completely, but Cm-cellulose degrading activity incompletely . Cm-cellulose increased p-nitrophenyl-beta-D-cellobioside degrading activity, and vice versa, whereas methylumbelliferyl-beta-D-cellobiose strongly inhibited p-nitrophenyl-beta-D-cellobioside degrading activity . The cellulase gene (cel gene), 1461 base pairs, of Bacillus sp . D04 was cloned . The nucleotide sequence of the cel gene was highly homologous to those of Bacillus subtilis DLG and B . subtilis BSE616 . The cel gene was overexpressed in Escherichia coli, and its product was purified . The substrate specificity and substrate competition pattern of the purified recombinant cellulase were the same as those of the purified cellulase from Bacillus sp . D04 . These results suggest that a single polypeptide cellulase had both endo- and exoglucanase activities and each activity exists in a separate site.

Science, 1995 Oct 27, 270(5236), 641 - 4
Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division; Duncan L et al.; Cell fate is determined by cell-specific activation of transcription factor sigma F after asymmetric division during sporulation by Bacillus subtilis . The activity of sigma F is governed by SpoIIAA, SpoIIAB, and SpoIIE, a membrane protein localized at the polar septum . SpoIIAB binds to and inhibits sigma F, and SpoIIAA inhibits SpoIIAB, which prevents SpoIIAB from binding to sigma F . SpoIIAB is also a serine kinase that inactivates SpoIIAA . Here, it is demonstrated that SpoIIE dephosphorylates SpoIIAA-P and overcomes SpoIIAB-mediated inhibition of sigma F . The finding that SpoIIE is a serine phosphatase links asymmetric division to the pathway governing cell-specific gene transcription.

Science, 1995 Oct 27, 270(5236), 637 - 40
Localization of protein implicated in establishment of cell type to sites of asymmetric division; Arigoni F et al.; Asymmetric division in Bacillus subtilis generates progeny cells with dissimilar fates . SpoIIE, a membrane protein required for the establishment of cell type, was shown to localize near sites of potential polar division . SpoIIE initially localizes in a bipolar pattern, coalescing at marks in the cell envelope at which asymmetric division can take place . Then, during division, SpoIIE becomes restricted to the polar septum and is lost from the distal pole . Thus, when division is complete, SpoIIE sits at the boundary between the progeny from which it dictates cell fate by the activation of a cell-specific transcription factor.

Gene, 1995 Oct 16, 164(1), 185 - 6
Construction of a recombinant cellulolytic Escherichia coli; Srivastava R et al.; A 1.2-kb DNA fragment from Bacillus subtilis CD4 encoding endo-beta-1,4-glucanase and cellobiase activities was cloned and expressed in Escherichia coli . Carboxymethylcellulase (CMCase) and cellobiase activities were detected in a cell-free lysate of recombinant (re-) E . coli (Ec) . The re-enzymes were functional in Ec, as it utilized carboxymethylcellulose, soluble cellulose and cellobiose as sole carbon sources for growth.

Gene, 1995 Oct 16, 164(1), 113 - 6
The Bacillus subtilis cell-division 135-137 degrees region contains an essential orf with significant similarity to murB and a dispensable sbp gene; Rowland SL et al.; Sequence similarity analysis has revealed that orf2, in the cell division 135-137 degrees region of the Bacillus subtilis (Bs) chromosome, is the probable homolog of Escherichia coli murB (encoding a reductase involved in peptidoglycan synthesis) . The amino-acid sequences of the two protein products show 24% identity (47% overall similarity), with several regions of higher similarity which may represent functional domains of the proteins . Attempts to insertionally inactivate orf2 were unsuccessful, strongly suggesting that it is an essential Bs gene . A small gene found in the same region as orf2, sbp (encoding the 'small basic protein'), was shown to be non-essential in Bs.

J Mol Biol, 1995 Oct 13, 253(1), 8 - 16
Sequence-specific interactions between promoter DNA and the RNA polymerase sigma factor E; Tatti KM et al.; In order to determine which amino acyl residues in a secondary sigma factor govern its specificity of recognition at the -35 region of promoters, we examined the effects of amino acid substitutions in sigma E in Bacillus subtilis that made the sequence of its putative -35 recognition region more similar to another sigma factor in B . subtilis, sigma K . We found that a single amino acid substitution at position 217 of sigma E resulted in a sigma factor that could direct transcription from sigma K-dependent promoters . Furthermore, we tested whether this amino acid substitution in sigma E had changed the specificity of interactions of the sigma with -35 region sequences by examining the activity of the mutant sigma E on derivatives of sigma E-dependent promoters that contained single base-pair substitutions . We found that this substitution in sigma E specifically suppressed the effect of a single base-pair substitution at position -31 in a sigma E-dependent promoter spoIIID . The amino acyl residue at another position (219) on sigma E affected the specificity of interaction with position -33 in spoIIID promoter . The amino acyl residues at the two positions in sigma E, 217 and 219, that determine the specificity of interactions between the sigma and base-pairs in the -35 region of its cognate promoters (positions -33 and -31, respectively, in the spoIIID promoter) probably closely contact these base-pairs.

J Mol Biol, 1995 Oct 13, 253(1), 151 - 67
Studies on the lumazine synthase/riboflavin synthase complex of Bacillus subtilis: crystal structure analysis of reconstituted, icosahedral beta-subunit capsids with bound substrate analogue inhibitor at 2.4 A resolution; Ritsert K et al.; The lumazine synthase/riboflavin synthase of Bacillus subtilis is a bifunctional enzyme complex catalysing the formation of riboflavin from 5-amino-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and L-3,4-dihydroxy-2-butanone-4-phosphate via 6,7-dimethyl-8-ribityllumazine . The complex is composed of 3 alpha (riboflavin synthase) subunits and 60 beta (lumazine synthase) subunits and has a relative mass of 1 MDa . The 60 beta subunits are arranged in an icosahedral capsid enclosing the alpha trimer in the central core . The protein was crystallised, and an X-ray structure of the icosahedral capsid was obtained at 3.3 A resolution with a crystallographic R-factor of 0.33 . Hollow, icosahedral capsids consisting of 60 beta subunits can be obtained by inhibitor-driven renaturation of isolated beta subunits . They catalyse the formation of 6,7-dimethyl-8-ribityllumazine at the same rate as the native alpha 3 beta 60 complex and can be crystallised in two different hexagonal and one monoclinic form . Crystallographic intensity data of the monoclinic crystals to a resolution of 2.4 A were obtained using synchrotron radiation and an image plate detector system . The orientation of the icosahedral molecules in the monoclinic cell was deduced by real space vector search procedures from a 3.5 A Patterson map . Phases were calculated from the model of the alpha 3 beta 60 protein and were extended by cyclic averaging exploring the 30-fold redundancy of the electron density . The 2.4 A map allowed us to refine the existing atomic model of lumazine synthase . The refined model includes 154 amino acid residues, one inhibitor molecule, 58 water molecules and one phosphate ion . Applying non-crystallographic-symmetry restraints the crystallographic R-factor is 16.7% for 100,092 reflections between 10 and 2.4 A . The chain folding of the beta subunits is closely similar to the native alpha 3 beta 60 enzyme . The lumazine synthase bears resemblance to the sugar binding proteins . The significantly higher resolution compared to the alpha 3 beta 60 structure determination allows a detailed description of the substrate analogue binding site . The environment of the 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione inhibitor is particularly rigid, and the chain segments involved in forming the active site are highly conserved for lumazine synthases of different species . A residual density feature in the final map is interpreted as a bound phosphate which mimics the binding of the second substrate . We discuss the reaction mechanism on this structural basis.

J Biol Chem, 1995 Oct 6, 270(40), 23533 - 9
Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties; Chakicherla A et al.; Biosynthesis of lantibiotics such as nisin and subtilin involves post-translational modifications, including dehydration of serines and threonines, formation of thioether cross-linkages, translocation, cleavage of a leader sequence, and release into the medium . We have studied the cellular machinery that performs the modifications by constructing and expressing nisin-subtilin chimeric prepeptides in a strain of Bacillus subtilis 168 that possesses all of the cellular machinery for making subtilin except for the presubtilin gene . The chimeras consisted of a normal subtilin leader region (SL), fused to nisin-subtilin chimeric structural regions, one of which was SL-Nis1-11-Sub12-32, in which the N-terminal portion of the structural region was derived from nisin, and the C-terminal portion derived from subtilin . This chimera was accurately and efficiently converted to the corresponding mature lantibiotic, as established by reverse phase high performance liquid chromatography profiles, proton NMR spectroscopy, mass spectral analysis, and biological activity . A succinylated form of the chimera was also produced . Another chimera was in the reverse sense, with subtilin sequence at the N terminus and nisin sequence at the C terminus of the structural region (SL-Sub1-11-Nis12-34) . It was processed into a heterogeneous mixture of products, none of which had the characteristics of a correctly processed polypeptide, but did contain a minor component that was active, with a specific activity that considerably exceeded nisin itself . These results, together with results published earlier, establish that processing requires specific recognition between the prelantibiotic peptide and the processing machinery, and in order for the processing to occur correctly, there must be an appropriate combination of the N-terminal part of the leader region and the C-terminal part of the structural region of the prepeptide.

Mol Microbiol, 1995 Oct, 18(2), 295 - 300
Bacillus subtilis MrgA is a Dps(PexB) homologue: evidence for metalloregulation of an oxidative-stress gene; Chen L et al.; Upon the cessation of exponential growth, Bacillus subtilis enters a transition phase leading to either sporulation or a non-sporulating stationary phase . During this transition period, cells secrete degradative enzymes, become competent for DNA transformation, are motile and acquire resistance to oxidative killing . We now report that mrgA, originally identified as a gene repressed by metal ions, encodes a member of the Dps/PexB family of general stress proteins . Like Escherichia coli Dps(PexB), MrgA forms highly stable, multimeric protein-DNA complexes which accumulate in stationary-phase cells and protect against oxidative killing . MrgA is part of an inducible oxidative stress response in B . subtilis: mrgA is induced by hydrogen peroxide, and a strain lacking MrgA displays increased sensitivity to oxidative killing . In addition, a hydrogen peroxide-resistant mutant, which constitutively overproduces catalase and alkyl hydroperoxide reductase, also overproduces MrgA . These results indicate a complex interplay between metal ions and the expression of the B . subtilis oxidative stress response.

Mol Microbiol, 1995 Oct, 18(2), 271 - 82
pAM beta 1 resolvase has an atypical recombination site and requires a histone-like protein HU; Petit MA et al.; The broad-host-range plasmid pAM beta 1 from Gram-positive bacteria encodes a resolvase, designated Res beta, which shares homology with the proteins of the resolvase-invertase family . Here we report the purification and in vitro characterization of Res beta . This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro . Res beta binds to two regions within its resolution site res . One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3) . The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E . coli histone-like protein HU . The possible mode of action of HU in the resolution process is discussed.

Mol Microbiol, 1995 Oct, 18(1), 1 - 12
Regulation of insecticidal crystal protein production in Bacillus thuringiensis; Baum JA et al.; The production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell . In most instances, the temporal and spatial regulation of ICP gene expression is determined at the transcriptional level by mother-cell-specific sigma factors that share homology with sigma E and sigma K from Bacillus subtilis . The cryIII ICP genes are a notable exception; these genes are transcribed from sigma A-like promoters during vegetative growth, are induced or derepressed at the onset of stationary phase, and are overexpressed in sporulation mutants of B . thuringiensis blocked in the phosphorylation of Spo0A, a key regulator of sporulation initiation . Transcription alone, however, cannot account for the impressive ability of this bacterium to accumulate insecticidal proteins . A variety of post-transcriptional and post-translational mechanisms also contribute to the efficient production of ICPs in B . thuringiensis, thus making this bacterium a cost-effective biological control agent.

Res Microbiol, 1995 Oct, 146(8), 643 - 57
Phage typing of Bacillus subtilis and B . thuringiensis; Ackermann HW et al.; Phage typing schemes for Bacillus subtilis and B . thuringiensis were constructed using 98 phages and 743 bacterial strains . Most phages were host-species-specific . Phages were classified by electron microscopy . The B . subtilis scheme includes 10 phages and 29 phage types . The B . thuringiensis scheme comprises 8 phages and 25 phage types and can be applied to B . cereus . There is no correlation between H antigen serotypes and phagovars in B . thuringiensis . Characteristics of typing phages are described for identity control.

IEEE Trans Biomed Eng, 1995 Oct, 42(10), 1038 - 43
Polarized light scattering: a biophysical method for studying bacterial cells; Diaspro A et al.; We outline a method which uses differentially polarized light scattering to study the properties of bacterial cell suspensions, i.e., spores of Bacillus Subtilis . The identification of bacterial cells of different strains, with and without plasmid insertion, was performed by means of differential polarization light scattering (DPLS) . The samples displayed distinct angular behaviors for the S14 and S34 normalized scattering parameters of the Mueller matrix depending on the strain and on a plasmid insertion in the chromosomal unit . These experiments, performed blindly, point out the possibility of achieving real time identification of micro-organisms by DPLS spectrometry.

Eur J Cell Biol, 1995 Oct, 68(2), 167 - 82
Phagocytic processing of the macrophage endoparasite, Mycobacterium avium, in comparison to phagosomes which contain Bacillus subtilis or latex beads; de Chastellier C et al.; The intraphagosomal survival strategy of pathogenic mycobacteria was studied in bone marrow-derived mouse macrophages . These bacteria survive inside phagosomes by interfering in an unknown manner with phagosome processing which normally would lead to digestion of the phagocytic particle in phagolysosomes . Here, phagosome processing was compared for different phagocytic particles: live Mycobacterium avium, degradable Bacillus subtilis, or indigestible latex beads . We show detailed electron microscopic morphological observations which characterize various phases of interaction between endocytic organelles and phagosomes . We measured fusion of phagosomes with early endosomes or with lysosomes by using newly internalized endocytic contents (horseradish peroxidase, HRP) and membrane marker (plasma membrane glycoconjugates labeled with {3H}galactose via exoglycosylation) . Morphometric analysis of these observations showed that the nature of the phagocytic particle affects phagosome processing: As long as particles remain undigested, maturation of phagosomes is prevented and they remain fusogenic towards early endosomes; concurrent to particle digestion, phagosome processing proceeds towards transfer of phagocytic contents to phagolysosomes which display kinetic and compositional characteristics of lysosomes . As an intact phagocytic particle, M . avium remains in non-matured phagosomes which fuse with early endosomes, but not with lysosomes . Fusion with early endosomes is reduced, thereby indicating the stage where this endoparasite exerts its effect.

Protein Expr Purif, 1995 Oct, 6(5), 679 - 84
Overexpression and purification of the trimeric aspartate transcarbamoylase from Bacillus subtilis; Baker DP et al.; A procedure has been developed for the overexpression and purification of milligram quantities of the Bacillus subtilis aspartate transcarbamoylase . The plasmid pEK171, carrying the B . subtilis pyrB structural gene under the control of the Escherichia coli pyrBI promoter, was transformed into the E . coli strain EK1104 and the enzyme overexpressed to approximately 50% of total soluble protein under extreme derepression of the pyrimidine pathway . The enzyme was subsequently purified by means of ammonium sulfate fractionation, anionic exchange chromatography using Q-Sepharose Fast Flow resin, negative chromatography on Matrex Gel Red A agarose, and hydrophobic interaction chromatography using Matrex Phenyl Cellufine . The purification yields approximately 60 mg of pure enzyme per liter of bacterial culture . Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties very similar to those of the enzyme purified from B . subtilis cells.

J Bacteriol, 1995 Oct, 177(20), 5906 - 11
Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis; Webb CD et al.; We report the use of the green fluorescent protein (GFP) of Aequorea victoria to visualize cell-specific gene expression and protein subcellular localization during sporulation in Bacillus subtilis . Sporangia bearing the gene (gfp) for the green fluorescent protein fused to genes under the control of the sporulation transcription factor sigma F exhibited a forespore-specific pattern of fluorescence . Forespore-specific fluorescence could be detected with fusions to promoters that are utilized with low (csfB) and high (sspE-2G) efficiency by sigma F-containing RNA polymerase . Conversely, a mother cell-specific pattern of fluorescence was observed in sporangia bearing a transcriptional fusion of gfp to a spore coat protein gene (cotE) under the control of sigma E and an in-frame fusion to a regulatory gene (gerE) under the control of sigma K . An in-frame fusion of gfp to cotE demonstrated that GFP can also be used to visualize protein subcellular localization . In sporangia producing the CotE-GFP fusion protein, fluorescence was found to localize around the developing spore, and this localization was dependent upon SpoIVA, a morphogenetic protein known to determine proper localization of CotE.

J Bacteriol, 1995 Oct, 177(20), 5899 - 905
Synthesis of sn-glycerol 3-phosphate, a key precursor of membrane lipids, in Bacillus subtilis; Morbidoni HR et al.; The Bacillus subtilis gpsA gene was cloned by complementation of an Escherichia coli gpsA strain auxotrophic for sn-glycerol 3-phosphate . The gene was sequenced and found to encode an NAD(P)H-dependent dihydroxyacetone phosphate reductase with a deduced molecular mass of 39.5 kDa . The deduced amino acid sequence showed strong conservation with that of the E . coli homolog and to other procaryotic and eucaryotic dihydroxyacetone phosphate reductases . The physical location of gpsA on the B . subtilis chromosome was at about 200 degrees . Disruption of the chromosomal gpsA gene yielded B . subtilis strains auxotrophic for glycerol, indicating that the gpsA gene product is responsible for synthesis of the sn-glycerol 3-phosphate required for phospholipid synthesis . We also found that transformation of the classical B . subtilis glycerol auxotrophs with a gpsA-containing genomic fragment yielded transformants that grew in the absence of glycerol . In agreement with prior work, our attempts to determine the reductase activity in B . subtilis extracts were unsuccessful . However, expression of the B . subtilis gpsA gene in E . coli gave reductase activity that was only slightly inhibited by sn-glycerol 3-phosphate . Since the E . coli GpsA dihydroxyacetone phosphate reductase is very sensitive to allosteric inhibition by sn-glycerol 3-phosphate, these results indicate that the B . subtilis gpsA-encoded reductase differs from that of E . coli . It seems that B . subtilis regulates sn-glycerol 3-phosphate synthesis at the level of gene expression rather than through the E . coli mechanism of strong allosteric inhibition of an enzyme produced in excess.

J Bacteriol, 1995 Oct, 177(20), 5756 - 61
Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters; Nickerson CA et al.; The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters . These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL . The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat . In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay . Hybrid promoters derived from lambda pR that contained curved DNA were bound by E . coli RNA polymerase more efficiently than was the original lambda pR . Binding of E . coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis . Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E . coli RNA polymerase binding than was the original lambda pL . The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E . coli RNA polymerase in a tight, nucleosomal-like fashion . The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.

J Appl Bacteriol, 1995 Oct, 79(4), 470 - 4
A note on the development of a non-aldehyde fixation technique for examining spores of Bacillus subtilis under the electron microscope; Knott AG et al.; Various techniques were studied for fixing spores of Bacillus subtilis prior to examining them by transmission or scanning electron microscopy . A non-aldehyde technique employing carbodiimide in cacodylate buffer produced excellent results and could be of value in studying the cytological changes produced in spores exposed to inimical treatments.

Comp Biochem Physiol B Biochem Mol Biol, 1995 Oct, 112(2), 287 - 93
2,3-Bisphosphoglycerate-independent phosphoglycerate mutase is conserved among different phylogenic kingdoms; Grana X et al.; We have previously demonstrated that maize (Zea mays) 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM-i) is not related to 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase . With the aid of specific anti-maize PGAM-i antibodies, we demonstrate here the presence of a closely related PGAM-i in other plants . We also describe the isolation and sequencing of a cDNA-encoding almond (Prunus amygdalus) PGAM-i that further demonstrates this relationship among plant PGAM-i . A search of the major databases for related sequences allowed us to identify some novel PGAM-i from different sources: plants (Arabidopsis thaliana, Oryza sativa and Antithamniom sp.), monera (Escherichia coli, Bacillus subtilis and Bacillus megaterium) and animals (Caenorhabditis elegans) . All of these amino acid sequences share a high degree of homology with plant PGAM-i . These observations suggest that the PGAM-i from several biological kingdoms constitute a family of protein different from other proteins with related enzymatic function and arose from a common ancestral gene that has diverged throughout its evolution.

Microbiology, 1995 Oct, 141 ( Pt 10), 2391 - 404
Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization; Rashid MH et al.; The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of Bacillus subtilis AC327, and then the corresponding gene was cloned into Escherichia coli in two inactive forms by standard procedures . Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated lytD = cwIG) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide . Moreover, another monocistronic operon (designated pmi = orfX), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene . Expression of a lytD-lacZ fusion gene, driven by lytD regulatory sequences, was observed during the exponential growth phase . The introduction of a sigD null mutation greatly reduced (by about 95%) the expression of the fusion . Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain . Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity . Processing of these proteins occurred between the N-terminal cell-wall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase . Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls . The lytD gene product, in either an intact or a truncated form, was found to be lethal for E . coli, and the N-terminally truncated glucosaminidase proteins, produced in E . coli, were very unstable . The partially purified glucosaminidase from B . subtilis was found to be very unstable at low ionic strength at 37 degrees C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.

Microbiology, 1995 Oct, 141 ( Pt 10), 2379 - 89
In Bacillus subtilis 168, teichoic acid of the cross-wall may be different from that of the cylinder: a hypothesis based on transcription analysis of tag genes; Mauel C et al.; Five of the genes known to encode the enzymes for the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons, tagAB and tagDEF . lacZ and gus transcriptional fusions to the first genes of these operons revealed that: (i) in media of different richness, higher growth rates were paralleled by lower transcription levels; (ii) upon transition to stationary phase, the transcription per unit mass of both operons increased abruptly by a factor of about two; and (iii) a rise in temperature was accompanied by decreased transcription of tagA and increased transcription of tagD . Mapping of transcription start points revealed two divergent sigma A-controlled promoters . Although tagD and the neighbouring downstream gene tagE are transcribed from the same promoter, the latter was expressed at a much lower level than the former . Moreover, expression of tagE, and of the translationally coupled tagF, did not increase at the onset of the stationary phase, indicating that additional regulatory signals may act in the intergenic tagD-tagE region . Optimal transcription of these operons appears to require the entire regulatory region, suggesting that tag gene expression may, among other factors, be regulated by the three-dimensional configuration of this segment . The biological implications of these results are discussed.

Curr Opin Biotechnol, 1995 Oct, 6(5), 517 - 22
Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins; Wong SL; During the past year, significant progress has been made using Bacillus subtilis to produce a wide range of foreign proteins . Through strain improvement and co-expression of molecular chaperones, secretory proteins can be produced at a higher level . Through protein engineering, target proteins can be redesigned to have better stability and solubility . A combination of these two strategies would be a useful approach to produce heterologous proteins from B . subtilis at high quality and with a high yield.

Plant Mol Biol, 1995 Oct, 29(1), 173 - 8
Characterisation of HEVER, a novel stress-induced gene from Hevea brasiliensis; Sivasubramaniam S et al.; A novel stress-induced gene, HEVER (Hevea ethylene-responsive) from the rubber tree, Hevea brasiliensis, has been isolated and characterised . HEVER is encoded by a multigene family . The HEVER transcript is expressed at basal levels in Hevea tissues and is developmentally regulated . In addition, the HEVER transcript and protein are induced by stress treatment with salicylic acid and ethephon . Sequence analysis shows that HEVER encodes a 33 kDa protein that has significant homology to the hypothetical protein SLEXORFA-1 from the plant, Stellaria longipes, and two bacterial proteins, BAC180K-75 from Bacillus subtilis and MVRNO3-1 from Methanococcus vannielii.

Br J Nutr, 1995 Oct, 74(4), 523 - 9
Effect of dried Bacillus subtilis culture on growth, body composition and hepatic lipogenic enzyme activity in female broiler chicks; Santoso U et al.; To investigate the effect of dried Bacillus subtilis culture on growth, body composition and hepatic lipogenic enzyme activity, female broiler chicks were fed on either no additive (control) or dried B . subtilis-culture-supplemented commercial diets (215 g crude protein/kg, 12.85 MJ metabolizable energy/kg) at 10 or 20 g/kg diet for 28 d from 14 to 42 d of age . Body weight, and moisture, fat, protein and ash contents of the body were not influenced by the B . subtilis culture . Feed efficiency, N utilization, the ratio of abdominal fat or liver to body weight, acetyl-coenzyme A carboxylase (EC 6.4.1.2) activity, liver and serum cholesterol contents were significantly lower in treatment groups, while fatty acid synthetase activity and serum cholesterol concentration were not significantly different, compared with the control group . Liver triacylglycerol concentration was decreased in chicks given 20 g culture/kg diet, while serum and carcass triacylglycerol concentrations were significantly lower in treatment groups than in the control group . Serum phospholipid concentration was increased but carcass phospholipid concentration was decreased in chicks given 20 g B . subtilis/kg diet, while liver phospholipid concentration was not significantly influenced . The advantages of inclusion of B . subtilis to the broiler diet included improved feed efficiency, less abdominal fat, reduced triacylglycerol concentrations in the liver, serum and carcass and reduced cholesterol concentrations in the liver and carcass.

Am Ind Hyg Assoc J, 1995 Oct, 56(10), 979 - 86
Factors affecting microbiological colony count accuracy for bioaerosol sampling and analysis; Chang CW et al.; The effects of the following variables on the occurrence of colony masking (the indistinguishable merging or overlap of sufficiently close colonies) were evaluated experimentally using the bacterium Bacillus subtilis: spore density on a collection surface, concentration of nutrients in the culture medium, sample incubation time, and ability of an observation system to distinguish overlapped colonies . Increasing spore surface density and incubation time increased colony masking, whereas lowering nutrient concentration decreased colony diameter and, therefore, masking but also limited spore germination and growth . Overall, full-strength medium was best for accurate counting of early microcolonies examined with the aid of a microscope, whereas half- or quarter-strength medium was better for counting older readily observable macrocolonies . Masking bias was determined for varying spore surface densities and colony diameters and was applied to two widely used slit-to-agar bioaerosol impactors . Appropriate collection times have been determined for these samplers to minimize colony masking for expected bioaerosol concentrations . It was found, for example, that 6-min samples collected from an environment with an air concentration of 10(3) CFU m-3 would result in colony surface densities, for 3-mm colonies, of 1.5 and 3.9 microorganisms cm-2 for the two samplers with respective masking biases of < 10% and < 20%.

J Bacteriol, 1995 Oct, 177(19), 5711 - 5
Replication through the terminus region of the Bacillus subtilis chromosome is not essential for the formation of a division septum that partitions the DNA; Wu LJ et al.; Germinated and outgrowing spores of a temperature-sensitive DNA initiation mutant of Bacillus subtilis were allowed to initiate a single round of replication by being shifted from 34 to 47 degrees C at the appropriate time . The DNA replication inhibitor 6-(parahydroxyphenylazo)-uracil was added to separate portions of the culture at various times during the round . Samples were collected from each around the time of the first division septation for measurements of the extent of the round completed, the level of division septation, the position of the septum within the outgrown cell, and the distribution of DNA (nucleoid) in relation to the septum . The extent of replication was measured directly through a hybridization approach . The results show clearly that a central division septum can close down onto a chromosome that is only partially replicated (to a minimum extent of about 60% of the round) such that DNA appears on both sides of the septum and frequently very close to it . It is concluded, as claimed previously on the basis of a less direct approach (T . McGinness and R.G . Wake, J . Mol . Biol . 134:251-264, 1979), that replication through the terminus region of the chromosome is not essential for the formation of a division septum that partitions the DNA.

J Bacteriol, 1995 Oct, 177(19), 5696 - 700
Mutations in GltC that increase Bacillus subtilis gltA expression; Belitsky BR et al.; Mutants with altered forms of GltC, a positive LysR-type regulator of Bacillus subtilis glutamate synthase gene expression, were isolated . The mutant GltC proteins stimulated expression from the wild-type gltA promoter region 1.5- to 2.0-fold and from mutant promoter regions up to 80-fold . Moreover, expression of gltA became much less dependent on a nitrogen source-associated signal.

J Bacteriol, 1995 Oct, 177(19), 5686 - 95
Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression; Belitsky BR et al.; The Bacillus subtilis gltAB genes, coding for the two subunits of glutamate synthase, are transcribed divergently from the gltC gene, encoding a LysR-type transcriptional activator of gltAB . The predicted gltA and gltC transcription start sites are separated by 51 to 52 bp . A 15-bp, consensus binding site (Box I) for LysR-type proteins was found centered at position -64 with respect to the gltA transcription start . This site was shown by mutational analysis to be required both for GltC-mediated activation of gltA and for autorepression of gltC . Box II, which is similar to Box I, is centered 22 bp downstream of Box I and overlaps the -35 region of the gltA promoter . Box II was found to be essential for activation of gltA but not for gltC autoregulation . Introduction of approximately one additional helical turn of DNA between Box I and Box II enhanced gltA expression 7- to 40-fold under nonactivating conditions and about 2-fold under activating conditions . Expression of gltA was dramatically decreased when the distance between Box I and Box II was altered by a nonintegral number of helical turns of DNA . gltC autorepression was abolished by most of the inserts between Box I and Box II but was augmented by adding one helical turn.

J Bacteriol, 1995 Oct, 177(19), 5628 - 35
Identification and characterization of sporulation gene spoVS from Bacillus subtilis; Resnekov O et al.; We report the identification and characterization of an additional sporulation gene from Bacillus subtilis called spoVS, which is induced early in sporulation under the control of sigma H . We show that spoVS is an 86-codon-long open reading frame and is capable of encoding a protein of 8,796 Da which exhibits little similarity to other proteins in the databases . Null mutations in spoVS have two contrasting phenotypes . In otherwise wild-type cells they block sporulation at stage V, impairing the development of heat resistance and coat assembly . However, the presence of a spoVS mutation in a spoIIB spoVG double mutant (which is blocked at the stage {II} of polar septation) acts as a partial suppressor, allowing sporulation to advance to a late stage . The implications of the contrasting phenotypes are discussed in the context of the formation and maturation of the polar septum.

J Bacteriol, 1995 Oct, 177(19), 5598 - 605
Cloning, nucleotide sequence, and regulation of katE encoding a sigma B-dependent catalase in Bacillus subtilis; Engelmann S et al.; A sigma B-dependent stress gene of Bacillus subtilis was localized downstream of the licS gene . The predicted amino acid sequence exhibited a significant similarity to the sequence of the katE-encoded catalase HPII of Escherichia coli, and we designated it the open reading frame katE . In a B . subtilis katE mutant, catalase 2 could not be detected . The amount of katE-specific mRNA was increased after heat, salt, or ethanol stress or after glucose starvation in a sigma B-dependent manner . As in E . coli, the transcription of the katE gene in B . subtilis was unaffected by the addition of H2O2 to exponentially growing cells . In contrast, the katA gene encoding catalase 1 of B . subtilis showed an induction pattern different from that of katE; katA expression was strongly increased by oxidative stress . The similarity between E . coli sigma S-dependent genes and B . subtilis sigma B-dependent genes suggests that both may confer multiple stress resistance to stationary-phase cells.

J Bacteriol, 1995 Oct, 177(19), 5590 - 7
Regulation of the putative bglPH operon for aryl-beta-glucoside utilization in Bacillus subtilis; Kruger S et al.; The expression of the putative operon bglPH of Bacillus subtilis was studied by using bglP'-lacZ transcriptional fusions . The bglP gene encodes an aryl-beta-glucoside-specific enzyme II of the phosphoenolpyruvate sugar:phosphotransferase system, whereas the bglH gene product functions as a phospho-beta-glucosidase . Expression of bglPH is regulated by at least two different mechanisms: (i) carbon catabolite repression and (ii) induction via an antitermination mechanism . Distinct deletions of the promoter region were created to determine cis-acting sites for regulation . An operatorlike structure partially overlapping the -35 box of the promoter of bglP appears to be the catabolite-responsive element of this operon . The motif is similar to that of amyO and shows no mismatches with respect to the consensus sequence established as the target of carbon catabolite repression in B . subtilis . Catabolite repression is abolished in both ccpA and ptsH1 mutants . The target of the induction by the substrate, salicin or arbutin, is a transcriptional terminator located downstream from the promoter of bglP . This structure is very similar to that of transcriptional terminators which regulate the induction of the B . subtilis sacB gene, the sacPA operon, and the Escherichia coli bgl operon . The licT gene product, a member of the BglG-SacY family of antitermination proteins, is essential for the induction process . Expression of bglP is under the negative control of its own gene product . The general proteins of the phosphoenolpyruvate-dependent phosphotransferase system are required for bglP expression . Furthermore, the region upstream from bglP, which reveals a high AT content, exerts a negative regulatory effect on bglP expression.

J Bacteriol, 1995 Oct, 177(19), 5582 - 9
Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis . ; Sekiguchi J et al.; DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively . The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B . subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB) . The gene was tentatively named cwlD . Bright spores produced by a B . subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild-type strain were observed . However, degradation of the cortex was completely blocked . Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8) . The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme . Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G-dependent promoter upstream of the orf1 gene . The cwlD mutant harboring a B . subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level.

J Bacteriol, 1995 Oct, 177(19), 5467 - 72
Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp . may contribute to regulation of xylose utilization; Dahl MK et al.; The xyl operons of several gram-positive bacteria are regulated at the level of transcription by xylose-responsive repressor proteins (XylR) . In addition, they are catabolite repressed . Here, we describe a mechanism by which glucose metabolism can affect both regulatory mechanisms . Glucose-6-phosphate appeared to be an anti-inducer of xyl operon transcription, since it could compete with xylose in interaction in vitro with XylR from Bacillus subtilis, B . megaterium, and B . licheniformis . On the other hand, glucose was a low-efficiency inactivator of XylR from B . subtilis and B . megaterium and a weak anti-inducer of XylR from B . licheniformis . Thus, the chemical nature of the substituent at C-5 of xylose and the primary structure of XylR determine the effect of these compounds on xyl operon transcription.

J Bacteriol, 1995 Oct, 177(19), 5427 - 33
Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE); Yuan G et al.; To study the regulatory mechanism controlling the heat-inducible expression of Bacillus subtilis groE, two regulatory elements, the sigma A-like promoter and the inverted repeat (IR {CIRCE}) in the control region, were characterized . The groE promoter was shown to be transcribed by the major RNA polymerase under both heat shock and non-heat shock conditions . The IR was found to have two functions . (i) It ensures the fast turnover of the groE transcript, and (ii) it serves as an operator . This IR acts as a negative heat shock regulatory element, since deletion of this sequence resulted in high-level expression of groE even at 37 degrees C . Although this IR is present in the 5' untranslated region of the groE transcript, groE transcripts under heat shock and non-heat shock conditions showed similar in vivo half-lives of 5 min . This rapid turnover at 37 degrees C requires the presence of the IR . Without the IR, the groE transcript showed a longer half-life of 17 min . Increasing the distance between the groE transcription start site and the IR systematically by inserting nucleotide sequences from 5 to 21 bp in length resulted in a gradual abolition of the negative regulatory effect mediated by the IR . This effect was not due to a significant change in transcript stability or the transcription start site and is consistent with the model that this IR serves as an operator.

J Antibiot (Tokyo), 1995 Oct, 48(10), 1159 - 64
Structure-activity relationship of pamamycins: effects of alkyl substituents; Natsume M et al.; Nine new pamamycin homologues were isolated from the culture broth of Streptomyces alboniger IFO 12738 using a combination of ODS and NH2 HPLCs, and their structures determined by GC-MS . The structural differences in these homologues are in the numbers and positions of methyl and ethyl groups . The aerial mycelium-inducing and growth-inhibitory activities in S . alboniger of these homologues and their antibiotic activity against Bacillus subtilis were examined . The effects of the alkyl substituents on these activities are discussed.

J Antibiot (Tokyo), 1995 Oct, 48(10), 1095 - 103
Bacillopeptins, new cyclic lipopeptide antibiotics from Bacillus subtilis FR-2; Kajimura Y et al.; Bacilopeptins, new iturin-group antifungal antibiotics, were isolated from the culture broth of Bacillus subtilis FR-2 obtained from the rhizosphere of garlic suffering from the basal rot caused by Fusarium oxysporum . Their structures were elucidated to be cyclic lipopeptides similar to bacillomycin L by NMR and mass spectral studies coupled with amino acid analysis . The absolute configuration of each amino acid residue was determined by chiral HPLC.

Appl Environ Microbiol, 1995 Oct, 61(10), 3633 - 8
Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins; Popham DL et al.; Spores of a Bacillus subtilis strain with an insertion mutation in the dacB gene, which codes for an enzyme involved in spore cortex biosynthesis, have a higher core water content than wild-type spores . Spores lacking the two major alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha-beta- spores) have the same core water content as do wild-type spores, but alpha-beta- dacB spores had more core water than did dacB spores . The resistance of alpha-beta-, alpha-beta- dacB, dacB, and wild-type spores to dry and moist heat, hydrogen peroxide, and UV radiation has been determined, as has the role of DNA damage in spore killing by moist heat and hydrogen peroxide . These data (i) suggest that core water content has little if any role in spore UV resistance and are consistent with binding of alpha/beta-type SASP to DNA being the major mechanism providing protection to spores from UV radiation; (ii) suggest that binding of alpha/beta-type SASP to DNA is the major mechanism unique to spores providing protection from dry heat; (iii) suggest that spore resistance to moist heat and hydrogen peroxide is affected to a large degree by the core water content, as increased core water resulted in large decreases in spore resistance to these agents; and (iv) indicate that since this decreased resistance (i.e., in dacB spores) is not associated with increased spore killing by DNA damage, spore DNA must normally be extremely well protected against such damage, presumably by the saturation of spore DNA by alpha/beta-type SASP.

J Mol Biol, 1995 Sep 29, 252(4), 386 - 98
The small subunit of the terminase enzyme of Bacillus subtilis bacteriophage SPP1 forms a specialized nucleoprotein complex with the packaging initiation region; Chai S et al.; Initiation of SPP1 DNA packaging requires the gene 1 and gene 2 products (G1P and G2P), which are different subunits of the terminase enzyme . G1P specifically recognizes the phage packaging initiation region (pac) . The apparent equilibrium constant for the G1P-pac-DNA complex was estimated to be 9 nM . DNase I footprinting experiments reveal that the pac region can be subdivided into three discrete sites (pacL, pacC and pacR) . G1P binds co-operatively to the non-adjacent pacL and pacR sites . Several G1P protomers bind to the target sequences which map close to the pac cleavage site (pacC site), but do not overlap with it . G1P interacts in a different fashion with the encapsidated (pacR site) and with the non-encapsidated (pacL site) end of the phage genome . G1P interaction with the intrinsically bent pacL DNA occurs only on one face of the DNA double helix . G1P binding to the pacL and in the pacR region results in a DNA loop . Electron microscopy of purified G1P shows that the protein is an oligomer in solution . G1P binding to the core region of the pacL site could facilitate the formation of a higher-order nucleoprotein structure . This specialized complex would allow the pac DNA to form a loop between binding sites brought together by interaction with G1P . The results presented here suggest that G1P could provide a tool to discriminate the first encapsidated end, which contains pacR, from the non-encapsidated pacL end.

Gene, 1995 Sep 22, 163(1), 69 - 74
Construction of gusA transcriptional fusion vectors for Bacillus subtilis and their utilization for studies of spore formation; Karow ML et al.; A series of gusA transcriptional fusion vectors is described for Bacillus subtilis (Bs) . The series includes a vector for use with the amyE system of Shimotsu and Henner {Gene 43 (1986) 85-94}, an integrative vector and vectors that provide gusA or gusA neo cassettes . The gusA fusions are compatible with lacZ fusion vectors that are widely used with Bs, and gusA and lacZ fusions are expressed at similar levels . beta-Glucuronidase (beta Glu) and beta-galactosidase (beta Gal) do not exhibit any cross-reactivity, there is very little endogenous beta Glu activity in Bs, and there is no indication of mutation to high-level expression . We have use strains containing both gusA and lacZ fusions to compare the times of expression of different genes during sporulation.

Mol Gen Genet, 1995 Sep 20, 248(5), 583 - 91
Analysis of an insertional operator mutation (gntOi) that affects the expression level of the Bacillus subtilis gnt operon, and characterization of gntOi suppressor mutations; Yoshida K et al.; The Bacillus subtilis gnt operon is negatively regulated via interaction of the gnt repressor (GntR) with an operator upstream of gntR, which is antagonized by gluconate . An 8 bp insertional operator mutation (gntOi) of the gnt operon was constructed which affected the expression level of this operon . Two suppressors of this gntOi mutation, exhibiting normal expression, were also isolated; one involved a threonine substitution for the Ala-48 residue (gntR48T) within the helix-turn-helix DNA-binding motif of GntR, and the other an adenine substitution for the guanine at nucleotide -4 within the gntOi operator (gntOiM4A) (+ 1 is the transcription initiation site) . The gntR48T mutation by itself rendered the gnt operon partially constitutive . When the gntR43L mutation, which renders the gnt operon fully constitutive, was introduced into the gntOi or gntOiM4A mutant, the operator mutations were found not to affect the promoter activity of the gnt operon . These in vivo results indicate that the gntOi mutation affects the operator interaction with GntR, causing a low expression level even in the presence of gluconate . In vitro gel retardation and DNase I footprint analyses demonstrated that even when gluconate was present, GntR still bound to the gntOi operator region.

FEMS Microbiol Lett, 1995 Sep 15, 131(3), 271 - 7
Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilis secA mutant strains; Klein M et al.; The Staphylococcus carnosus secA gene was cloned using the Bacillus subtilis secA gene as a probe . The S . carnosus secA encodes a polypeptide of 844 amino acid residues which is homologous to other known SecA proteins . The S . carnosus SecA functionally complemented the growth and secretion defects of a temperature-sensitive B . subtilis secA mutant at the non-permissive temperature . In contrast, the growth defect of an Escherichia coli secA mutant could not be complemented by the S . carnosus SecA protein . Our results suggest that the interactions of SecA with precursor proteins and/or other components of bacterial preprotein translocase are optimized within each organism.

J Mol Biol, 1995 Sep 15, 252(2), 189 - 202
Structural analysis of the Bacillus subtilis delta factor: a protein polyanion which displaces RNA from RNA polymerase; Lopez de Saro FJ et al.; RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (beta beta' alpha 2), the 20.4 kDa delta (delta) protein, and of one of several sigma (sigma) specificity factors . The delta protein, together with several truncated variants, has been overproduced and purified from Escherichia coli . It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region . The purified amino-terminal domain (delta N) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis . In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (delta C) is largely unstructured and highly charged (net charge of -47) . RNA polymerase purified from a B . subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function . Addition of delta, but not sigma A or delta N, displaces RNA bound to RNA polymerase in a binary complex . The ability of delta to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region . Although delta C can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced . This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase.

Biochemistry, 1995 Sep 5, 34(35), 11080 - 9
Transmembrane topology and axial ligands to hemes in the cytochrome b subunit of Bacillus subtilis succinate:menaquinone reductase; Hagerhall C et al.; The membrane-anchoring subunit of Bacillus subtilis succinate:menaquinone reductase is a protein of 202 residues containing two protoheme IX groups with bis-histidine axial ligation . Residues His13, His28, His70, His113, and His155 are the possible heme ligands . The transmembrane topology of this cytochrome was analyzed using fusions to alkaline phosphatase . The results support a proposed model with five transmembrane polypeptide segments and the N-terminus exposed to the cytoplasm . Mutant B . subtilis cytochromes containing a His13-->Tyr, a His28-->Tyr, and a His113-->Tyr mutation, respectively, were produced in Escherichia coli, partially purified, and analyzed . In addition, succinate: menaquinone reductase containing the His13-->Tyr mutation in the anchor subunit was overproduced in B . subtilis, purified, and characterized . The data demonstrate that His13 is not an axial heme ligand . Thermodynamic and spectroscopic properties of the cytochrome are, however, affected by the His13-->Tyr mutation; compared to wild type, the redox potentials of both hemes are negatively shifted and the gmax signal in the EPR spectrum of the high-potential heme is shifted from 3.68 to 3.50 . From the combined results we conclude that His28 and His113 function as axial ligands to the low-potential heme, which is located in the membrane near the outer surface of the cytoplasmic membrane . Residues His70 and His155 ligate the high-potential heme, which is positioned close to His13 in the protein, near the inner surface of the membrane.

Biochem Mol Biol Int, 1995 Sep, 37(1), 89 - 99
Over-expression, purification and phosphorylation of the Bacillus subtilis CheA protein; Fuhrer DK; The CheA (Bacillus subtilis) protein was over-expressed in a CheA minus E . Coli strain . Cells induced for the over-expression of CheA were lysed by passage through a French press device . The French press lysate was partially purified by ammonium sulfate precipitation and the resulting lysate was loaded onto a cibacron blue column and eluted with a NaCl gradient . CheA was finally purified by separation with a G-150 column . The purity and identity of CheA was verified by SDS-PAGE, CnBr digestion and amino acid analysis . Purified material autophosphorylated at the expected molecular weight of CheA.

Mol Microbiol, 1995 Sep, 17(5), 953 - 60
Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr; Fujita Y et al.; Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis-acting catabolite-responsive element (CRE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR-Lacl family of bacterial transcriptional repressors/activators, and the seryl-phosphorylated form of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system . Footprinting experiments revealed that the purified CcpA protein interacted with P-ser-HPr to cause specific protection of the gnt CRE against DNase I digestion . The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to T at position +154 (+1 is the gnt transcription initiation nucleotide) . The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/P-ser-HPr complex in footprinting experiments . Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B . subtilis mediated by CcpA and P-ser-HPr.

Antimicrob Agents Chemother, 1995 Sep, 39(9), 2141 - 4
Macrolide antibiotics inhibit 50S ribosomal subunit assembly in Bacillus subtilis and Staphylococcus aureus; Champney WS et al.; Macrolide antibiotics are clinically important antibiotics which are effective inhibitors of protein biosynthesis in bacterial cells . We have recently shown that some of these compounds also inhibit 50S ribosomal subunit formation in Escherichia coli . Now we show that certain macrolides have the same effect in two gram-positive organisms, Bacillus subtilis and Staphylococcus aureus . Assembly in B . subtilis was prevented by erythromycin, clarithromycin, and azithromycin but not by oleandomycin . 50S subunit formation in S . aureus was prevented by each of seven structurally related 14-membered macrolides but not by lincomycin or two streptogramin antibiotics . Erythromycin treatment did not stimulate the breakdown of performed 50S subunits in either organism . The formation of the 30S ribosomal subunit was also unaffected by these compounds . Assembly was also inhibited in a B . subtilis strain carrying a plasmid with the ermC gene that confers macrolide resistance by rRNA methylation . These results suggest that ribosomes contain an additional site for the inhibitory functions of macrolide antibiotics.

Protein Sci, 1995 Sep, 4(9), 1801 - 14
1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR; Feher VA et al.; Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR . The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA . Assignments for many sidechain proton resonances are also reported . The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold . Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY . This surface may define a region of binding specificity for response regulators . Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM . Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.

Biosci Biotechnol Biochem, 1995 Sep, 59(9), 1613 - 8
Construction, purification, and properties of a truncated alkaline endoglucanase from Bacillus sp . KSM-635; Ozaki K et al.; Part of a 2.4-kb DNA fragment that encoded the amino-terminal 584 residues (65 kDa) of an alkaline endoglucanase from Bacillus sp . KSM-635 (941 amino acid residues; 105 kDa) was spontaneously deleted during subcloning of the fragment . The remaining 1.1-kb insert of the deleted plasmid encoded amino acids from Ala228 to Leu584 of the enzyme . However, Escherichia coli HB101 cells harboring this plasmid produced an active endoglucanase . After addition of a termination codon, TAA, immediately downstream of the codon for Leu584, the 1.1-kb fragment was inserted into an expression vector, pHSP64 . The resultant plasmid was introduced into Bacillus subtilis ISW1214 for extracellular production of the truncated endoglucanase . The enzyme was then purified to homogeneity from a culture of the recombinant B . subtilis cells . Amino-terminal sequencing of the enzyme showed that the enzyme consisted of 7 amino acid residues encoded by the vector and 357 amino acid residues encoded by the truncated gene, with a molecular mass of 40.2 kDa . The purified enzyme was very active against carboxymethylcellulose and its pH and temperature profiles were almost identical to those of the enzyme produced by Bacillus sp . KSM-635.

J Bacteriol, 1995 Sep, 177(17), 5193 - 6
A partially functional 245-amino-acid internal deletion derivative of Escherichia coli sigma 70; Kumar A et al.; Two hundred forty-five consecutive amino acids of the sigma 70 subunit of Escherichia coli RNA polymerase are not conserved in the homologous protein of Bacillus subtilis . We show that their deletion from a sigma 70-32 hybrid protein caused no severe loss of function in vivo, while sigma 70 itself retained considerable function in vitro.

J Bacteriol, 1995 Sep, 177(17), 5148 - 50
Characterization of a new cell-bound alpha-amylase in Bacillus subtilis 168 Marburg that is only immunologically related to the exocellular alpha-amylase; Haddaoui E et al.; Immunoblot analysis of Bacillus subtilis cell extracts with polyclonal antibodies, raised against purified exocellular alpha-amylase, revealed one protein species of 82,000 Da . This protein was found even in cells in which the amyE gene, encoding exocellular alpha-amylase, was disrupted . Isolated from the membrane fraction, the 82,000-M(r) protein displayed an alpha-amylase activity in vitro.

J Bacteriol, 1995 Sep, 177(17), 5129 - 34
Specificity of DNA binding activity of the Bacillus subtilis catabolite control protein CcpA; Kim JH et al.; CcpA was purified from Escherichia coli BL21 (lambda DE3)/pLysS carrying plasmid pTSC5, which was constructed by inserting the ccpA gene into the polycloning site of pGEM4 . The purified protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mass of 38 kDa but was eluted from a calibrated Bio-Gel P-100 column with an apparent mass of 75 kDa . Western blot (immunoblot) analysis revealed the presence of CcpA in E . coli BL21 (lambda DE3)/pLysS/pTSC5, which carries ccpA, and in wild-type Bacillus subtilis 168 but not in E . coli BL21 (lambda DE3)/pLysS/pGEM4 or in B . subtilis WLN-29, in which ccpA is inactivated by transposon Tn917 insertion . Purified CcpA bound to DNA containing amyO and retarded its mobility in electrophoretic mobility shift analysis . Complete retardation of the DNA required 75 ng of CcpA per assay . In DNase protection analysis, CcpA bound to DNA containing amyO and protected a region spanning amyO when either DNA strand was labeled . Mutant forms of amyO not effective in catabolite repression were not retarded by CcpA.

J Biol Chem, 1995 Sep 1, 270(35), 20329 - 36
Genetic and transcriptional organization of the region encoding the beta subunit of Bacillus subtilis RNA polymerase; Boor KJ et al.; The gene encoding the beta subunit of Bacillus subtilis RNA polymerase was isolated from a lambda gt11 expression library using an antibody probe . Gene identity was confirmed by the similarity of its predicted product to the Escherichia coli beta subunit and by mapping an alteration conferring rifampicin resistance within the conserved rif coding region . Including the rif region, four colinear blocks of sequence similarity were shared between the B . subtilis and E . coli beta subunits . In E . coli, these conserved blocks are separated by three regions that either were not conserved or were entirely absent from the B . subtilis protein . The B . subtilis beta gene was part of a cluster with the order rplL (encoding ribosomal protein L7/L12), orf23 (encoding a 22,513-dalton protein that is apparently essential for growth), rpoB (beta), and rpoC (beta') . This organization differs from the corresponding region in E . coli by the inclusion of orf23 . Experiments using promoter probe vectors and site-directed mutagenesis located a major rpoB promoter overlapping the 3'-coding region of orf23, 250 nucleotides upstream from the beta initiation codon . Thus, the B . subtilis rpoB region differs from its E . coli counterpart in both genetic and transcriptional organization.

Mutat Res, 1995 Sep, 337(2), 97 - 110
Diverse capacities for the adaptive response to DNA alkylation in Bacillus species and strains; Morhoshi F et al.; Our previous studies of Bacillus subtilis showed that the genes responsible for the adaptive response to DNA alkylation were organized as a divergent regulon, in contrast to scattered operons in Escherichia coli ada regulon . To study the generality and diversity of gene organization, several species and strains of Bacillus were examined for the responsiveness to DNA alkylation . B . cereus cells exhibited the highest resistance to MNNG treatment . When the cells were grown in the presence of MNNG, 3-methyladenine DNA glycosylase and two species of DNA methyltransferase were induced as in B . subtilis 168 cells . B . licheniformis 749 and B . amyloliquefaciens H cells exhibited a partial response that manifested itself as the induction of one species of DNA methyltransferase . On the other hand, B . thuringiensis var . Tohokuensis, B . megaterium KMT, and B . subtilis W23 cells were totally deficient in this response, and were hypersensitive to alkylating agents . To determine the cause of this deficiency in strain W23, we examined the genomic structure of the corresponding region where three genes (alkA, adaA, and adaB) were located in 168 . No homologues for the three genes were detected in W23 DNA by Southern hybridization . Two genes (glmS and ndhF) flanking the adaptive response regulon in 168 were also present in W23 . A sequence of about 2750 bp that carried the entire regulon in 168 was replaced with a sequence of about 250 bp that was unique to W23 . At the ends of the conserved segments, palindromic sequences corresponding to the transcriptional termination sites of the adaB and glmS genes were observed . The regulon in 168 could be artificially replaced by the W23 sequence, and be regained through DNA-mediated transformation.

Nat Struct Biol, 1995 Sep, 2(9), 752 - 7
Nucleotide mimicry in the crystal structure of the uracil-DNA glycosylase-uracil glycosylase inhibitor protein complex; Savva R et al.; The Bacillus subtilis bacteriophages PBS-1 and PBS-2 protect their uracil-containing DNA by expressing an inhibitor protein (UGI) which inactivates the host uracil-DNA glycosylase (UDGase) base-excision repair enzyme . Also, PBS1/2 UGI efficiently inactivates UDGases from other biological sources, including the enzyme from herpes simplex virus type-1 (HSV-1) . The crystal structure of the HSV-1 UDGase-PBS1 UGI complex at 2.7 angstrum reveals an alpha-beta-alpha sandwich structure for UGI which interacts with conserved regions of UDGase involved in DNA binding, and directly mimics protein-DNA interactions observed in the UDGase-oligonucleotide complex . The inhibitor completely blocks access to the active site of UDGase, but makes no direct contact with the uracil-binding pocket itself.

Microbiology, 1995 Sep, 141 ( Pt 9), 2219 - 22
Identical amino acid sequence of the aroA(G) gene products of Bacillus subtilis 168 and B . subtilis Marburg strain; Bolotin A et al.; A DNA fragment containing the aroA(G) gene of Bacillus subtilis 168, encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase-chorismate mutase, was cloned and sequenced . The N-terminus of the protein encoded by aroA(G) showed homology with chorismate mutase encoded by aroH of B . subtilis and with the chorismate mutase parts of proteins encoded by the pheA and tyrA genes of Escherichia coli . The C-terminus of the aroA(G) product has sequence similarity with 3-deoxy-D-manno-octulosonate 8-phosphate synthase of E . coli . It was shown that the proteins encoded by the aroA(G) gene of B . subtilis 168 and the aroA gene of B . subtilis ATCC 6051 Marburg strain are identical, so the observed differences in DAHP synthase activity from these two strains must result from other changes.

Genetika, 1995 Sep, 31(9), 1201 - 9
{Determination and comparative analysis of the nucleotide sequence for minireplicon fragments of the cryptic plasmid p1414 from the soil strain of Bacillus subtilis}; Kanapina ASh et al.; Nucleotide sequences of two minireplicon fragments of the p1414 cryptic plasmid of Bacillus subtilis were determined . The fragments corresponded to the region containing ori(+) and the gene coding for Rep protein . Comparing sequences of the fragments with corresponding sequences of other ss+ plasmids suggested that ori(+) of p1414 belongs to the family of endogenous cryptic B . subtilis plasmids, which form an individual, closely related subgroup in the group of ori(+) sequences of the pC194 type . It was found that the amino acid sequence of a conservative FLTLTV motif located, together with its flanking sequences, at the N ends of Rep proteins encoded by different ss+ plasmids, is similar to those of several transmembrane proteins and signal peptides . These results, together with computer data on predicting the secondary and tertiary structure of the N-terminal domain of the p1414 Rep protein, suggest that the domain can serve as a "membrane anchor" during plasmid replication.

Acta Pharm Hung, 1995 Sep, 65(5), 157 - 62
{Polymer-containing eyedrops . IV . Microbiological study of eyedrops containing antibiotics}; Koczka-Kiss C et al.; Authors studied microbiological activities of eye-drops containing neomycin sulphate, gentamicin-sulphate and chloramphenicol, respectively . Effects of viscous eye-drops on Bacillus subtilis were measured by comparing them with resistest disc in the function of the type and concentration of macromolecule, pH and electrolyte content of the solution . It was found that viscosity increasing solution does not have any significant effect on the microbiological activity of the eye-drop in the studied range of concentration . Effectivity of the solution does not change either during storage.

Mol Gen Genet, 1995 Aug 30, 248(4), 391 - 8
Overproduction of the ATP-dependent nuclease AddAB improves the structural stability of a model plasmid system in Bacillus subtilis; Meima R et al.; The effect of the ATP-dependent exonuclease AddAB complex on the structural stability of plasmid pGP1 in Bacillus subtilis was studied . Using deletion mutagenesis and gene amplification techniques, B . subtilis strains were constructed either lacking or overproducing the AddAB complex, a key enzyme in homologous recombination . The deletion mutant possessed no residual ATP-dependent nuclease activity; in contrast, the nuclease activity was up to 30 times higher in lysates of strains carrying multiple copies of the addAB genes in the chromosome . Southern blot analyses of these strains indicated that a linear relationship exists between the number of chromosomal gene copies and the level of AddAB activity . The structural stability of pGP1 was analyzed in the AddAB-deficient and over-producing backgrounds . Frequencies of deletion formation in the plasmid, as monitored by the expression of the pGP1-encoded penP-lacZ fusion on media containing X-gal, were shown to be increased at least 25-fold in the addAB knock-out mutant, whereas the stability of pGP1 was improved up to 15-fold in strains overproducing the AddAB enzyme . A possible explanation for these findings is that interactions between AddAB and plasmid molecules prevent the formation of secondary structures that constitute potential deletion target sites, and thereby enhance the structural stability of plasmids.

Proc Natl Acad Sci U S A, 1995 Aug 29, 92(18), 8190 - 4
Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions; Chen L et al.; The Bacillus subtilis mrgA gene encodes an abundant DNA-binding protein that protects cells against the lethal effects of H2O2 . Transcription of mrgA is induced by H2O2 or by entry into stationary phase when manganese and iron levels are low . We have selected for strains derepressed for transcription of mrgA in the presence of Mn(II) . The resulting cis-acting mutants define an operator site just upstream of the mrgA promoter . Similar sequences flank the promoters for the catalase gene, katA, and the heme biosynthesis operon, hemAXCDBL . Like mrgA, transcription of the katA and hem genes is repressed by Mn(II), which thereby potentiates the killing action of H2O2 . We identified two classes of trans-acting mutants derepressed for mrgA transcription in the presence of Mn(II): some exhibit a coordinate derepression of MrgA, catalase, heme biosynthesis, and alkyl hydroperoxide reductase and are H2O2 resistant, while others have reduced catalase activity and are H2O2 sensitive . These data indicate that the peroxide stress response of B . subtilis is regulated by a repressor that senses both metal ion levels and H2O2.

Nucleic Acids Res, 1995 Aug 25, 23(16), 3214 - 23
Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon; Meijer WJ et al.; A 3.1 kb fragment of the large (approximately 55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced . In contrast to the parental plasmid, derived minireplicons were unstably maintained . Using deletion analysis the fragment essential and sufficient for replication was delineated to 1.1 kb . This 1.1 kb fragment is located between two divergently transcribed genes, denoted orfA and orfB, neither of which is required for replication . orfA shows homology to the B.subtilis chromosomal genes rapA (spoOL, gsiA) and rapB (spoOP) . The 1.1 kb fragment, which is characterized by the presence of several regions of dyad symmetry, contains no open reading frames of more than 85 codons and shows no similarity with other known plasmid replicons . The structural organization of the pLS20 minimal replicon is entirely different from that of typical rolling circle plasmids from Gram-positive bacteria . The pLS20 minireplicons replicate in polA5 and recA4 B.subtilis strains . Taken together, these results strongly suggest that pLS20 belongs to a new class of theta replicons.

Nucleic Acids Res, 1995 Aug 25, 23(16), 3290 - 4
Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution; Bakin A et al.; The number and location of all of the pseudouridine (phi) residues in Saccharomyces cerevisiae small subunit (SSU) ribosomal RNA have been determined by a reverse transcriptase sequencing method {Bakin, A . and Ofengand, J., 1993, Biochemistry, 32, 9754-9762} . Thirteen residues were found in addition to the previously described m1acp3 phi 1189 . The residues were scattered throughout the molecule with three being in expansion segments . No phi was found in the three highly conserved single-stranded sequence elements common to all SSU RNAs . Specifically, phi 563, the analog of phi 516 (Escherichia coli) and phi 517 (Bacillus subtilis) were not found . Eight of the phi were located identically to those in mammalian SSU RNA and three were near to mammalian phi residues in the secondary structure . There was no discernible correlation between the sites for phi and the known locations of the methylated nucleosides as exists in large subunit (LSU) RNAs . Comparison of the structural context in which phi was found in SSU RNA with that in LSU RNA showed a differential bias suggestive of possible different roles for phi in the two rRNAs . This work also identified the locations of three putative new modified bases in SSU rRNA, and revealed 15 sequence differences between the yeast strain used here and the reported sequence.

Biochim Biophys Acta, 1995 Aug 15, 1231(1), 111 - 6
Raman detection of a peroxy intermediate in the hydroquinone-oxidizing cytochrome aa3 of Bacillus subtilis; Varotsis C et al.; When the mixed valence, carbon monoxide-bound form of the hydroquinone-oxidizing cytochrome aa3-600 of Bacillus subtilis is illuminated in the presence of O2, it forms a species that corresponds to 'Compound C', first described for the mitochondrial cytochrome c oxidase by Chance, Saronio and Leigh (J . Biol . Chem . 250 (1975) 9226-9237) . Resonance Raman spectra of the this species show a mode at 366 cm-1 that shifts to 342 cm-1 when the experiment is repeated with 18O2 . The appearance of this mode is insensitive to deuteration exchange within the limits of resolution . High- (1200-1700 cm-1) and low-frequency (200-500 cm-1) data, allow us to assign the 366 cm-1 mode to the Fe(3+)-O stretching vibration of a peroxide adduct where the iron is either low or intermediate spin . This is to our knowledge the first time an 18O2-sensitive iron-oxygen stretching mode has been reported for 'Compound C', providing strong support for the notion that this species is a peroxide adduct . The observed 366 cm-1 v(Fe(3+)-O(-)-O-) frequency is 8 cm-1 higher than that previously found for a transient peroxy intermediate in the reaction between the fully reduced mitochondrial enzyme and O2 . Our observation indicates that, while similar, the metastable peroxyheme a3 species reported here differs in the fine details of geometry, protonation state, and/or hydrogen bond status.

Biochemistry, 1995 Aug 15, 34(32), 10245 - 55
Structure of CuB in the binuclear heme-copper center of the cytochrome aa3-type quinol oxidase from Bacillus subtilis: an ENDOR and EXAFS study; Fann YC et al.; We have studied the structure of the CuB site in the binuclear heme-copper center of the fully oxidized form of the quinol-oxidizing cytochrome aa3-600 from Bacillus subtilis by EXAFS and ENDOR spectroscopy . This enzyme is member of the large superfamily of heme-copper respiratory oxidases, which catalyze the reduction of dioxygen to water and link it to translocation of protons across the bacterial or mitochondrial membrane . The EXAFS of the CuB site strongly suggests tetragonal coordination by two or three histidines with one or two O/N donor ligands . There are some indications that a Cl- ion might fractionally occupy substitution-labile sites, although the majority of enzyme molecules did not contain any heavy (second row) scatters, indicative of a Cl- (or S) bridge between the heme iron and CuB {cf . Powers, L., et al . (1994) Biochim . Biophys . Acta 1183, 504-512} . Proton ENDOR spectroscopy of the CuB site in 1H2O and 2H2O media showed evidence of an oxygenous copper ligand with an exchangeable proton . 14N ENDOR revealed three inequivalent nitrogenous ligands with hyperfine coupling constants consistent with histidines . Together, these results strongly suggest that the fully oxidized enzyme has a low-symmetry, tetragonal CuB site with three histidine nitrogens and one oxygen as ligands, the latter with an exchangeable proton(s) . The identity and assignment of these ligands are discussed.

Structure, 1995 Aug 15, 3(8), 781 - 90
Structural evidence for the evolutionary divergence of mycoplasma from gram-positive bacteria: the histidine-containing phosphocarrier protein; Pieper U et al.; BACKGROUND: The three-dimensional structures of histidine-containing phosphocarrier protein (HPr), a member of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), have been determined from Gram-negative and Gram-positive bacteria . The structure of HPr reported here for Mycoplasma capricolum is the first protein structure to be determined for this class of organism . Comparative structural studies with the bacterial proteins highlight sequence-structure correlations relevant to proposals about the evolutionary origin of mycoplasmas . RESULTS: The crystal structure of HPr from M . capricolum has been determined and refined at 1.8 A resolution, revealing the same overall fold as that of other HPrs of known structure . However, mycoplasma HPr resembles HPrs from Gram-positive bacteria more closely than those from Gram-negative bacteria . As in HPrs from Bacillus subtilis and Escherichia coli, the phosphoryl group carrier (His15) forms the N-terminal cap of a helix, but in contrast to the other crystal structures, the side chain of the adjacent Arg17 is conformationally disordered . A sulfate ion interacts with Ser46, a residue known to be phosphorylated in a regulatory manner . CONCLUSIONS: The greater degree of structural similarity of the M . capricolum HPr to HPrs from Gram-positive rather than Gram-negative bacteria is consistent with the proposal that mycoplasma evolved from Gram-positive bacteria . The proposal that no major conformational transition is required for phosphorylation of the active-site histidine is reinforced by comparing the crystal structures with and without an anion in the active site . The conformational disorder of the Arg17 side chain suggests that its guanidinium group does not have to form specific interactions with other protein groups before phosphorylation at His15 . The association of a sulfate ion with Ser46 serves as a model for HPr(Ser46-P) . As there is no evidence of a conformational change accompanying Ser46 phosphorylation, the inhibitory effect of this event may be attributable to altered surface electrostatics.

Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7916 - 20
TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides; Babitzke P et al.; The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences . The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs . RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats . A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly . In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex . TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA.

Nucleic Acids Res, 1995 Aug 11, 23(15), 2900 - 8
A frameshift error detection algorithm for DNA sequencing projects; Fichant GA et al.; During the determination of DNA sequences, frameshift errors are not the most frequent but they are the most bothersome as they corrupt the amino acid sequence over several residues . Detection of such errors by sequence alignment is only possible when related sequences are found in the databases . To avoid this limitation, we have developed a new tool based on the distribution of non-overlapping 3-tuples or 6-tuples in the three frames of an ORF . The method relies upon the result of a correspondence analysis . It has been extensively tested on Bacillus subtilis and Saccharomyces cerevisiae sequences and has also been examined with human sequences . The results indicate that it can detect frameshift errors affecting as few as 20 bp with a low rate of false positives (no more than 1.0/1000 bp scanned) . The proposed algorithm can be used to scan a large collection of data, but it is mainly intended for laboratory practice as a tool for checking the quality of the sequences produced during a sequencing project.

J Biol Chem, 1995 Aug 11, 270(32), 18975 - 82
Identification of the magnesium-binding domain of the high-affinity ATP-binding site of the Bacillus subtilis and Escherichia coli SecA protein; van der Wolk JP et al.; The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane . The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA . This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J . (1982) EMBO J . 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction . Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine . Mutant SecA proteins were unable to growth-complement an E . coli secA amber mutant strain, and the E . coli SecA mutant interfered with the translocation of precursor proteins in vivo . B . subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity . The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant . Both SecA proteins were defective in lipid-stimulated ATPase activity . Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states . These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

Gene, 1995 Aug 8, 161(1), 69 - 73
Identification of a fliG homologue in Treponema denticola; Heinzerling HF et al.; Using a bacteriophage lambda library of Treponema denticola (Td) ATCC 35405 DNA, and, as a reagent, sera derived from individuals with advanced adult periodontal disease, a variety of recombinant clones producing antigens of this oral spirochete have been isolated . Nucleotide sequence analysis of a clone expressing three immunoreactive antigens has revealed the presence of an open reading frame highly homologous to the flagellar switch/motor protein, FliG, which is known to be essential for flagellar assembly and rotation, and chemotaxis in enteric bacteria . The deduced amino-acid sequence of the treponemal FliG protein had 73% similarity (55% identity) to the Bacillus subtilis FliG protein, and showed significant, but lesser homologies to Gram- FliG proteins . Sequence analysis of regions flanking fliG indicated that this gene is immediately preceded by a fliF homologue, further supporting that the cloned DNA encodes FliG of Td . The findings imply that although the signals for control of chemotaxis may be distinctly different in spirochetes, at least some of the molecules involved in torque generation, control of flagellar rotation and signal transduction are highly conserved with other bacteria . The stronger homology of the spirochete FliG with those of Gram+ bacteria is also consistent with recent analyses of other spirochetal genes.

Gene, 1995 Aug 8, 161(1), 51 - 6
Bacillus subtilis glnR mutants defective in regulation; Schreier HJ et al.; The Bacillus subtilis glnR gene (part of the glnRA operon) encodes a 135-amino-acid (aa) repressor, GlnR, that regulates glnRA transcription in response to nitrogen levels in the growth medium . Two glnR mutants unable to repress under nitrogen excess conditions were obtained by mutagenesis . Lesions were found at Leu77 and Ala80, aa that lie within a region (between aa 59-83) thought to form the alpha-helix-turn-alpha-helix (HTH) motif common among a class of regulatory proteins . Alteration of Gly72 by site-directed mutagenesis also affected regulation, suggesting that aa within the putative HTH region are critical for GlnR function and may be involved in DNA binding . However, other replacements within the aa 59-83 sequence failed to support the HTH structure proposed for this region . Mutations within the C-terminal region of GlnR were also found to affect regulation . Introduction of an ochre stop codon at aa 110, 116, 123 and 129 resulted in the production of truncated proteins that were constitutively repressed, strongly suggesting that a signal recognition site residues within the last seven aa of GlnR . Substituting Asp129 with Asn led to loss of repression, indicating that Asp129 may be directly involved in interacting with either positive or negative effector molecules, or is a target for post-translational modification.

Gene, 1995 Aug 8, 161(1), 45 - 9
Efficient secretion of Bacillus subtilis levanase by Saccharomyces cerevisiae; Wanker E et al.; The secretion of Bacillus subtilis (Bs) levanase (Lev) was studied in the yeast Saccharomyces cerevisiae . A set of different yeast expression plasmids, based on the constitutive PGK promoter and harbouring the Bs Lev-encoding gene (sacC), was constructed . In these plasmids, the original Bs signal sequence was either intact, partially deleted or entirely missing . With all constructs, Lev was produced from yeast transformants . However, only when the intact bacterial signal peptide was present was the synthesized enzyme secreted; around 20% was found in the periplasm and 30% in the culture medium . The secreted protein found in the periplasmic space was mainly core-glycosylated and unglycosylated, and had a size of 80-90 and 74 kDa, respectively . In contrast, Lev found in the culture medium was mainly hyper-glycosylated and had a size of 180-200 kDa . Yeast transformants harbouring sacC, but lacking parts of the bacterial signal sequence, only produced cytoplasmic protein which was not glycosylated and had a size of about 74 kDa . The deletion of the entire signal peptide and a further 22 amino acids at the N terminus of mature Lev resulted in a 71-kDa cytoplasmic protein which was not active.

Mol Microbiol, 1995 Aug, 17(4), 621 - 31
The endogenous Bacillus subtilis (natto) plasmids pTA1015 and pTA1040 contain signal peptidase-encoding genes: identification of a new structural module on cryptic plasmids; Meijer WJ et al.; Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism . Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules . A new structural module was identified on the B . subtilis plasmids pTA1015 and pTA1040 . It is composed of two genes: one specifies an unidentified protein with a putative signal peptide; and the other (sipP) specifies a functional type 1 signal peptidase (SPase) . The homologous, but non-identical, sipP genes of the two plasmids are the first identified plasmid-specific SPase-encoding genes . With respect to structure and activity, the corresponding enzymes (denoted SipP) are highly similar to the chromosomally encoded SPase, SipP, of B . subtillis and several newly identified SPases of other bacilli . Our findings suggest that plasmid-encoded SPases have evolved because, of under certain conditions, SPase can be a limiting factor for protein secretion in B . subtilis.

J Bacteriol, 1995 Aug, 177(16), 4825 - 7
Transcription of spoIVB is the only role of sigma G that is essential for pro-sigma K processing during spore formation in Bacillus subtilis; Gomez M et al.; Activation of pro-sigma K processing in the mother cell at late stages of sporulation in Bacillus subtilis requires the presence of active sigma G in the forespore . Placing the spoIVB gene under the control of sigma F, the early forespore transcription factor, allows sigma K to become active in the absence of sigma G . Therefore, transcription of spoIVB is the only role of sigma G that is essential for the signaling pathway between sigma G and sigma K.

J Bacteriol, 1995 Aug, 177(16), 4813 - 6
Bacillus subtilis gnt repressor mutants that diminish gluconate-binding ability; Yoshida K et al.; The Bacillus subtilis gnt operon is negatively regulated by GntR, which is antagonized by gluconate . Three GntR mutants with diminished gluconate-binding ability were obtained . Two were missense mutants (Met-209 to Ile and Ser-230 to Leu), whereas the third had a deletion of the C-terminal 23 amino acids . The mutant GntR proteins were unable to become properly detached from the gnt operator even in the presence of gluconate.

J Bacteriol, 1995 Aug, 177(16), 4721 - 9
The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration; Popham DL et al.; Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB . Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance . The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores . These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration . This defect in spore core dehydration was exaggerated by the additional loss of dacB expression . However, loss of dacB alone did not alter the spore core water content . Spores produced by spmA and spmB mutants germinated faster than did those of the wild type . Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth . Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant . Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants . These results are consistent with spore core water content being the major determinant of spore heat resistance . The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.

J Bacteriol, 1995 Aug, 177(16), 4619 - 27
The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A; Mandic-Mulec I et al.; SinR is a pleiotropic DNA binding protein that is essential for the late-growth processes of competence and motility in Bacillus subtilis and is also a repressor of others, e.g., sporulation and subtilisin synthesis . In this report, we show that SinR, in addition to being an inhibitor of sporulation stage II gene expression, is a repressor of the key early sporulation gene spo0A . The sporulation-specific rise in spo0A expression at time zero is absent in a SinR-overproducing strain and is much higher than normal in strains with a disrupted sinR gene . This effect is direct, since SinR binds specifically to spo0A in vitro, in a region overlapping the -10 region of the sporulation-specific Ps promoter that is recognized by E-sigma H polymerase . Methyl interference and site-directed mutagenesis studies have identified guanine residues that are important for SinR recognition of this DNA sequence . Finally, we present evidence that SinR controls sporulation through several independent genes, i.e., sp0A, spoIIA, and possibly spoIIG and spoIIE.

Lett Appl Microbiol, 1995 Aug, 21(2), 117 - 20
Effects of chlorhexidine gluconate on the development of spores of Bacillus subtilis; Knott AG et al.; The effects of sublethal concentrations of the membrane-active agent chlorhexidine gluconate (CHG) on the growth rate and sporulation of Bacillus subtilis vegetative MB2 cells have been investigated . CHG increased the mean generation time (Mgt) of vegetative cells in casein medium . It also affected spore development: as CHG concentrations increased, spore index (SI) values decreased and sensitivity to both toluene and heat increased.

Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7455 - 9
Identification of the Bacillus subtilis pur operon repressor; Weng M et al.; Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine . We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon . B . subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region . The PurR binding site which overlaps the promoter encompasses approximately 110 bp . The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate . A mutation that deletes the repressor binding site or one that disrupts purR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal . These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool . In addition, purR is autoregulated . There is no structural or mechanistic similarity between the B . subtilis and Escherichia coli purine repressors.

J Bacteriol, 1995 Aug, 177(15), 4557 - 61
Tetracycline/H+ antiport and Na+/H+ antiport catalyzed by the Bacillus subtilis TetA(L) transporter expressed in Escherichia coli; Guffanti AA et al.; The properties of TetA(L)-dependent tetracycline/proton and Na+/proton antiport were studied in energized everted vesicles of Escherichia coli transformed with a cloned tetA(L) gene (pJTA1) from Bacillus subtilis . Inhibition patterns by valinomycin and nigericin indicated that both antiports were electrogenic, in contrast to the tetracycline/proton antiport encoded by gram-negative plasmid tet genes . Tetracycline uptake in the everted system was dependent upon a divalent cation, with cobalt being the preferred one . The apparent Km for tetracycline was markedly increased at pH 8.5 versus pH 7.5, whereas the Vmax was unchanged . The much higher apparent Km for Na+ decreased at pH 8.5 relative to that at pH 7.5, as did the Vmax . Na+ did not affect tetracycline uptake, nor did Co2+ and/or tetracycline affect Na+ uptake; complex patterns of inhibition by amiloride and analogs thereof were observed.

J Bacteriol, 1995 Aug, 177(15), 4532 - 6
In vitro binding affinity of the Bacillus subtilis AbrB protein to six different DNA target regions; Strauch MA; AbrB is a transcriptional regulator of many Bacillus subtilis genes . A number of AbrB-binding sites have previously been delimited by DNase I footprinting studies, but the heterogeneity of the protected sequences and sizes has not led to a determination of a possible consensus motif for recognition . We have examined the affinity of AbrB for binding to six known target regions when the regions were placed in DNA fragments of various sizes . The sites are shown to vary dramatically in AbrB-binding affinity when they are present in smaller fragments, but the differences are smaller when the affinities of larger fragments are compared . Additional observations that indicate that AbrB binding may be a multistep cooperative process are reported.

J Bacteriol, 1995 Aug, 177(15), 4402 - 9
Molecular dissection of mutations in the Bacillus subtilis spore photoproduct lyase gene which affect repair of spore DNA damage caused by UV radiation; Fajardo-Cavazos P et al.; In response to UV irradiation, Bacillus subtilis spore DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, or spore photoproduct (SP) . SP is broken down into monomers during spore germination by the product of the spl gene which has been proposed to encode the enzyme SP lyase . The wild-type spl gene was cloned by complementation of a mutation designated spl-1; the putative spl gene product is a 40-kDa protein whose deduced amino acid sequence contains regions homologous to DNA photolyases . During phenotypic characterization of spl subclones using transformation crosses between the cloned wild-type spl gene and an spl-1 mutant recipient, in addition to the expected transformant classes exhibiting UV-resistant (type I) and UV-sensitive (type III) spores, an additional recombinant class was observed (called type II), spores of which exhibited slower germination kinetics following UV irradiation . The results suggested that the spl-1 allele consisted of at least two separable mutations . The DNA region which could rescue the spl-1 allele was localized to a 511-bp region within the spl coding sequence; this region was amplified from the spl-1 mutant chromosome by PCR and sequenced . The region contained two amino acid substitutions, an Arg replacing Gly-168 (G168R) and an Asp replacing Gly-242 (G242D) in the deduced SP lyase sequence, as well as 18 silent mutations . PCR amplification of chromosomal DNA from a selected type II recombinant and sequence analysis of the amplification product confirmed that recombination had indeed occurred between codons 168 and 242 and further localized the point of crossover by using the 18 silent mutations as molecular markers throughout the region . By in vitro mutagenesis, alleles of spl containing all combinations of single and double amino acid substitutions were introduced into the cloned wild-type spl gene . When integrated into the B . subtilis chromosome at the amyE locus, it was observed that although both amino acid substitutions contribute to the spl-1 phenotype, the G168R mutation exerted a much greater effect than did the G242D mutation.

J Bacteriol, 1995 Aug, 177(15), 4342 - 9
Amino acid efflux in response to chemotactic and osmotic signals in Bacillus subtilis; Wong LS et al.; We observed a large efflux of nonvolatile radioactivity from Bacillus subtilis in response to the addition of 31 mM butyrate or the withdrawal of 0.1 M aspartate in a flow assay . The major nonvolatile components effluxed were methionine, proline, histidine, and lysine . In studies of the release of volatile radioactivity in chemotaxis by B . subtilis cells that had been labeled with {3H}methionine, the breakdown of methionine to methanethiol can contribute substantially to the volatile radioactivity in fractions following addition of 0.1 M aspartate . However, methanol was confirmed to be released after aspartate addition and, in lesser quantities, after aspartate withdrawal . Methanol and methanethiol were positively identified by derivitization with 3,5-dinitro-benzoylchloride . Amino acid efflux but not methanol release was observed in response to 0.1 M aspartate stimulation of a cheR mutant of B . subtilis that lacks the chemotaxis methylesterase . The amino acid efflux could be reproduced by withdrawal of 0.1 M NaCl, 0.2 M sucrose, or 0.2 M xylitol and is probably the result of changes in osmolarity . Chemotaxis to 10 mM alanine or 10 mM proline resulted in methanol release but not efflux of amino acids . In behavioral studies, B . subtilis tumbled for 16 to 18 s in response to a 200 mosM upshift and for 14 s after a 20 mosM downshift in osmolarity when the bacteria were in perfusion buffer (40 mosM) . The pattern of methanol release was similar to that observed in chemotaxis . This is consistent with osmotaxis in B . subtilis away from an increase or decrease in the osmolarity of the incubation medium . The release of methanol suggests that osmotaxis is correlated with methylation of a methyl-accepting chemotaxis protein.

J Bacteriol, 1995 Aug, 177(15), 4321 - 6
Escherichia coli genes required for cytochrome c maturation; Thony-Meyer L et al.; The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor . In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes . A deletion mutant of E . coli which lacked all of these genes was constructed . Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant . The biogenesis of foreign cytochromes, such as the soluble B . japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated . None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type . The results suggest that the E . coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.

J Virol, 1995 Aug, 69(8), 5024 - 32
Sequential interactions of structural proteins in phage phi 29 procapsid assembly; Lee CS et al.; The mechanism of viral capsid assembly is an intriguing problem because of its fundamental importance to research on synthetic viral particle vaccines, gene delivery systems, antiviral drugs, chimeric viruses displaying antigens or ligands, and the study of macromolecular interactions . The genes coding for the scaffolding (gp7), capsid (gp8), and portal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually or in combination to study the mechanism of phi 29 procapsid assembly . When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex . Circular dichroisin spectrum analysis indicated that gp7 is mainly composed of alpha helices . When two of the proteins were coexpressed, gp7 and gp8 assembled into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact . These results suggested that gp7 served as a bridge for gp8 and gp10 . When gp7, gp8, and gp10 were coexpressed, active procapsids were produced . Complementation of extracts containing one or two structural components could not produce active procapsids, indicating that no stable intermediates were formed . A dimeric gp7 concatemer promoted the solubility of gp8 but was inactive in the assembly of procapsid or procapsid-like particles . Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be important for interaction with gp8 . Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8 . Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles . Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly . These results indicate that phi 29 procapsid assembly deviated from the single-assembly pathway and that coexistence of all three components with a threshold concentration was required for procapsid assembly . The trimolecular interaction was so rapid that no true intermediates could be isolated . This finding is in accord with the result of capsid assembly obtained by the equilibrium model proposed by A . Zlotnick (J . Mol . Biol . 241:59-67, 1994).

J Appl Bacteriol, 1995 Aug, 79(2), 213 - 8
Factors affecting the electroporation of Bacillus subtilis; McDonald IR et al.; Bacillus subtilis 168 trp- was found to be transformable with the tetracycline resistance plasmid pAB124 by electroporation of whole cells, inconsistently and at very low frequencies . Supplementation of the growth medium with glycine, or particularly DL-threonine, produced cells that could be electrotransformed much more efficiently at frequencies up to 2.5 x 10(3) transformants per microgram plasmid DNA . Transformation was optimal with cells grown in medium containing a racemic mixture of the D- and L-isomers of threonine, and no transformants were obtained when pure forms of the D- and L-threonine isomers were used . The cell walls of B . subtilis grown in the presence or absence of D-, L- and DL-threonine had a similar amino acid composition which did not include threonine . A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is discussed . Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants . The effects of buffer composition and culture incubation time were also determined and the electroporation protocol optimized accordingly . The response of a range of other B . subtilis strains to electroporation by the method produced was found to be variable . In all cases, transformation was verified by recovery of the plasmid DNA from putative transformants.

J Appl Bacteriol, 1995 Aug, 79(2), 171 - 7
The effects of glucosinolates and their hydrolysis products on microbial growth; Brabban AD et al.; Rapemeal, which contains potentially toxic compounds such as glucosinolates, was assessed as a substrate for the growth of micro-organisms . The effects of glucosinolates and their degradation products were tested on a range of industrially important microbial species . Sinigrin (2-propenyl glucosinolate) was found to be relatively innocuous to all of the organisms tested but its hydrolysis to yield isothiocyanates, thiocyanates and nitriles resulted in inhibition of growth . The initial inhibitory sinigrin concentration before its hydrolysis was found to be species-dependent with Bacillus subtilis being the most resistant (80 micrograms ml-1) and Saccharomyces cerevisiae (40 micrograms ml-1) the most sensitive . Three Gram-positive organisms tested were found to be more resistant to hydrolysis products than other micro-organisms . Similar results were observed with phenylisothiocyanate for which inhibition was found to be inhibitor and cell concentration-dependent . Addition of thioglucoside glucohydrolase during active growth of Escherichia coli in a sinigrin-containing liquid medium reduced the number of viable cells . Similar effects were also observed in rapemeal media in which growth inhibition was dependent on the glucosinolate content of the rapemeal.

Cytometry, 1995 Aug 1, 20(4), 324 - 33
Flow cytometric study of differentiating cultures of Bacillus subtilis; Chung JD et al.; We report on 1) the development of a flow cytometry-based technique for detecting beta-galactosidase in differentiating cultures of Bacillus subtilis and 2) the application of this technique in the study of early developmental gene expression . The problems associated with generating detectable signals (despite the small size of B . subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-beta-D-galactopyranoside (C8-FDG) . Additionally, to control for background fluorescence during the staining process, we included a control population in the C8-FDG staining mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26 . The distinct emission spectra of C8-fluorescein and PKH26 allow nonspecific C8-FDG staining in this control population to be monitored using two-color analysis . This technique has been applied in the study of developmental gene expression in sporulating cultures of B . subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations . One population is induced for expression of early sporulation genes, which is determined using lacZ fusions, whereas the other remains uninduced . These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.

Nat Struct Biol, 1995 Aug, 2(8), 663 - 73
Extremely rapid protein folding in the absence of intermediates; Schindler T et al.; Here we used the cold-shock protein CspB from Bacillus subtilis to study protein folding at an elementary level . The thermodynamic stability of this small five-stranded beta-barrel protein is low, but unfolding and refolding are extremely rapid reactions . In 0.6 M urea the time constant of refolding is about 1.5 ms, and at the transition midpoint (4 M urea) the folded and unfolded forms equilibrate in less than 100 ms . Both the equilibrium unfolding transition and the folding kinetics are perfectly described by a N<-->U two-state model . The validity of this model was confirmed by several kinetic tests . Folding intermediates could neither be detected at equilibrium nor in the folding kinetics . We suggest that the extremely rapid folding of CspB and the absence of folding intermediates are related phenomena.

Biosci Biotechnol Biochem, 1995 Aug, 59(8), 1433 - 7
Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation; Ohse M et al.; Plasmid DNAs in the range from 2.9 to 12.6 kbp were transferred into Bacillus subtilis ISW1214 intact cells by the use of electroporation . The transformation efficiency (transformants per microgram plasmid DNA) decreased with increases of size of the DNA . However that of 2.0 x 10(3) transformants per microgram of DNA were done routinely, by using a plasmid with a large molecular size of 12.6 kbp . The size of plasmid DNA in the range of 3.7 to 12.6 kbp did not affect the molecular efficiency (transformants per molecule input DNA) . The transformation efficiency as high as 9.3 x 10(4) transformants per microgram of purified plasmid pUB110 was obtained, using a cell concentration of 7.6 x 10(10) cells/ml and DNA concentration of 4 micrograms/ml in buffer containing 0.3 M sucrose, 1 mM CaCl2, and 1 mM sodium citrate (pH 6.0) under optimal pulse conditions of an electric field strength of 7 kV/cm and a duration of 500 mus with a single squared pulse at 0 degrees C . The gene expression for antibiotic resistance after electroporation was completed within 1.5 h . The transformants were confirmed to harbor the same intact plasmid by agarose gel electrophoretic analysis.

J Bacteriol, 1995 Aug, 177(15), 4520 - 3
Effects of spo0 mutations on spo0A promoter switching at the initiation of sporulation in Bacillus subtilis; Chibazakura T et al.; Transcriptional analyses of the Bacillus subtilis sporulation initiator gene spo0A revealed that promoter switching from the vegetative (Pv) to the sporulation-specific (Ps) promoter did not occur in the spo0A, spo0B, spo0E, spo0F, and spo0H mutants . The sof-1 mutation in spo0A restored the promoter switching in the spo0F mutant . These results strongly suggest that Spo0A plays a central role in the regulation of its own promoter switching.

J Bacteriol, 1995 Aug, 177(15), 4327 - 32
Substrate requirements for ErmC' methyltransferase activity; Zhong P et al.; ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli) . The gene for ErmC' was cloned and expressed to a high level in E . coli, and the protein was purified to virtual homogeneity . Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B . subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085 . Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA . In addition, the Vmax for this fragment also rose sevenfold . A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.

J Bacteriol, 1995 Aug, 177(15), 4272 - 8
Translation of trpG in Bacillus subtilis is regulated by the trp RNA-binding attenuation protein (TRAP); Yang M et al.; trpG of Bacillus subtilis encodes a glutamine amidotransferase subunit that is involved in the synthesis of both folic acid and L-tryptophan . Expression of trpG is negatively regulated by tryptophan even though this gene is located within a folic acid biosynthetic operon . Examination of both transcriptional and translational gene fusions to lacZ involving trpG and direct measurements of trpG mRNA levels and TrpG polypeptide accumulation demonstrated that translation of trpG is regulated by tryptophan whereas transcription is not . these studies also show that this regulation is mediated by the trp RNA-binding attenuation protein . Deletion and point mutations indicated that regulation is dependent on a series of G/UAG trinucleotide repeats surrounding the putative ribosome-binding site for trpG . Our results are consistent with a model in which the tryptophan-activated trp RNA-binding attenuation protein and ribosomes compete for binding to trpG mRNA.

Mol Gen Genet, 1995 Jul 28, 248(2), 121 - 5
Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis in Bacillus subtilis; Chen CL et al.; A total of 20 Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis . Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome . We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes . One of the clones obtained, pFC660, was 46 kb long . Eight transposon insertion sites were mapped within this plasmid . Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase . Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.

Mol Gen Genet, 1995 Jul 22, 248(1), 9 - 16
Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome; Itaya M; A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined . An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B . subtilis . The lambda DNA was first subcloned in four segments, having partially overlapping regions . Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling . In the B . subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation . Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration . The remaining gap was sealed by gap-filling . The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B . subtilis chromosome under non-selective conditions . The present method can be used to accommodate larger DNAs in the B . subtilis chromosome and possible applications of this technique are discussed.

Mol Gen Genet, 1995 Jul 22, 248(1), 114 - 20
sigma B-dependent regulation of gsiB in response to multiple stimuli in Bacillus subtilis; Maul B et al.; The expression of the gsiB gene of Bacillus subtilis in response to a wide variety of stress conditions was analysed, and the results provide evidence that gsiB is subject to a sigma B-dependent regulation . Primer extension experiments established identical start points for gsiB transcription during growth and after the induction by heat shock, salt or ethanol stress, and glucose limitation . The sequence upstream of the transcription start point shows great similarity to the sequences of sigma B-dependent promoters of B . subtilis . sigma B was absolutely required for the increase in gsiB mRNA level and in the synthesis rate of GsiB in response to various stimuli . Measurements of the ATP pool indicated that changes in the level of ATP might be one of the signals that regulate the activity of sigma B in B . subtilis.

Biochemistry, 1995 Jul 18, 34(28), 8950 - 9
The Escherichia coli adenylyl cyclase complex: requirement of PTS proteins for stimulation by nucleotides; Peterkofsky A et al.; GTP, as well as other nucleoside triphosphates, stimulates the activity of Escherichia coli adenylyl cyclase in permeable cells; the stimulatory effect is lost when the cells are disrupted by passage through a French pressure cell . These data suggested that the allosteric regulation by GTP of adenylyl cyclase activity requires an interaction of the enzyme with other protein factors . Strains deleted for genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) failed to show an activity stimulation by GTP . With a view to localizing the site of interaction of GTP with the adenylyl cyclase complex, a variety of studies using purified PTS proteins were performed using the photoaffinity labeling reagent, 8-azidoGTP . These studies showed that 8-azidoGTP bound specifically to HPr . A species specificity study showed that the photoaffinity reagent labeled E . coli HPr but not HPr proteins from Mycoplasma capricolum or Bacillus subtilis . A variety of site-directed mutations of E . coli HPr were evaluated for interaction with GTP by photoaffinity labeling as well as by nuclear magnetic resonance; the results of these studies indicate that the lysine residues at positions 24 and 27, serine-46, the threonine at position 36, and the aspartate at position 69 are important for the binding of GTP to HPr . Molecular modeling has been used to formulate a model for the binding of GTP to HPr involving electrostatic interaction of the phosphate groups of the nucleotide with the side chains of lysine residues 27 and 45 and serine-43, interaction of the sugar with serine-46, and interaction of the base with lysine-24 . From these data, it is hypothesized that the binding of GTP to HPr is required for the GTP-dependent stimulation of the activity of the adenylyl cyclase complex.

J Biol Chem, 1995 Jul 14, 270(28), 16840 - 7
Tertiary structure of uracil-DNA glycosylase inhibitor protein; Beger RD et al.; The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that inactivates uracil-DNA glycosylase from diverse organisms . The secondary structure of Ugi consists of five anti-parallel beta-strands and two alpha-helices (Balasubramanian, S., Beger, R.D., Bennett, S.E., Mosbaugh, D.W., and Bolton, P.H . (1995) J . Biol . Chem . 270, 296-303) . The tertiary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance . The Ugi structure contains an area of highly negative electrostatic potential produced by the close proximity of a number of acidic residues . The unfavorable interactions between these acidic residues are apparently accommodated by the stability of the beta-strands . This negatively charged region is likely to play an important role in the binding of Ugi to uracil-DNA glycosylase.

J Biol Chem, 1995 Jul 14, 270(28), 16788 - 95
Substrate channeling in the lumazine synthase/riboflavin synthase complex of Bacillus subtilis; Kis K et al.; The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of three alpha subunits . The beta subunits catalyze the condensation of 5-amino-6-ribityl-amino-2,4(1H,3H)-pyrimidinedione (PYR) with 3,4-dihydroxy-2-butanone 4-phosphate (DHB) yielding 6,7-dimethyl-8-ribityllumazine . This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation . The second product of this reaction is PYR, which is also a substrate of the beta subunits and can be recycled in the catalytic process . Sigmoidal kinetics would be expected for the formation of riboflavin from PYR and DHB and are indeed observed with mixtures of artifactual beta 60 capsids and alpha subunit trimers . In contrast, the formation of riboflavin from PYR and DHB by the native alpha 3 beta 60 is characterized by a finite initial rate, which is similar to the rate of lumazine formation . Most notably, the rate of riboflavin formation has its maximum value at t = 0 and decreases dramatically after the consumption of PYR and DHB despite the presence of transiently formed lumazine . These data suggest that a significant fraction of DHB is converted to riboflavin by substrate channeling, which is conducive to an improved overall catalytic rate of riboflavin formation at low substrate concentrations . The channel is leaky, and the intermediate lumazine is therefore transiently accumulated in the bulk solution . The partitioning factor relating the direct formation of riboflavin via substrate channeling and the formation of transient 6,7-dimethyl-8-ribityl-lumazine increases at low concentrations of the substrates PYR and DHB and has a maximum value at pH 7.5 . Channeling appears to result from the compartmentalization of the alpha subunits inside the icosahedral beta subunit capsid whose catalytic sites are located close to the inner capsid surface.

J Biol Chem, 1995 Jul 14, 270(28), 16701 - 13
OpuA, an osmotically regulated binding protein-dependent transport system for the osmoprotectant glycine betaine in Bacillus subtilis; Kempf B et al.; Exogenously provided glycine betaine can efficiently protect Bacillus subtilis from the detrimental effects of high osmolarity environments . Through functional complementation of an Escherichia coli mutant deficient in glycine betaine uptake with a gene library from B . subtilis, we have identified a multicomponent glycine betaine transport system, OpuA . Uptake of radiolabeled glycine betaine in B . subtilis was found to be osmotically stimulated and was strongly decreased in a mutant strain lacking the OpuA transport system . DNA sequence analysis revealed that the components of the OpuA system are encoded by anoperon (opuA) comprising three structural genes: opuAA, opuAB, and opuAC . The products of these genes exhibit features characteristic for binding protein-dependent transport systems and in particular show homology to the glycine betaine uptake system ProU from E . coli . Expression of the opuA operon is under osmotic control . The transcriptional initiation sites of opuA were mapped by high resolution primer extension analysis, and two opuA mRNAs were detected that differed by 38 base pairs at their 5' ends . Synthesis of the shorter transcript was strongly increased in cells grown at high osmolarity, whereas the amount of the longer transcript did not vary in response to medium osmolarity . Physical and genetic mapping experiments allowed the positioning the opuA operon at 25 degrees on the genetic map of B . subtilis.

Nucleic Acids Res, 1995 Jul 11, 23(13), 2351 - 60
Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA; Helmann JD; Sequence analysis of 236 promoters recognized by the Bacillus subtilis sigma A-RNA polymerase reveals an extended promoter structure . The most highly conserved bases include the -35 and -10 hexanucleotide core elements and a TG dinucleotide at position -15, -14 . In addition, several weakly conserved A and T residues are present upstream of the -35 region . Analysis of dinucleotide composition reveals A2- and T2-rich sequences in the upstream promoter region (-36 to -70) which are phased with the DNA helix: An tracts are common near -43, -54 and -65; Tn tracts predominate at the intervening positions . When compared with larger regions of the genome, upstream promoter regions have an excess of An and Tn sequences for n > 4 . These data indicate that an RNA polymerase binding site affects DNA sequence as far upstream as -70 . This sequence conservation is discussed in light of recent evidence that the alpha subunits of the polymerase core bind DNA and that the promoter may wrap around RNA polymerase.

Science, 1995 Jul 7, 269(5220), 69 - 72
Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains; Stachelhaus T et al.; Peptide synthetases involved in the nonribosomal synthesis of peptide secondary metabolites possess a highly conserved domain structure . The arrangement of these domains within the multifunctional enzymes determines the number and order of the amino acid constituents of the peptide product . A general approach has been developed for targeted substitution of amino acid-activating domains within the srfA operon, which encodes the protein templates for the synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis . Exchange of domain-coding regions of bacterial and fungal origin led to the construction of hybrid genes that encoded peptide synthetases with altered amino acid specificities and the production of peptides with modified amino acid sequences.

Biochemistry, 1995 Jul 4, 34(26), 8465 - 73
Pathway of promoter melting by Bacillus subtilis RNA polymerase at a stable RNA promoter: effects of temperature, delta protein, and sigma factor mutations; Juang YL et al.; Bacillus subtilis RNA polymerase (RNAP) contains a catalytic core (beta beta' alpha 2; or E) associated with one of several sigma factors, which determine promoter recognition, and delta protein, which enhances promoter selectivity . We have shown previously that specific mutations in sigma A region 2.3, or addition of delta, decrease the ability of RNAP to melt the ilv-leu promoter . Here we extend these studies to a stable RNA promoter, PtmS, which controls transcription of seven tRNA genes . KMnO4 footprinting was used to visualize DNA melting at PtmS as a function of both temperature and the protein composition of the RNAP holoenzyme . We propose that the pathway leading to productive initiation includes several intermediates: a closed complex (RPc), a complex in which DNA melting has nucleated within the conserved TATA element (RPn), and an open complex in which DNA-melting extends to at least -4 (RPo1) . RNAP reconstituted with either of two mutant sigma A proteins, Y189A and W192A, was defective for both the nucleation and propagation of the transcription bubble while a third sigma A mutant, W193A, allows normal nucleation of DNA-melting, but does not efficiently propagate the melted region downstream.

Biochemistry, 1995 Jul 4, 34(26), 8458 - 64
Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P identified by in vitro selection; Pan T; Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P (P RNA) have been obtained by in vitro selection . The selection method involves cleavage of a circular RNA library by the P RNA, isolation of the linear cleavage product, and regeneration of circular RNA to allow amplification and multiple cycles of selection . The use of circular RNA ensures that potential substrates can be selected without restricted location of the cleavage site . Such a selection method has been used previously to isolate RNA motifs that undergo autolytic cleavage with Pb2+ {Pan, T., & Uhlenbeck, O . (1992) Biochemistry 31, 3887-3895} . The circular RNA pool after eight cycles of selection was cleaved by the B . subtilis P RNA as efficiently as a pre-tRNA(Phe) substrate, estimated to be more than 10 orders of magnitude better than the unselected RNA library . Kinetic analysis of individual variants showed that the kcat/KM of the selected RNA was up to 4-fold higher than that of the pre-tRNA(Phe) . When cleavage was carried out with Escherichia coli P RNA, the selected RNA was 10-60-fold less reactive than the reaction of the pre-tRNA(Phe) . Two distinct classes of variants are selected, both of which appear to differ significantly from the known P RNA substrates . Terminal truncation experiments suggest that a large number of nucleotides in the class I variants can be deleted without affecting the cleavage activity . The resulting minimal class I substrates contain a short stem-loop with no other apparent helical structures . The class II substrates are cleaved within a putative helical stem that is formed entirely by the primer sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Dent Mater, 1995 Jul, 11(4), 231 - 3
Failure of ethylene oxide to sterilize extracted human teeth; White RR et al.; OBJECTIVES: The purpose of this study was to determine if ethylene oxide could sterilize extracted human teeth to be used in research . METHODS: An occlusal preparation was cut in freshly extracted molars and a small hole was drilled into the pulp chamber . A suspension of Bacillus subtilis (globigii) endospores, the standard biological monitor used for ethylene oxide sterilization, was injected into the pulp cavity, and the pulp cavity access was filled with composite material and sealed with a light-cured sealant . The teeth were exposed to either a 30 degrees C or 63 degrees C ethylene oxide sterilization process . Following exposure, the teeth were aseptically split and cultured to reveal viable spores . RESULTS: Sixty-four percent of the teeth exposed to "cold" ethylene oxide treatment and 80% of the teeth exposed to the "warm" treatment still contained viable spores . SIGNIFICANCE: Ethylene oxide cannot be relied on to sterilize extracted human teeth . Therefore, before they are used in research, other methods should be used to ensure killing of bloodborne pathogens that may be present within the teeth.

Biotech Histochem, 1995 Jul, 70(4), 175 - 84
A color image analysis method for assessment of germination based on differential fluorescence staining of bacterial spores and vegetative cells using acridine orange; Bruno JG et al.; Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var . niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose . These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods . Large green rods were rarely observed . An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature . This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination . In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation . Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time . Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose . The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator . These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs . vegetative cells.

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