Mol Microbiol, 1991 Sep, 5(9), 2181 - 90 Separate promoters direct expression of phoAIII, a member of the Bacillus subtilis alkaline phosphatase multigene family, during phosphate starvation and sporulation; Chesnut RS et al.; Alkaline phosphatase (APase) expression can be induced in Bacillus subtilis by phosphate starvation or by sporulation . We have recently shown that there are multiple APase structural genes contributing to the total alkaline phosphatase expression in B . subtilis . The expression of the alkaline phosphatase III gene (phoAIII) was analysed under both phosphate-starvation induction and sporulation induction conditions . phoAII is transcribed from two promoter regions, PV and PS . The PV promoter initiated transcription 37 bp before the translation initiation codon and was used to transcribe phoAIII during phosphate-starvation induction in vegetative cells . The PS promoter initiated transcription 119 bp before the translation initiation codon and was used during sporulation induction . Genes which have previously been shown to affect total vegatative APase, pho regulon genes phoP, phoR and phoS, affected expression of phoAIII during phosphate starvation . Genes known to affect expression of total sporulation APase, i.e . spoIIA, spoIIG and spoIIE, affected phoAIII expression during sporulation induction . Our data show that one member of the APase multigene family, phoAIII, contributes to the total APase expression both during phosphate-starvation induction and sporulation induction, and that the mechanism of regulation includes two promoters, each requiring different regulatory genes. J Gen Microbiol, 1991 Sep, 137 ( Pt 9), 2073 - 83 Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis; van Dijl JM et al.; The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism . When present on a low-copy-number plasmid, the amount of E . coli SPase I produced (per mg cell protein) in B . subtilis was half that produced in wild-type E . coli cells . The production of E . coli SPase I in B . subtilis was increased approximately fivefold by cloning the lep gene into a high-copy-number plasmid . The expression of E . coli SPase I in B . subtilis did not appear to increase the rate of processing of two hybrid secretory precursor proteins . Two observations may explain the failure of E . coli SPase I to stimulate processing of exported proteins in B . subtilis . First, the E . coli SPase I was apparently not exposed on the outside of the B . subtilis cytoplasmic membrane, indicating its incorrect insertion into the membrane . Second, in vitro processing studies, using cell-free extracts of B . subtilis producing E . coli SPase I, suggested that the enzyme was not active . A further outcome of this study was that conditions favouring processing of precursors by SPase I in cell-free extracts of E . coli did not favour processing by the corresponding enzyme in B . subtilis cell-free extracts . This suggests that significant differences exist between the two enzymes . The observation that antibodies directed against E . coli SPase I did not cross-react with B . subtilis membrane proteins supports this idea. Mol Gen Mikrobiol Virusol, 1991 Sep, (9), 21 - 4 {Study of CAT gene expression in Sa and pC194 plasmids in Escherichia coli, Francisella tularensis, and Bacillus subtilis cells}; Pomerantsev AP et al.; The unit activities were defined for chloramphenicol-acetyltransferases coded for by the cat-genes of the plasmids Sa and pC194 in Francisella tularensis, Escherichia coli and Bacillus subtilis cells . Francisella tularensis cells were shown to hold intermediate position between Escherichia coli and Bacillus subtilis cells in their ability to express the genes of the different taxonomic origin . The direct dependence was found between the dose of the gene coding for chloramphenicol-acetyltransferase synthesis and efficiency of the gene expression, minimal inhibiting concentration of the antibiotic and colony size on the media containing chloramphenicol. Mol Gen Genet, 1991 Sep, 228(3), 393 - 400 Molecular analysis of the Bacillus subtilis recF function; Alonso JC et al.; recF resides between the dnaN and gyrB genes of Bacillus subtilis . The recF15 mutation results in replacement of a glutamate residue in the wild type with a lysine residue in the mutant RecF protein . We investigated the in vivo regulation of recF using a transcriptional fusion to the xylE gene and assaying mRNA production . We found that novobiocin leads to a four-fold induction in recF gene expression, but this is not observed in a gyrB mutant strain . Enhancement of expression of the recF gene in the presence of novobiocin is unrelated to the SOS response . The RecF protein, which has a predicted molecular mass of 42.2 kDa, does not seem to be involved in its own regulation. J Bacteriol, 1991 Sep, 173(18), 5685 - 93 Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis; Weinrauch Y et al.; The sequence and properties of the comQ gene are described . comQ was predicted to encode a 34,209-Da protein, and the product of comQ was shown to be required for the development of genetic competence . The apparent transcriptional initiation and termination sites of comQ were mapped, and the location of a likely E sigma A promoter was inferred . The expression of comQ was maximal early in growth and declined as the cells approached the stationary phase . This expression was not dependent on any of the competence regulatory genes tested (comA, comP, sin, abrB, degU, and spo0A) . Disruption of comQ in the chromosome prevented the development of competence as well as the transcription of comG, a late competence operon . This disruption also decreased the expression of srfA, a regulatory operon needed for the expression of competence . These and other results suggest a role for ComQ early in the hierarchy of competence regulatory genes, probably as a component of a signal transduction system. J Bacteriol, 1991 Sep, 173(17), 5487 - 93 Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis; Nakano MM et al.; srfA is an operon required for the production of the lipopeptide antibiotic surfactin, competence development, and efficient sporulation in Bacillus subtilis . The expression of srfA is induced after the end of exponential growth and is dependent on the products of late-growth regulatory genes comP, comA, and spo0K . To begin to understand the mechanism of srfA regulation, the srfA promoter region was identified and characterized . To examine srfA promoter activity, the srfA promoter was fused to lacZ and inserted into the B . subtilis chromosome as a single copy at the SP beta prophage . The location of the transcription start site of srfA was determined by primer extension analysis and shown to be preceded by a sequence that resembles the consensus promoter recognized by the sigma A form of RNA polymerase . The srfA operon was found to have a sequence corresponding to a long, untranslated leader region of the srfA mRNA (300 bp) . A nucleotide sequence and mutational analysis of the promoter identified a region of dyad symmetry required for srfA-lacZ expression . A similar sequence is found in the region upstream of the degQ promoter, transcription from which is also regulated by ComA . This region of dyad symmetry found upstream of these promoters may be the target for ComA-dependent transcriptional activation. Res Microbiol, 1991 Sep-Oct, 142(7-8), 831 - 9 The role of negative control in sporulation; Smith I et al.; Negative controls play an important role in the regulation of differentiation in many organisms . Sporulation in Bacillus subtilis is also regulated by DNA-binding proteins which exert a repressive effect on genes which are essential for this process . AbrB represses spo0H, coding for sigma H . One of the earliest events in the initiation of sporulation is the lifting of this repression so that more sigma H can be made . As part of an RNA polymerase holoenzyme, this positive transcription factor is responsible for the elevated synthesis of sufficient phosphorylated Spo0A to activate the expression of several stage II genes . Sin, another DNA-binding protein, represses the same genes, spoIIA, spoIIE and spoIIG, that are activated by Spo0A . Thus sporulation is controlled at the two earliest stages by at least two repressors . Sin and AbrB are repressors of other late growth functions but are essential for competence development . Sin is also a positive regulator for motility and autolysin production . These results suggest that AbrB and Sin act as developmental switches, enabling cells at the beginning of stationary growth to choose different developmental fates. Res Microbiol, 1991 Sep-Oct, 142(7-8), 815 - 23 Control of the initiation of sporulation in Bacillus subtilis by a phosphorelay; Trach K et al.; Sporulation in Bacillus subtilis is a developmental process induced as a response to nutritional stress . Activation of sporulation-specific gene transcription is under the control of the spoOA gene product . The SpoOA protein and the SpoOF protein are both homologous to response regulator proteins of two-component regulatory systems which control bacterial responses to a variety of environmental challenges . Response regulators are activated by specific kinases which phosphorylate them . In this study, it was shown that phosphorylation of SpoOA occurs via a phosphotransferase which is the product of the spoOB locus . The phosphodonor in this reaction is the phosphorylated form of SpoOF . It is postulated that SpoOF acts as a secondary messenger that can be phosphorylated by a variety of kinases depending on the particular environmental stress . The series of phosphate transfer reactions in this system is called a phosphorelay . The end product of this series of reactions is SpoOA approximately P which is shown to have greater affinity for the DNA target, the OA box, of SpoOA on the abrB promoter than the unphosphorylated form . SpoOA approximately P, but not SpoOA, was shown to be an activator of transcription of the spoIIA operon which codes for the sporulation-specific sigma factor sigma F . Thus, the initiation of sporulation is dependent on SpoOA approximately P which arises through the phosphorelay and which acts as a transcription factor to repress certain genes, e.g . abrB, and activate others, e.g . spoIIA. Agric Biol Chem, 1991 Sep, 55(9), 2367 - 74 Use of a triple protease-deficient mutant of Bacillus subtilis as a host for secretion of a B . subtilis cellulase and TEM beta-lactamase; Nakamura A et al.; The mature portion of the TEM beta-lactamase (BLA) gene (bla) derived from pBR322 was fused with the promoter and signal region of Bacillus subtilis cellulase (BSC) gene (bsc), and the productivity was compared with that of the cloned native bsc gene, using a wild-type B . subtilis strain and a strain deficient in three proteases (i.e., extracellular serine protease, extracellular neutral protease, and the major intracellular serine protease) as hosts . The effects of the sen, sacQ, and prtR genes, carried on the same plasmids, were tested as for the productivities of BSC and BLA . The production of BSC was increased 9-fold by using the combination of the triple-protease deficient strain and the sacQ gene, compared with that by using the wild-type strain as a host, and no degradation of BSC was observed in the triple-protease deficient strain . On the other hand, though the production of the BSC-BLA fusion protein increased 2.5-fold in the triple-protease deficient strain, the BLA activity was decreased after the cell reached the stationary phase of growth, possibly due to some proteolysis . These observations show different sensitivity of secretary proteins to cellular proteases, and suggest that BLA is decomposed by remaining minor proteases. Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1212 - 8 The primary structure of rat ribosomal protein S18; Chan YL et al.; The amino acid sequence of the rat 40S ribosomal subunit protein S18 was deduced from the sequence of nucleotides in a recombinant cDNA . S18 has 152 amino acids and has a molecular weight of 17,707 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 10-13 copies of the S18 gene . The mRNA for the protein is about 600 nucleotides in length . Rat S18 is identical to mouse S18 (also referred to as KE3) and is related to Escherichia coli S13 and to other S13-like ribosomal proteins from Bacillus subtilis, from Bacillus stearothermophilus, and from plant mitochondria (Nicotiana tabacum and Zea mays). FEBS Lett, 1991 Aug 5, 287(1-2), 153 - 6 Mutant analysis of interaction of the Bacillus subtilis transcription regulator AbrB with the antibiotic biosynthesis gene tycA; Furbass R et al.; The AbrB protein of B . subtilis represses the transcription of various postexponentially expressed genes, such as the antibiotic biosynthesis gene tycA . Recently, we have shown that ArbB binds to the tycA promoter region at two A + T-rich sites; the 'promoter site' (-60 to -35) and the 'leader site' (+169 to +231) . In this study we demonstrate that a Ptyc-lacZ fusion missing the leader region is constitutively expressed in wild-type B . subtilis cells and in B . subtilis cells carrying spoOA or abrB mutations . We also show that substitution mutations within the recently reported potential helix-turn-helix DNA binding motif of AbrB did not affect its specific DNA binding ability. J Mol Biol, 1991 Aug 5, 220(3), 631 - 48 Complete physical map of the Bacillus subtilis 168 chromosome constructed by a gene-directed mutagenesis method; Itaya M et al.; All the SfiI sites and most of the NotI sites were located precisely on the chromosome of Bacillus subtilis 168 by a novel method, termed gene-directed mutagenesis . The stepwise elimination of these restriction sites by this method allowed not only the physical connection of the restriction fragments but also the accurate determination of the position of the restriction sites themselves . The resulting physical map of the 4165 x 10(3) base-pair B . subtilis chromosome has been correlated with the genetic map by determination of the exact location of known genes . The complete physical map provides a rapid and accurate way for mapping of new genes as well as analysis of large DNA rearrangements on the chromosome . The novel strategy is, in principle, applicable to the analysis of the genome of other organisms. J Bacteriol, 1991 Aug, 173(16), 4952 - 8 Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases; Wu XC et al.; We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins . This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors . With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity . No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride . By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme . To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene . This gene was placed under the control of a strong, constitutively expressed promoter, P43 . With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed . An artificial operon, P43-sacY-degQ, was also constructed . However, only a partial additive enhancement effect (24-fold enhancement) was observed . Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application . The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield. J Bacteriol, 1991 Aug, 173(15), 4827 - 35 Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro; Tovar-Rojo F et al.; alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light . Three mutations have been introduced into the B . subtilis sspC gene, which codes for the alpha/beta-type wild-type SASP, SspCwt . One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP . The other mutations changed residues conserved in all alpha/beta-type SASP . In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), residue 57 (Lys) was changed to Gln . The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B . subtilis spores and Escherichia coli as well as in vitro with use of purified protein . Both SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type . In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt . Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro . These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance. J Bacteriol, 1991 Aug, 173(15), 4646 - 52 Analysis of transcriptional control of the gerD spore germination gene of Bacillus subtilis 168; Kemp EH et al.; The gerD locus of Bacillus subtilis comprises a single gene whose function is essential for the germination of B . subtilis spores in media containing asparagine, glucose, and fructose . The expression of gerD has been characterized by using a chromosomal lacZ fusion to the gerD promoter . The promoter is switched on at the same time as the synthesis of glucose dehydrogenase, 2.5 h after sporulation has been initiated in the developing forespore . The gerD gene is not expressed in spoIIB or spoIIIA, -IIIB, -EIII, -FIII, or -IIIG mutants, but it is expressed in spoIIIC and -IIID and spoIVA mutant backgrounds . The in vivo transcriptional start point of the gene has been mapped by primer extension analysis, and sequences upstream from the start point show considerable homology with the promoter consensus sequences recognized by RNA polymerase containing the forespore-specific sigma factor sigma G (E sigma G) . gerD is transcribed in vitro by E sigma G with a similar if not identical start point to that found in vivo, and expression of the gene can be rapidly induced in vegetative cells following the induction of sigma G synthesis . These results indicate that gerD is another member of the sigma G regulon, which includes a number of genes expressed only in the forespore compartment of sporulating cells of B . subtilis. J Bacteriol, 1991 Aug, 173(15), 4595 - 602 The rpsD gene, encoding ribosomal protein S4, is autogenously regulated in Bacillus subtilis; Grundy FJ et al.; Although the mechanisms for regulation of ribosomal protein gene expression have been established for gram-negative bacteria such as Escherichia coli, the regulation of these genes in gram-positive bacteria such as Bacillus subtilis has not yet been characterized . In this study, the B . subtilis rpsD gene, encoding ribosomal protein S4, was found to be subject to autogenous control . In E . coli, rpsD is located in the alpha operon, and S4 acts as the translational regulator for alpha operon expression, binding to a target site in the alpha operon mRNA . The target site for repression of B . subtilis rpsD by protein S4 was localized by deletion and oligonucleotide-directed mutagenesis to the leader region of the monocistronic rpsD gene . The B . subtilis rpsD leader exhibits little sequence homology to the E . coli alpha operon leader but may be able to form a pseudoknotlike structure similar to that found in E . coli. J Bacteriol, 1991 Aug, 173(15), 4877 - 88 Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription; Wu L et al.; We cloned and sequenced a fragment of the Bacillus stearothermophilus NUB36 chromosome that contains two open reading frames (ORFs) whose products were detected only in cells of cultures grown in complex medium at high temperature . The nucleotide sequence of the two ORFs exhibited significant identity to the sequence of the glnQ and glnH loci of the glutamine transport system in enteric bacteria . In addition, growth response to glutamine, sensitivity to the toxic glutamine analog gamma-L-glutamylhydrazide, and glutamine transport assays with parental strain NUB3621 and mutant strain NUB36500, in which the ORF1 coding segment in the chromosome was interrupted with the cat gene, demonstrated that glnQ and glnH encode proteins that are active in the glutamine transport system in B . stearothermophilus . The inferred promoter for the glnQH operon exhibited a low homology to the -35 and -10 regions of the consensus promoter sequences of Bacillus subtilis and Escherichia coli genes . In addition, the inferred promoter for the glnQH operon also exhibited a low homology with the consensus promoter sequence deduced from the sequences of the promoters of nine different genes from B . stearothermophilus . Transcription of the glnQH operon was activated in a nitrogen-rich medium at high temperature and inhibited under the same conditions at low temperature . Transcription of the glnQH operon was partially activated in a nitrogen-poor medium at low temperature . The region upstream from glnQ contains sequences that have a low homology with the nitrogen regulator I-binding sequences and the nitrogen-regulated promoters of enteric bacteria . The effect of temperature on the regulation of the glnQH operon is discussed. Appl Environ Microbiol, 1991 Aug, 57(8), 2392 - 4 Characteristics of an inulinase produced by Bacillus subtilis 430A, a strain isolated from the rhizosphere of Vernonia herbacea (Vell Rusby); Vullo DL et al.; Bacillus subtilis 430A, isolated from the Vernonia herbacea (Vell Rusby) rhizosphere, produced an exocellular inulinase that fits the requirements for the production of syrups on an industrial scale . The partially purified enzyme, obtained by acetone precipitation, displayed a higher specificity for inulin (Km, 8 mM) than for sucrose (56 mM) and a total invertase/total inulase ratio of 0.62 . In addition, it is stable at an optimal temperature of 45 to 50 degrees C for at least 7 h and is inhibited by the end product, fructose, at 14 mM. Mol Microbiol, 1991 Aug, 5(8), 1927 - 40 The spoIIIA operon of Bacillus subtilis defines a new temporal class of mother-cell-specific sporulation genes under the control of the sigma E form of RNA polymerase; Illing N et al.; We have cloned and characterized a 5 kbp region of the Bacillus subtilis chromosome and show that it contains the promoter-proximal part of the spoIIIA locus . The locus consists of a polycistronic operon containing at least three genes . We show that the operon is regulated at the transcriptional level, from a promoter that is first activated about 80 minutes after the induction of sporulation, immediately after septation . Expression of spoIIIA in different spo mutant backgrounds correlates with the ability of each strain to synthesize the sporulation-specific sigma factor, sigma E . Moreover, synthesis of sigma E in vegetative cells by use of an inducible promoter causes expression of mother-cell-specific genes spoIID, spoIIIA, and spoIIID, but not the prespore-specific genes, spoIIIG and spoVA . We suggest that sigma E may be the primary determinant of mother-cell-specific gene expression and that the SpoIIID protein exerts an additional level of regulation on spoIIIA, apparently by acting as a transcriptional repressor . Since the onset of spoIIID expression occurs about 10 minutes after that of spoIIIA, spoIIIA expression is transient . Thus spoIIIA defines a third temporal class of gene controlled by the sigma E form of RNA polymerase. Mol Microbiol, 1991 Aug, 5(8), 1915 - 25 Transcriptional regulation of a Bacillus subtilis dipeptide transport operon; Slack FJ et al.; The Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation . The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase . Post-exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant . This implicated AbrB, another known regulator, as a repressor of dciA . In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion . Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB. Bioorg Khim, 1991 Aug, 17(8), 1066 - 73 {Enzymatic synthesis of acyl peptides containing p-nitroanilides of basic amino acids}; Voiushina TL et al.; A method is suggested for synthesis of acylpeptides, containing arginine or lysine p-nitroanilides at the C-terminus, via the acyl transfer reaction catalyzed by the Bacillus subtilis serine proteinase . Acyl-di- and acyltripeptide ethers with L- and D-amino acids were used as the carboxyl component taken in a twofold excess . When the concentration of dimethylformamide increases, the hydrolysis of the initial ether and the reaction product diminishes . Because of the enzyme inactivation by dimethylformamide the latter's optimal concentration is 70-80%. Mol Microbiol, 1991 Aug, 5(8), 2063 - 72 Bacillus subtilis cytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa3-type oxidases; van der Oost J et al.; The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127 degrees on the Bacillus subtilis chromosome . The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker . The mutant was unable to oxidize N,N,N',N'-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate . Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B . subtilis . Another mutant, with a spontaneous deletion upstream from ctaC, was found to express neither of these enzymes . Radioactive haem-labelling was used to identify subunit II, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B . subtilis. J Gen Microbiol, 1991 Aug, 137 ( Pt 8), 1987 - 98 Cloning, expression, sequence analysis and biochemical characterization of an autolytic amidase of Bacillus subtilis 168 trpC2; Foster SJ; By use of a functional assay for activity, an autolysin structural gene was cloned on a 3 kb EcoRI DNA fragment from a lambda gt11 expression library of Bacillus subtilis 168 trpC2 genomic DNA . Sequencing of the fragment showed five open reading frames, the central one of which encoded the lytic enzyme as found by subclone activity mapping and its homology to a recently sequenced autolysin gene . The protein had a deduced sequence of 272 amino acids and a molecular mass of 29957 Da . When expressed in Escherichia coli DH5 alpha, the protein was processed to a 21 kDa form, as estimated by renaturing SDS-PAGE . The autolysin was an N-acetylmuramyl-L-alanine amidase and its activity was MgCl2-dependent (20 mM optimum) and LiCl-sensitive . The enzyme could bind to and hydrolyse a wide range of peptidoglycan substrates isolated from Gram-positive bacteria; the binding was also MgCl2-dependent . Initial mapping experiments located the autolysin gene near aroD on the B . subtilis 168 chromosome. Mol Gen Mikrobiol Virusol, 1991 Aug, (8), 23 - 5 {The role of DNA-gyrase from Bacillus subtilis during intermolecular irregular recombination}; Zhvingila DIu et al.; The location of oxolinis acid-induced gyrase cleavage sites on pBR322 and pUB110 plasmid DNA in Bacillus subtilis cells has been studied and established . The treated Bacillus subtilis protoplasts were used in the study . Coordinates of the gyrase cleavage sites were compared to the location of the illegitimate recombination sites precisely mapped on the plasmid genomes . The obtained data indicate involvement of the DNA gyrase in formation of a fraction of recombinants in Bacillus subtilis. J Appl Bacteriol, 1991 Aug, 71(2), 147 - 53 Catabolite repression vs derepression, an approach to differentiation during sporulation in Bacillus subtilis; Bhaduri S et al.; Glutamine, like glucose, repressed sporulation and the synthesis of mycobacillin and dipicolinic acid by Bacillus subtilis, and these syntheses were depressed by dibutyryl cyclic GMP but not by dibutyryl cyclic AMP . Neither of these dibutyryl cyclic nucleotides affected sporulation or a number of spore-associated parameters in the strain under normal physiological conditions . Mutants insensitive to glutamine repression were indifferent to the addition of either of the dibutyryl cyclic nucleotides both in the presence and in the absence of glutamine . Sporulation resulted from the remission of repression obtained under the catabolically active state. Agric Biol Chem, 1991 Aug, 55(8), 2099 - 103 Dye-sensitized photooxidation of neutral protease from Bacillus subtilis var . amylosacchariticus: assignment of histidine residue oxidized; Morikawa S et al.; The neutral protease of Bacillus subtilis var . amylosacchariticus was photooxidized in the presence of methylene blue, by which treatment the enzyme was rapidly inactivated . The inactive enzyme was digested with endoproteinase Asp-N, the resultant peptides were separated by HPLC, and their amino acid sequences were compared with those obtained from the unmodified enzyme . Of four peptides that contained histidine residues, only the recovery of one peptide was found to be decreased by the photooxidation with the appearance of a new peptide . Comparisons of amino acid compositions and sequences between these two peptides showed that the latter peptide lacked His228 of the former one, indicating that His228 was photooxidized . This result suggests that His228 is involved in the catalytic reaction of the neutral protease or interaction with substrates. Agric Biol Chem, 1991 Aug, 55(8), 2075 - 82 Transfer reaction catalyzed by exo-beta-1,4-galactanase from Bacillus subtilis; Nakano H et al.; A transfer reaction catalyzed by an exo-beta-1,4-galactanase from Bacillus subtilis was studied . The enzyme had a broad acceptor specificity and transferred galactobiosyl residues to acceptors such as various alcohols, including hydroxy benzenes and saccharides . Transfer products of glycerol formed by the enzyme were compared with those formed by Escherichia coli beta-galactosidase and by Penicillium citrinum endo-galactanase . E . coli enzyme transferred 90% of galactose residues to the primary hydroxyl groups of glycerol and P . citrinum endo-enzyme transferred 80% of saccharide residues to the secondary hydroxyl group . The B . subtilis exo-galactanase was less specific than the other two enzymes and formed two products (1-DG and 2-DG) with a 2-DG/1-DG ratio of about 2 . The structures of the saccharides were examined by 13C-nuclear magnetic resonance analysis and by enzymatic hydrolysis . 1-DG and 2-DG were elucidated to be O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----1)-glycerol and O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----2)-glycerol, respectively . The efficiency of the transfer reaction was measured at various concentrations of glycerol using galactotriose as a donor . About 40-75% of galactobiosyl residues were transferred at an acceptor concentration range of 20-100 mg/ml. Biochemistry, 1991 Jul 16, 30(28), 6896 - 907 Polypeptide backbone resonance assignments and secondary structure of Bacillus subtilis enzyme IIIglc determined by two-dimensional and three-dimensional heteronuclear NMR spectroscopy; Fairbrother WJ et al.; The enzyme IIIglc-like domain of Bacillus subtilis IIglc (IIIglc, 162 residues, 17.4 kDa) has been cloned and overexpressed in Escherichia coli . Sequence-specific assignment of the backbone 1H and 15N resonances has been carried out with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional (3D) NMR spectroscopy . Amide proton solvent exchange rate constants have been determined from a series of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra acquired following dissolution of the protein in D2O . Major structural features of IIIglc have been inferred from the pattern of short-, medium- and long-range NOEs in 3D heteronuclear 1H nuclear Overhauser effect 1H-15N multiple-quantum coherence (3D NOESY-HMQC) spectra, together with the exchange rate constants . IIIglc contains three antiparallel beta-sheets comprised of eight, three, and two beta-strands . In addition, five beta-bulges were identified . No evidence of regular helical structure was found . The N-terminal 15 residues of the protein appear disordered, which is consistent with their being part of the Q-linker that connects the C-terminal enzyme IIIglc-like domain to the membrane-bound IIglc domain . Significantly, two histidine residues, His 68 and His 83, which are important for phosphotransferase function, are found from NOE measurements to be in close proximity at the ends of adjacent strands in the major beta-sheet. J Mol Biol, 1991 Jul 20, 220(2), 241 - 53 Identification of DNA sequences involved in regulating Bacillus subtilis glnRA expression by the nitrogen source; Schreier HJ et al.; The DNA binding protein, GlnR, encoded by glnR, is believed to be directly responsible for regulating glnRA expression in Bacillus subtilis . Identification of cis-acting loci involved in glnRA control is the focus of this study . Analysis of glnRA-lacZ transcriptional fusions harboring deletions extending into the promoter region demonstrated that sequences upstream from position -35, relative to the transcription start-point, were necessary for nitrogen source regulation . These sequences included a 21 base-pair (bp) element, from positions -40 to -60, having 2-fold symmetry; the element shares homology to certain binding sites utilized by proteins having the alpha-helix-turn-alpha-helix motif, of which GlnR is a member . Involvement of this element in regulation was examined by using synthetic DNA fragments containing the promoter and upstream sequences driving lacZ expression . Fragments extending from positions -63 to -8 and from positions -52 to -8 yielded full and partial regulation, respectively . Regulation from a fragment containing a 5 bp insertion between positions -36 and -37 was impaired . A T.A to A.T transversion mutation at position -41 did not have any detectable effect on regulation, whereas a T.A to C.G transition mutation at the same site resulted in constitutive expression . Using a gel electrophoresis mobility shift assay, it was found that purified GlnR bound to a glnRA restriction fragment that extended from positions -104 to +83; binding was abolished after digestion with HinfI, which cleaves between positions -52 and -48 . Furthermore, HinfI digestion was inhibited by the presence of GlnR . Thus, the GlnR binding site extends from the vicinity of position -35 upstream to position -63 . We suggest that the glnRA operator is the 21 bp sequence lying within this region. Gene, 1991 Jul 15, 103(1), 107 - 11 Parameters affecting plasmid stability in Bacillus subtilis; Leonhardt H et al.; For the analysis of parameters affecting plasmid stability in Bacillus subtilis, we used a pUB 110-derived shuttle plasmid containing direct and inverted nucleotide repeats (DRs and IRs) . Deletions of up to 6 kb were found to occur between DRs of 7 to 16 bp . IRs as small as 43 or 58 bp were shown to stimulate the formation of these deletions in their neighbourhood . However, these structural features (DRs and IRs) per se were not responsible for plasmid instability . The unstable recombinant plasmids, but not their deletion-carrying (delta) derivatives, were found to impair the growth of the host and to accumulate high amounts of linear plasmid multimers {high mol . wt . (hmw) DNA} . We propose that the accumulation of hmw DNA may be the major reason for the selective pressure against recombinant plasmids, and the enrichment of delta-plasmids . Host mutations and other parameters increasing the stability of recombinant plasmids in B . subtilis are described. J Biol Chem, 1991 Jul 15, 266(20), 13411 - 7 The transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein; Kallio PT et al.; The synthesis of a variety of proteins, including the well characterized degradative enzymes, which occurs during the transition state between vegetative growth and the onset of sporulation in Bacillus subtilis is controlled by a class of molecules known as transition state regulators . One of these regulators is the product of the hpr gene, first identified by mutations affecting the synthesis of extracellular proteases . We have purified the Hpr protein and found that it binds specifically to DNA fragments carrying the promoters and the upstream regions of the alkaline (aprE) and neutral (nprE) protease genes of B . subtilis . DNase I protection experiments revealed that the Hpr protein is able to bind at four and two regions of the aprE and nprE promoters, respectively . We have also located two Hpr binding sites in the promoter region of a gene of unknown function which is nevertheless known to be developmentally regulated during the transition state and which occurs in the same operon as the gene encoding another transition state regulator, Sin . The location of one of the Hpr binding sites on the aprE gene occurs adjacent to a region to which the Sin protein binds . However, in mixing competition experiments we have shown that Hpr and Sin binding occurred independently, and no visible alterations of protected regions were detected. Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6087 - 91 Molecular structure of Bacillus subtilis aspartate transcarbamoylase at 3.0 A resolution; Stevens RC et al.; The three-dimensional structure of Bacillus subtilis aspartate transcarbamoylase (ATCase; aspartate carbamoyltransferase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) has been solved by the molecular replacement method at 3.0 A resolution and refined to a crystallographic R factor of 0.19 . The enzyme crystallizes in the space group C2 with unit cell dimensions a = 258.5, b = 153.2, and c = 51.9 A and beta = 97.7 degrees . The asymmetric unit is composed of three monomers related by noncrystallographic threefold symmetry . A total of 295 of 304 amino acid residues have been built into the monomer . The last 9 residues in the C terminus were not included in the final model . Each monomer consists of 34% alpha-helix and 18% beta-strand . Three solvent-exposed loop regions (residues 69-84, 178-191, and 212-229) are not well defined in terms of electron density . The catalytic trimer of ATCase from B . subtilis shows great similarity to the catalytic trimer in Escherichia coli ATCase, which was used in constructing the model for molecular replacement . The unliganded trimer from B . subtilis, which is not cooperative, resembles the T (inactive) state slightly more than the R (active)-state form of the E . coli trimer . However, certain regions in the B . subtilis trimer exhibit shifts toward the E . coli R-state conformation. J Biol Chem, 1991 Jul 5, 266(19), 12301 - 5 Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis; Bischoff DS et al.; The Bacillus subtilis gene encoding CheB, which is homologous to Escherichia coli CheY, the regulator of flagellar rotation, has been cloned and sequenced . It has been verified, using a phage T7 expression system, by showing that a small protein, the same size as E . coli CheY, is actually made from this DNA . Despite the fact that the two proteins are 36% identical, with many highly conserved residues, they appear to play different roles . Unlike CheY null mutants, which swim smoothly, CheB null mutants tumble incessantly . However, a CheB point mutant swims smoothly, even in the presence of a plasmid bearing cheB, which restores the null mutants to wild type . Expression of CheB in wild type B . subtilis makes the cells exhibit more tumbling . Since both absence of CheB and presence of high levels of CheB cause tumbling, CheB appears to be required, in certain circumstances, for both smooth swimming and tumbling . Expression in wild type E . coli makes the cells smooth swimmers and strongly inhibits chemotaxis. Biochemistry, 1991 Jul 2, 30(26), 6503 - 8 Cell-free biosynthesis of surfactin, a cyclic lipopeptide produced by Bacillus subtilis; Ullrich C et al.; The lipopeptide antibiotic surfactin is a potent extracellular biosurfactant produced by various Bacillus subtilis strains . Biosynthesis of surfactin was studied in a cell-free system prepared from B . subtilis ATCC 21332 and OKB 105, which is a transformant producing surfactin in high yield {Nakano, M . M., Marahiel, M . A., & Zuber, P . (1988) J . Bacteriol . 170, 5662-5668} . Cell material was disintegrated by treatment with lysozyme and French press . A cell-free extract was prepared by ammonium sulfate fractionation, which catalyzed the formation of surfactin at the expense of ATP . Lipopeptide biosynthesis required the L-amino acid components of surfactin and D-3-hydroxytetradecanoyl-coenzyme A thioester . D-Leucine which is present in surfactin was not utilized but inhibited the biosynthetic process . The structure of surfactin, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses . An enzyme fraction was prepared by gel permeation chromatography which catalyzed ATP/pyrophosphate exchange reactions dependent on the component amino acids of surfactin . This enzyme fraction was capable of binding substrate amino acids covalently, probably via thioester linkages . The formation of these intermediates was inhibited by various thiol blocking reagents and phenylmethanesulfonyl fluoride . De novo synthesis of the lipopeptide was not observed with this partially purified enzyme fraction most likely due to the lack of an acyltransferase activity required for linking the beta-hydroxy fatty acid to the peptide moiety. J Bacteriol, 1991 Jul, 173(13), 4107 - 15 A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product; van den Burg B et al.; By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically active phages on plates supplemented with 0.8% skim milk, chromosomal B . caldolyticus DNA fragments that specified proteolytic activity were obtained . Subcloning of one of these fragments in a protease-deficient Bacillus subtilis strain resulted in protease proficiency of the host . The nucleotide sequence of a 2-kb HinfI-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids . This ORF was denoted as the B . caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene . Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B . caldolyticus enzyme was a neutral protease . The B . sterothermophilus and B . caldolyticus enzymes differed at only three amino acid positions . Nevertheless, the thermostability and optimum temperature of the B . caldolyticus enzyme were 7 to 8 degrees C higher than those of the B . stearothermophilus enzyme . In a three-dimensional model of the B . stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions . The role of these residues in thermostability was analyzed by using site-directed mutagenesis . It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B . stearothermophilus and B . caldolyticus. J Bacteriol, 1991 Jul, 173(13), 3977 - 80 Characterization of Bacillus subtilis recombinational pathways; Alonso JC et al.; Recombination in Bacillus subtilis requires the products of numerous rec loci . To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele . On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups . Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations . Group gamma consists of the recH and recP mutations . These results suggest that B . subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways . The RecA protein is required in all three pathways of intermolecular recombination. Virology, 1991 Jul, 183(1), 366 - 73 Regulation of the phage phi 29 prohead shape and size by the portal vertex; Guo PX et al.; Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded DNA into a preformed prohead during morphogenesis . The prohead is composed of the scaffold protein gp7, the capsid protein pg8, the portal protein gp10, and the dispensable head fiber protein gp8.5 . Our objective was to elucidate the phi 29 prohead assembly pathway and to define the factors that determine prohead shape and size . The structural genes of the phi 29 prohead were cloned and expressed in Escherichia coli individually or in combination to study form determination . The scaffold protein was purified from E . coli as a soluble monomer . In vivo and in vitro studies showed that the scaffolding protein interacted with both the portal vertex and capsid proteins . When the scaffold protein interacted only with the capsid protein in vivo, particles were formed with variable size and shape . However, in the presence of the portal vertex protein, particles with uniform size and shape were produced in vivo . SDS-PAGE analysis showed that the latter particles contained the proteins of the scaffold, capsid, head fiber, and portal vertex . These results suggest that the scaffolding protein serves as the linkage between the portal vertex and the capsid proteins, and that the portal vertex plays a crucial role in regulating the size and shape of the prohead. Appl Environ Microbiol, 1991 Jul, 57(7), 1893 - 8 Nature of Escherichia coli cell lysis by culture supernatants of Bacillus species; Dean CR et al.; Escherichia coli cells were found to be sensitive to lysis by the supernatants of a variety of protease-positive Bacillus species when treated at 45 degrees C but not when treated at 37 degrees C . Different E . coli strains manifested different lysis sensitivities when treated at 45 degrees C . When the lysis rates of E . coli cells at various stages of growth were investigated, post-exponential-phase cells were shown to be most sensitive to lysis . The lysis rate was inversely related to cell viability, and susceptibility appeared to be at least partly due to lysis of dead E . coli cells . A close relation was observed between levels of lysis activity and proteolytic activity . A Bacillus subtilis mutant lacking alkaline and neutral protease activity failed to lyse E . coli cells . It was concluded that Bacillus proteases played a major role in the observed E . coli lysis. J Bacteriol, 1991 Jul, 173(13), 4240 - 2 Isolation and characterization of Bacillus subtilis mutants blocked in the synthesis of pantothenic acid; Baigori M et al.; We have produced and characterized by physiological and enzymatic analyses pantothenate (pan) auxotrophs of Bacillus subtilis . panB auxotrophs are deficient in ketopantoate hydroxymethyltransferase, whereas panE mutants lack ketopantoic acid reductase . The pan mutations were mapped by phage PBS1-mediated two-factor crosses and found to be located in the interval purE-tre of the genetic map of B . subtilis. J Parenter Sci Technol, 1991 Jul-Aug, 45(4), 187 - 92 Airborne microbial challenges of Blow/Fill/Seal equipment: a case study; Bradley A et al.; Controlled microbial challenges, comprising air-dispersed spores of Bacillus subtilis var niger, have been generated within a containment room (around 54 m3 in volume) housing a Blow/Fill/Seal machine . 'Stirred-settling' conditions were created throughout the room and the airborne spore challenge was monitored to ensure homogeneity within the room for extended periods of time . The Blow/Fill/Seal machine was set to fill 2 cm3 ampoules with Tryptone Soya Broth at each of three airborne challenge levels of 10(4), 10(6) and 10(7) spores m-3 (about 10(2), 10(4) and 10(5) spores ft-3 respectively) . A relationship has been established between the level of airborne micro-organisms in the machine operating environment and the extent of product contamination . This relationship allows prediction of operating conditions under which a level of sterility assurance, equal to that demanded of terminal sterilization, is attained . It is stressed that the findings apply only to the particular Blow/Fill/Seal machine and to the specific conditions of machine operation. Genetika, 1991 Jul, 27(7), 1264 - 8 {Excision repair does not protect Bacillus subtilis cells from inactivation by sunlight}; Lotareva OV et al.; Exponentially growing Bacillus subtilis cells are highly sensitive to inactivating action of sunlight, the strain deficient in excision repair being every more sensitive than the uvr1 mutant . The inactivating effect is connected with the action of irradiation located in the left part of the spectrum (the whole UV region and some zones of the visible one) . Increased sensitivity to sunlight disappeared when cells were exposed to sunlight in a liquid medium with casaminoacids (2 g/l) . The inactivating effect was probably of photodynamic nature and was caused either by DNA lesions that were not removed by excision repair or by the damage which arose not in DNA. Mol Gen Mikrobiol Virusol, 1991 Jul, (7), 3 - 8 {Diaminopimelate pathway for lysine synthesis in Escherichia coli and bacilli}; Kal'cheva EO et al.; The diaminopimmelate (DAP) pathway for lysine biosynthesis in Escherichia coli and some species of Bacillus are presented in the review . It was shown that the major variations of the DAP pathway of Bacillus subtilis from that described and extensively studied in Escherichia coli exist. Mol Gen Mikrobiol Virusol, 1991 Jul, (7), 22 - 5 {Subcloning and study of the GTP-cyclohydrolase gene of Bacillus subtilis}; Boretskii IuR et al.; Bacillus subtilis GTP-cyclohydrolase gene and its deletion derivatives were subcloned in Escherichia coli cells . The position of the gene within the riboflavine operon was defined . The deletion of the 14 kDa fragment from the N-end of GTP-cyclohydrolase gene did not affect the enzyme activity. Biochimie, 1991 Jul-Aug, 73(7-8), 1163 - 70 Suppression of defective-sporulation phenotypes by mutations in transcription factor genes of Bacillus subtilis; Ng C et al.; Mutations in the Bacillus subtilis major RNA polymerase sigma factor gene (rpoD/crsA47) and a sensory receiver gene (spoOA/rvtA11) are potent intergenic suppressors of several stage 0 sporulation mutations (spoOB, OE, OF & OK) . We show here that these suppressors also rescue temperature-sensitive sporulation phenotypes (Spots) caused by mutations in RNA polymerase, ribosomal protein, and protein synthesis elongation factor EF-G genes . The effects of the crsA and rvtA suppressors on RNA polymerase and ribosomal protein spots mutations are similar to those previously described for mutations in another intergenic suppressor gene rev . We have examined the effects of rvtA and crsA mutations on the expression of sporulation-associated membrane proteins, including flagellin and penicillin binding protein 5* (PBP 5*) . Both suppressors restored sporulation and synthesis of PBP 5* in several spoO mutants . However, only rvtA restored flagellin synthesis in spoO suppressed backgrounds . The membrane protein phenotypes resulting from the presence of crsA or rvtA suppressors in spoO strains suggests that these suppressors function via distinct molecular mechanisms . The rvtA and crsA mutations are also able to block the ability of ethanol to induce spoO phenocopies at concentrations of ethanol which prevent sporulation in wild type cells . The effects of ethanol on sporulation-associated membrane protein synthesis in wild type and suppressor containing strains have been examined. Biokhimiia, 1991 Jul, 56(7), 1209 - 14 {Dependence of Bacillus subtilis cell respiration on monovalent cations}; Samuilov VD et al.; Nigericin, monensin, valinomycin + carbonyl-cyanide-m-chlorophenylhydrazone and gramicidin inhibit the respiration of Bacillus subtilis cells incubated with NAD-dependent substrates or succinate, but not with ascorbate + N,N,N',N'-tetramethyl-p- phenylene-diamine . The level of inhibition was decreased by potassium ions and, in a lower degree, by sodium or ammonium ions . The results obtained suggest that the respiration of Bacillus subtilis depends on the presence of monovalent cations whose effects seem to be directed at complexes I, III and probably complex II of the respiratory chain. J Bacteriol, 1991 Jul, 173(14), 4341 - 6 Role and expression of the Bacillus subtilis rodC operon; Wagner PM et al.; The role of the rodC operon in Bacillus subtilis was investigated . The operon encodes two genes (rodD and rodC) necessary for the synthesis of the cell wall teichoic acid . Transcription of this operon is responsive to levels of phosphate and to concentrations of magnesium ions in the growth medium . This regulation of mRNA production corresponds to conditions that dictate the type of polymer that will be synthesized for the cell wall, i.e., teichoic or teichuronic acid . While the introduction of multiple copies of rodC was tolerated by the cells, multiple copies of rodD appeared to be lethal . The lethality of the rodD fragment was not exhibited if multiple copies of rodC were also present. FEMS Microbiol Lett, 1991 Jul 1, 65(3), 329 - 34 Detection and characterization of naturally occurring plasmids in Bacillus licheniformis; Parini C et al.; Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA . Among these strains, 14 were shown to harbor one or more plasmids of different size . Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B . licheniformis plasmid previously isolated . Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector . The resulting chimeras were able to transform Bacillus subtilis . The fragment with high homology probably contains the region with the replicative functions of plasmids from B . licheniformis species. Gene, 1991 Jun 30, 102(2), 277 - 82 Optimization of the signal-sequence cleavage site for secretion from Bacillus subtilis of a 34-amino acid fragment of human parathyroid hormone; Saunders CW et al.; We have effected the secretion from Bacillus subtilis of a 34-amino acid (aa) fragment of human parathyroid hormone (PTH,1-34), using a Bacillus amyloliquefaciens neutral protease signal sequence . The secretion efficiency depended on the aa sequence near the signal-sequence cleavage site . We constructed a series of gene fusions encoding different pairs of aa between the signal sequence and PTH,1-34 . There was a correlation between those polypeptides which were efficiently secreted and the potential for a beta-turn in the region just beyond the signal-sequence cleavage site . Based on this correlation, we constructed a gene fusion which specified Gly rather than Ala at the C terminus of the signal sequence, thus creating a beta-turn potential at the end of the signal sequence . The change provided a slight increase in secretion efficiency. Biochem Biophys Res Commun, 1991 Jun 28, 177(3), 998 - 1005 Identification of amino acid substitutions in the lipopeptide surfactin using 2D NMR spectroscopy; Baumgart F et al.; It is generally accepted that surfactin, being produced by various Bacillus subtilis strains, is a cyclic lipopeptide built from the heptapeptide L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu and a beta-hydroxy fatty acid with variable chain length of 13 - 15 carbon atoms . We investigated surfactin from Bacillus subtilis ATCC 21332 and OKB 105, dissolved in pyridine and methanol, with two-dimensional H NMR spectroscopy . In the NH-fingerprint region, 21 well resolved cross peaks are observed instead of the expected seven cross peaks for the given heptapeptide . We were able to assign all proton signals to 21 amino acids, to identify three heptapeptides, and thus to prove the existence of structural analogues of surfactin . In the major fraction A, the peptide sequence is as given above . In fractions B and C, the C-terminal leucine is replaced by valine and isoleucine, respectively. J Biol Chem, 1991 Jun 25, 266(18), 11628 - 31 Active site-directed inhibition by optically pure epoxyalkyl cellobiosides reveals differences in active site geometry of two 1,3-1,4-beta-D-glucan 4-glucanohydrolases . The importance of epoxide stereochemistry for enzyme inactivation; Hoj PB et al.; 1,3-1,4-beta-D-Glucan 4-glucanohydrolases (EC 3.2.-1.73) from Bacillus subtilis and barley (Hordeum vulgare) with identical substrate specificities but unrelated primary structures have been probed with (R,S)-epoxyalkyl (-propyl, -butyl, -pentyl) beta-cellobiosides and with optically pure (3S)- and (3R)-3,4-cellobiosides as active site-directed inhibitors . The optimal aglycon length for inactivation differs for the two enzymes, and they are differentially inhibited by the pure epoxybutyl beta-cellobioside diastereoisomers . The (3S)-epoxybutyl beta-cellobioside inactivates the B . subtilis enzyme much more efficiently than does the (3R)-isomer, whereas the reverse is true for the barley enzyme . Both enzymes are inactivated by a mixture of the stereoisomers at a rate intermediate of that observed with the individual isomers . The two beta-glucan endohydrolases may therefore employ sterically different mechanisms to achieve glycoside bond hydrolysis in their common substrate . The efficiency and specificity of epoxide-based "suicide" inhibitors may be enhanced significantly by the use of inhibitors bearing only one stereoisomeric form of the epoxide group. J Mol Biol, 1991 Jun 20, 219(4), 605 - 13 The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli; Levine A et al.; When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 degrees C, DNA replication is synchronized . Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate . We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin . On the left arm, replication continues for at least 190 x 10(3) base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene . When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B . subtilis . Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB . We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response . In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself . No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC . Post-initiation control in B . subtilis therefore results in duplication of many key genes involved in growth and sporulation . We discuss the possibility that such a control might be linked to differentiation in this organism. J Bacteriol, 1991 Jun, 173(11), 3282 - 90 Genetic evidence for interaction of sigma A with two promoters in Bacillus subtilis; Kenney TJ et al.; The specificity of promoter binding by RNA polymerase is governed by the sigma subunit . Recent studies, in which single-amino-acid substitutions in sigma factors have been found to suppress the effects of specific base pair substitutions in promoters, support the model that these sigma factors make sequence-specific contacts with nucleotides at the -10 and -35 regions of promoters . We found that single-amino-acid substitutions in the putative -35 region and -10 region recognition domains of sigma A specifically suppressed the effects of mutations in the -35 and -10 regions, respectively, of two promoters that are expressed in exponentially growing Bacillus subtilis . These mutations change the specificity of sigma A, the primary sigma factor in growing B . subtilis, and demonstrate that this sigma factor interacts with promoters in a manner similar to that of its homolog in Escherichia coli, sigma 70 . These mutant derivatives of sigma A also provide a tool that may be useful for determining whether sigma A uses specific promoters in vivo. Carbohydr Res, 1991 Jun 10, 212, 229 - 44 Investigation of the fine structure of alpha-dextrins derived from amylopectin and their relation to the structure of waxy-maize starch; Bertoft E; Alpha-dextrins, obtained by fractional precipitation with methanol of the products of the action of Bacillus subtilis alpha-amylase on waxy-maize amylopectin, were debranched with isoamylase and the distributions of the unit chains were analysed by gel-permeation chromatography . The large alpha-dextrins still contained long B-chains after hydrolysis for 60 min, but these were absent from the small dextrins with chain numbers of approximately 11 or less . The small dextrins contained increased amounts of chains with lengths intermediate of those of the long B-chains and the main part of the short chains . After hydrolysis for 210 min, almost all of the long B-chains had disappeared and the chains with intermediate lengths had been shortened further . The distributions of the unit chains of the internal chains, obtained by debranching of the phosphorolysis (phi)-limit dextrins, gave similar results and showed that the ratio of A- to B-chains was unchanged during the alpha-amylolysis . Models for the fine structure of the intermediate alpha-dextrins are proposed. J Mol Biol, 1991 Jun 5, 219(3), 403 - 14 Transcription activation at a distance by phage phi 29 protein p4 . Effect of bent and non-bent intervening DNA sequences; Serrano M et al.; Protein p4 of the Bacillus subtilis phage phi 29 switches on the transcription of the viral late genes by binding to the viral late promoter at a region close to the RNA polymerase binding site . Gel retardation and DNase I footprinting assays show that the presence of protein p4 is required for RNA polymerase recognition of the late promoter . The protein p4 and RNA polymerase DNA binding sites have been separated by the insertion of bent and non-bent DNA sequences of different lengths . These mutant promoters were used to study in vitro their protein p4-dependent transcriptional activity and their interaction with both protein p4 and RNA polymerase . The results indicate that protein p4 is able to function at longer DNA distances from the RNA polymerase binding site than in the natural promoter . The extent of protein p4 activity depended on the length and conformation of the inserted DNA . Activation of transcription and RNA polymerase binding was favoured when the relative orientation of protein p4 and RNA polymerase was conserved and when the intervening DNA had a bent conformation . These data, together with the DNase I footprints, suggest that activation at distance by protein p4 involves a DNA loop held by the interaction of protein p4 and RNA polymerase. J Bacteriol, 1991 Jun, 173(11), 3514 - 22 Protein synthesis in vitro by Micrococcus luteus; Farwell MA et al.; Bacillus subtilis and related gram-positive bacteria which have low to moderate genomic G + C contents are unable to efficiently translate mRNA derived from gram-negative bacteria, whereas Escherichia coli and other gram-negative bacteria are able to translate mRNA from both types of organisms . This phenomenon has been termed translational species specificity . Ribosomes from the low-G + C-content group (low-G + C group) of gram-positive organisms (B . subtilis and relatives) lack an equivalent to Escherichia ribosomal protein S1 . The requirement for S1 for translation in E . coli (G . van Dieijen, P . H . van Knippenberg, J . van Duin, B . Koekman, and P . H . Pouwels, Mol . Gen . Genet . 153:75-80, 1977) and its specific role (A.R . Subramanian, Trends Biochem . Sci . 9:491-494, 1984) have been proposed . The group of gram-positive bacteria characterized by high genomic G + C content (formerly Actinomyces species and relatives) contain S1, in contrast to the low-G + C group (K . Mikulik, J . Smardova, A . Jiranova, and P . Branny, Eur . J . Biochem . 155:557-563, 1986) . It is not known whether members of the high-G + C group are translationally specific, although there is evidence that one genus, Streptomyces, can express Escherichia genes in vivo (M . J . Bibb and S . N . Cohen, Mol . Gen . Genet . 187:265-277, 1985; J . L . Schottel, M . J . Bibb, and S . N . Cohen, J . Bacteriol . 146:360-368, 1981) . In order to determine whether the organisms of this group are translationally specific, we examined the in vitro translational characteristics of a member of the high-G + C group, Micrococcus luteus, whose genomic G + C content is 73% . A semipurified coupled transcription-translation system of M . luteus translates Escherichia mRNA as well as Bacillus and Micrococcus mRNA . Therefore, M . luteus is translationally nonspecific and resembles E . coli rather than B . subtilis in its translational characteristics. Antimicrob Agents Chemother, 1991 Jun, 35(6), 1264 - 6 Use of high-performance liquid chromatography to monitor stability of tetracycline and chlortetracycline in susceptibility determinations; Ray A et al.; Trypticase soy broth and nutrient broth were used in the antimicrobial susceptibility testing of tetracycline and chlortetracycline with Bacillus subtilis . The stability of the antibiotics in both media at an incubation temperature of 37 degrees C was monitored by high-performance liquid chromatography . It was found that Trypticase soy broth has limited application in susceptibility testing for the tetracycline congeners tested and that chlortetracycline is more unstable than tetracycline. J Dairy Sci, 1991 Jun, 74(6), 1773 - 8 Specificity, inhibitory studies, and oligosaccharide formation by beta-galactosidase from psychrotrophic Bacillus subtilis KL88; Rahim KA et al.; beta-Galactosidase from psychotrophic Bacillus subtilis KL88 was specific to the beta-D-glycosidic linkage normally present in lactose . The enzyme was completely inhibited by transition metal ions (Cu2+, Fe3+, Fe2+, Zn2+) and partially inhibited by high concentrations of glucose and galactose as well as Ca2+ . It was activated by most of the alkaline earth metal ions (NA+, K+, Li+) . Oligosaccharides were formed at the different levels of lactose concentrations reaching more than 20% for high lactose concentration (20%) . Three types of oligosaccharides were formed in significant concentrations detected by HPLC analysis. FEMS Microbiol Lett, 1991 Jun 1, 65(1), 9 - 13 Purification and characterization of a cell wall hydrolase encoded by the cwlA gene of Bacillus subtilis; Kuroda A et al.; A cell wall hydrolase of Bacillus subtilis was prepared from Escherichia coli cells harboring a plasmid containing the B . subtilis cwlA gene and purified by hydroxyapatite column chromatography and HPLC through TSK-gel G3000SWXL . In contrast to the molecular mass of 29,919 Da deduced from its nucleotide sequence, the purified CWLA is a 23 kDa protein . Characterization of the specific substrate bond cleaved by CWLA indicated the enzyme is an N-acetylmuramyl-L-alanine amidase . A 32-kDa precursor protein was detected on zymography of a crude cell homogenate . Some of the enzymatic properties of CWLA are also described. Genetics, 1991 Jun, 128(2), 215 - 23 DNA repair and the evolution of transformation in Bacillus subtilis . III . Sex with damaged DNA; Hoelzer MA et al.; Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination . The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome . Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged . In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells . In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA . We document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA . These results suggest that sites of transformation are often damaged sites in the recipient cell's genome. J Med Chem, 1991 Jun, 34(6), 1871 - 9 Streptonigrin and related compounds . 5 . Synthesis and evaluation of some isoquinoline analogues; Rao KV et al.; A series of analogues of streptonigrin, in which the quinoline of ring B is replaced by isoquinoline and the substituted pyridine of ring C is replaced by phenyl, nitrophenyl, aminophenyl, or benzyl functions, have been prepared . Thus, 1-substituted isoquinoline-5,8-diones with 7-amino or 6-(alkylamino) groups were prepared . The various quinones were evaluated for antimicrobial activity against Bacillus subtilis and root-growth inhibitory activity against Lepidium sativum . The effect of specific structural changes on these activities was examined with streptonigrin for comparison . The necessity of an aminoquinone function for activity is confirmed . With regard to the antibacterial activity, the isoquinoline analogues appear to be less active compared to the quinoline derivatives . However, the higher degree of antibacterial activity of the 1-benzylisoquinolines and the 1-nitrophenylisoquinolines compared to the 1-phenyl isoquinolines is noteworthy . In contrast to the results seen with the antibacterial activity, most of the isoquinoline analogues showed activity comparable to, or even higher than, that of streptonigrin in the root-growth inhibition assay . The 1-nitrophenylisoquinolines again appear to be the most active . The equal or greater potency of the benzyl analogue in comparison with the phenyl analogue was unexpected and questions the need for the extended conjugation and the geometry required for metal binding as considered earlier . It also opens up new possibilities for structural variation. J Bacteriol, 1991 Jun, 173(12), 3846 - 54 Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter; Adams LF et al.; Two sigma factors, sigma 35 and sigma 28, direct transcription from the Bt I and Bt II promoters of the cryIA(a) gene of Bacillus thuringiensis; this gene encodes a lepidopteran-specific crystal protoxin . These sigma factors were biochemically characterized in previous work (K . L . Brown and H . R . Whiteley, Proc . Natl . Acad . Sci . USA 85:4166-4170, 1988; K . L . Brown and H . R . Whiteley, J . Bacteriol . 172:6682-6688, 1990) . In this paper, we describe the cloning of the genes encoding these two sigma factors, as well as their nucleotide and deduced amino acid sequences . The deduced amino acid sequences of the sigma 35 and sigma 28 genes show 88 and 85% identity, respectively, to the sporulation-specific sigma E and sigma K polypeptides of Bacillus subtilis . Transformation of the sigma 35 and sigma 28 genes into B . subtilis shows that the respective B . thuringiensis sigma factor genes can complement spoIIG55 (sigma E) and spoIIIC94 (sigma K) defects . Further, B . thuringiensis core polymerase reconstituted with either the sigma 35 or sigma 28 polypeptide directs transcription from B . subtilis promoters recognized by B . subtilis RNA polymerase containing sigma E and sigma K, respectively . Thus, sigma 35 and sigma 28 of B . thuringiensis appear to be functionally equivalent to sigma E and sigma K of B . subtilis . However, unlike the situation for sigma K in B . subtilis, the homologous sigma 28 gene in B . thuringiensis does not result from a late-sporulation-phase chromosomal rearrangement of two separate, partial genes. Appl Environ Microbiol, 1991 Jun, 57(6), 1838 - 41 Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1; Hara T et al.; The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B . subtilis chromosomal DNA as a selective marker . The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor . The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B . amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181. J Bacteriol, 1991 Jun, 173(11), 3580 - 3 Genetic analysis of the flaA locus of Bacillus subtilis; Hauser PM et al.; We isolated two clones of recombinant lambda bacteriophage with overlapping inserts of Bacillus subtilis chromosomal DNA corresponding to part of the flaA locus . The flaA4 and flaA15 mutations were localized on the physical map by marker rescue experiments . The flaA locus and the flaB (sigD) gene were mapped in transduction crosses, and the order glnA polC flaB flaA was determined . FlaB was linked to polC in transformation crosses. Boll Soc Ital Biol Sper, 1991 Jun, 67(6), 561 - 8 {Determination of antibiotic chemicals using microbiological tests: evaluation of the limits of sensitivity}; De Santis EP et al.; Sensitivity of Bacillus subtilis BGA and Bacillus stearothermophilus var . calidolactis disc assays to 53 chemio-antibiotics was tested . Test-microorganisms were sown in two different mediums: PM Indicator Agar, Difco U.S.A., and Standard II Nutrient Agar, Merck Germany, modified according to Nouws . The mediums were used with or without addition of Trimethoprim (at a concentration of 0.12 or 0.024 mcg/ml of medium for B . subtilis and for B . stearothermophilus respectively) . B . stearothermophilus did not grow in Standard II . However, the B . subtilis assay gave the best results with Standard II, apart for aminoglycosides . The B . stearothermophilus disc assay was the most sensitive to penicillins (Minimum Inhibiting Concentration in mcg/ml, MIC, between 0.001 and 0.004), cephalosporins (MIC between 0.003 and 0.09, apart from Ceftazidime, 0.3) and aminoglycosides (MIC between 0.03 and 0.6) . The B.subtilis disc assay showed better sensitivity to quinolines (MIC between 0.05 and 4) and to some tetracyclines (oxytetracycline and chlortetracycline, MIC 0.03) . Trimethoprim, where added, determined a higher sensitivity to sulfonamides (MIC between 0.025 and 0.25). Mol Microbiol, 1991 Jun, 5(6), 1363 - 73 Sequential activation of dual promoters by different sigma factors maintains spoVJ expression during successive developmental stages of Bacillus subtilis; Foulger D et al.; The spoVJ gene of Bacillus subtilis encodes a 36 kDa protein and is expressed only in the mother cell . spoVJ has an interesting pattern of regulation during sporulation because it is expressed from sequentially activated promoters . These promoters, designated P1 and P2, are under the control of different sigma factors, sigma E and sigma K, which become active at separate times during sporulation . Removal of promoter P1, leaving promoter P2 active, resulted in about a 30-minute delay in the formation of heat-resistant spores and demonstrated that the expression of spoVJ from both promoters is essential for normal sporulation . A comparison is made between the sequences of the spoVJ promoters and the promoters of other genes dependent upon sigma E and sigma K. Genetika, 1991 Jun, 27(6), 983 - 90 {A new pleiotropic mutation affecting purine metabolism, sporulation and biosynthesis of exoenzymes in Bacillus subtilis}; Maznitsa II et al.; A pleiotropic mutation (cpm) which is localised in the vicinity of the spoA gene of Bacillus subtilis chromosome has been described . The mutation inhibits spore formation, renders bacteria auxotrophic for adenine and tyrosine, increases sensitivity to antibiotics, decreases cell motility and the ability to grow on D-ribose and D-xylose, inhibits growth of bacteriophages PBS1 and AR9 as well as enhances activity of alkaline proteinase and alpha-amylase . At the same time, the cpm mutants acquire the ability to produce inosine . Inosine excretion is connected with more than 50- and 5-fold increase in activity of 5'-nucleotidase in respect to IMP and AMP, accordingly, and 10-fold decrease in activity of purine nucleoside phosphorylase . Biosynthesis of inosine and Ade- phenotype of the cpm mutant are not mediated by the change in activity of sAMP synthetase . The nature and mechanism of action of the cpm mutation are under discussion. Biochem Int, 1991 Jun, 24(3), 517 - 25 Thermostability of Bacillus subtilis neutral protease; Eijsink VG et al.; The thermostability of the B . subtilis neutral protease was studied under various conditions . At elevated temperatures the enzyme was inactivated as a result of autolysis . The rate of inactivation did not depend on the enzyme concentration and the enzyme was most stable near its pH optimum . The rate of inactivation was unaffected by the presence of a second protease during the incubation at high temperatures . The results indicate that the rate of thermal inactivation of the neutral protease is determined by the kinetics of local unfolding processes that precede autolysis rather than by the catalytic rate of the autodigestion reaction or an irreversible unfolding step. J Bacteriol, 1991 Jun, 173(12), 3831 - 45 Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase; Sun DX et al.; L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced . A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase . These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspH1 mutation at 215 degrees . Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium . In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t1 to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73 . The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present . In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript . Depending on the growth medium, the levels of asparaginase and aspartase were from 2- to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspH1 mutation codes for a trans-acting transcriptional regulatory factor . In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by t0 of sporulation but then showed a small increase, which was mirrored by changes in the level of beta-galactosidase from an ansB-lacZ fusion . The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore . However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulation-specific RNA polymerase tested yielded a -73 transcript in vitro. J Bacteriol, 1991 Jun, 173(12), 3732 - 40 Effect of ermC leader region mutations on induced mRNA stability; Hue KK et al.; Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence . Another result of ribosome stalling is stabilization of ermC mRNA . The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region . Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure . Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability . A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation . This mutation was used to measure the effect of mRNA stabilization on ermC gene expression. J Struct Biol, 1991 Jun, 106(3), 211 - 20 Electron microscopy study of GroEL chaperonin: different views of the aggregate appear as a function of cell growth temperature; Carazo JM et al.; We have studied two members of the family of morphogenetic factors or chaperonins, the GroEL-like factors from Escherichia coli and Bacillus subtilis, in order to determine the possible structural basis of their related function in promoting the correct and efficient assembly of biological oligomers . The main objective of this work has been to study by transmission electron microscopy the possible changes that these factors may undergo when subjected to a number of different conditions such as changes in temperature in vivo and in pH in vitro . We applied both rotational and multivariate statistical analyses of single particles to images of GroEl-like aggregates from the two bacteria . The most striking result is the finding of two distinct "front views" of these aggregates, from both E . coli and B . subtilis . One view, which has not been described earlier, shows a sixfold symmetry and is most abundant at growing temperatures below 37 degrees C . After heat shock, a view showing seven morphological units becomes dominant . On the basis of our analysis it is clear that GroEL-like morphogenetic factors from two unrelated bacteria such as E . coli and B . subtilis present two distinct views: one sixfold and the other sevenfold . Their relative percentage of appearance is related to the temperature at which the cells were grown and also to the storage conditions (pH). Mol Microbiol, 1991 Jun, 5(6), 1447 - 57 Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon; Blomfield IC et al.; To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome . We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette . The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences . We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration . The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon . The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques . In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing . Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci . This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E . coli chromosome. Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4781 - 5 Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system; Neyfakh AA et al.; Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells . Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells . The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein . The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria . On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters . These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters. J Bacteriol, 1991 Jun, 173(12), 3644 - 55 Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis; Kooistra J et al.; The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned . The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment . Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity . Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA protein), and 28 kDa . Only the AddB and AddA proteins are required for ATP-dependent exonuclease activity . Both the AddB and AddA proteins contained a conserved amino acid sequence for ATP binding . In the AddA protein, a number of small regions were present showing a high degree of sequence similarity with regions in the E . coli RecB protein . The AddA protein contained six conserved motifs which were also present in the E . coli helicase II (UvrD protein) and the Rep helicase, suggesting that these motifs are involved in the DNA unwinding activity of the enzyme . When linked to the T7 promoter, a high level of expression was obtained in E . coli. Biochem Biophys Res Commun, 1991 May 31, 177(1), 48 - 53 A simple convenient biological dosimeter for monitoring solar UV-B radiation; Wang TC; The use of dry Bacillus subtilis spores as a biological dosimeter for the monitoring of solar UV-B (290-330 nm) radiation was described . Our field tests had supported the utility of this dosimeter as a reproducible and reliable sunlight dosimeter. Gene, 1991 May 15, 101(1), 113 - 6 Transcription of the Bacillus subtilis spoIIA locus; Wu JJ et al.; The spoIIA operon encodes three genes, including the structural gene for a sporulation-induced sigma factor sigma F . We used deletion analysis of spoIIA-lacZ fusions to define the location of the spoIIA promoter . We found that sigma H-RNA polymerase transcribes spoIIA accurately in vitro and propose that sigma H directs transcription of spoIIA during sporulation. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4533 - 7 Spo0A binds to a promoter used by sigma A RNA polymerase during sporulation in Bacillus subtilis; Satola S et al.; Examination of the effects of 56 single-base-pair substitutions in the spoIIG promoter and studies of the interaction of the spo0A product (Spo0A) with this promoter in vitro demonstrated that Spo0A acts directly to enable this promoter to be used by sigma A-associated RNA polymerase (EC 2.7.7.6) . The spoIIG operon from Bacillus subtilis is transcribed during sporulation by a form o RNA polymerase containing sigma A, the primary sigma factor in vegetative cells . The spoIIG promoter is unusual in that it contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs . We found that single-base-pair substitutions in the around the -35-like sequence, and substitutions in a region upstream from this position, around position -87, reduced promoter activity . DNase I protection and electrophoretic gel mobility shift assays were used to demonstrate that Spo0A binds specifically to these regions in vitro . Evidently, the -35-like sequence is part of a Spo0A binding site and therefore is possibly not a sigma A-recognition sequence . These results support a model in which Spo0A activates the spoIIG promoter after the onset of endospore formation. Nucleic Acids Res, 1991 May 25, 19(10), 2623 - 7 Transcription of genes involved in the earliest steps of actinorhodin biosynthesis in Streptomyces coelicolor; Parro V et al.; A 170bp long BamHI-Sau3A DNA fragment from the actIII-actI intergenic region of the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2) contains two promoters directing transcription in a divergent manner . One of them, the actIII promoter, is responsible for the transcription of the actIII gene and the other controls transcription of the adjacent actI region in the opposite direction . Weak activity of the actIII promoter can be detected in Streptomyces lividans and Bacillus subtilis in the absence but not in the presence of glucose . Neither promoter seems to function in Escherichia coli. Gene, 1991 May 15, 101(1), 15 - 21 An SfiI restriction map of the Bacillus subtilis 168 genome; Amjad M et al.; A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome . The combined sizes of the fragments indicate a genome size of approx . 4.2 Mb . The SfiI fragments range in size from 7-730 kb . Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map. J Biol Chem, 1991 May 15, 266(14), 9113 - 27 Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon; Quinn CL et al.; A 12.5-kilobase segment of Bacillus subtilis chromosomal DNA containing the entire pyrimidine biosynthetic (pyr) gene cluster has been cloned and sequenced . The sequenced DNA has seven cistrons encoding the six enzymes of de novo pyrimidine nucleotide biosynthesis and two open reading frames of unknown function . Based on the sequence and mapping of transcripts, the genes in this cluster appear to be transcribed on one large polycistronic message in the order ORF1, pyrB, pyrC, pyrAA, pyrAB, ORF2, pyrD, pyrF, pyrE . The deduced amino acid sequences for six pyrimidine biosynthetic enzymes from B . subtilis and comparisons with the corresponding sequences from numerous other species are presented . The 3' ends of the reading frames overlap the 5' ends of the downstream open reading frames for all cistrons in the cluster except ORF1 and pyrB, which are separated by a 145-base pair intercistronic region . The start of transcription was mapped by primer extension to a G residue 158 nucleotides upstream from the translation initiation codon of ORF1 . This site is preceded by a typical B . subtilis sigma A-dependent promoter . A promoter indicator plasmid was used to show that this region carried a promoter from which transcription was regulated by pyrimidines . Transcription from this promoter was not detected in primer extension experiments using mRNA prepared from B . subtilis cells grown in the presence of excess uracil . No transcripts initiating from the intercistronic space between ORF1 and pyrB were detected with S1 nuclease mapping; however, a transcription terminator was detected in this region that reduced but did not fully block transcriptional readthrough . This terminator was not regulated by pyrimidines in the growth medium under the conditions tested . The region immediately following the promoter and 5' to ORF1 has a potential transcription terminator sequence . The roles, if any, of these transcription terminators in the regulation of pyr operon expression are yet to be determined . However, deletion of the terminator immediately following the promoter abolished pyrimidine regulation of transcription in the indicator plasmid. Mol Microbiol, 1991 May, 5(5), 1081 - 9 Analysis of the gluconate (gnt) operon of Bacillus subtilis; Reizer A et al.; The gluconate (gnt) operon of Bacillus subtilis includes the gntR, gntK, gntP, and gntZ genes, respectively encoding the transcriptional repressor of the operon, gluconate kinase, the gluconate permease, and an unidentified open reading frame (Fujita and Fujita, 1987) . We have compared the proteins encoded by the gnt operon of B.subtilis with published sequences and showed that (i) the gluconate repressor is homologous to several putative regulatory proteins in Escherichia coli, (ii) the gluconate kinase of B . subtilis is homologous to xylulose kinase, glycerol kinase and fucose kinase in E . coli (20-26% identity; 12-59 S.D.), (iii) the gluconate permease exhibits a C-terminal domain which is homologous to a hydrophobic protein encoded by an unidentified open reading frame (dsdAp) which precedes the dsdA gene of E . coli (39% identity; 19 S.D.), and (iv) the gntZ gene product is homologous to 6-phosphogluconate dehydrogenases of other bacteria and of animals (48-56%; 82-178 S.D.), thereby suggesting that the B . subtilis gntZ encodes 6-phosphogluconate dehydrogenase . Several conserved regions of the sequenced 6-phosphogluconate dehydrogenases can serve as signature patterns of this protein . Computer analyses have indicated that the previously reported sequences of the porcine and ovine 6-phosphogluconate dehydrogenases, as well as the hypothetical DsdAp protein, are probably erroneous . The probable reasons for the errors are reported along with the proposed revised sequences. Nucleic Acids Res, 1991 May 11, 19(9), 2337 - 42 Identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the RNA polymerase; Nuez B et al.; Expression of Bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4 . This activator binds to a region of the late A3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending Tin the DNA . In this work the target sequences recognized by protein p4 in the phage phi 29 late A3 promoter have been characterized . The binding of protein p4 to derivatives of the late A3 promoter harbouring deletions in the protein p4 binding site has been studied . When protein p4 recognition sequences were altered, the activator could only bind to the promoter in the presence of RNA polymerase . This strong cooperativity in the binding of protein p4 and RNA polymerase to the promoter suggests the presence of direct protein-protein contacts between them. Eur J Biochem, 1991 May 8, 197(3), 733 - 40 Analysis of the spc ribosomal protein operon of Thermus aquaticus; Jahn O et al.; The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5'-portion of the Escherichia coli spc operon was cloned, using the E . coli gene for the ribosomal protein L5 as hybridization probe . The gene arrangement was found to be identical to E . coli, i.e . S17, L14, L24, L5, S14, S8 and L6 . Stop and start regions of contiguous cistrons overlap, except for the S14-S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E . coli and Bacillus subtilis . A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T . aquaticus analyzed here . The stop codon of gene S17 (the last gene of the S10 operon in E . coli) and the start codon of gene L14 (the first gene of the spc operon in E . coli) overlap in T . aquaticus, thus leaving no space to accommodate an intergenic promoter preceding spc-operon-encoded genes in T . aquaticus . A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 micrograms/ml) to chloramphenicol in E . coli and therefore could be largely excluded as the main promoter for spc-operon-encoded genes . We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E . coli, in the corresponding region or any other region in the cloned T . aquaticus spc DNA. Biochemistry, 1991 May 7, 30(18), 4594 - 9 Structural and functional roles of cysteine residues of Bacillus polymyxa beta-amylase; Uozumi N et al.; Bacillus polymyxa beta-amylase contains three cysteine residues at positions 83, 91, and 323, which can react with sulfhydryl reagents . To determine the role of cysteine residues in the catalytic reaction, cysteine residues were mutated to construct four mutant enzymes, C83S, C91V, C323S, and C-free . Wild-type and mutant forms of the enzyme were expressed in, and purified to homogeneity from, Bacillus subtilis . A disulfide bond between Cys83 and Cys91 was identified by isolation of tryptic peptides bearing a fluorescent label, IAEDANS, from wild-type and C91 V enzymes followed by amino acid sequencing . Therefore, only Cys323 contains a free SH group . Replacement of cysteine residues with serine or valine residues resulted in a significant decrease in the kcat/Km value of the enzyme . C323S, containing no free SH group, however, retained a high specific activity, approximately 20% of the wild-type enzyme . None of the cysteine residues participate directly in the catalytic reaction. J Bacteriol, 1991 May, 173(9), 2800 - 8 Tobramycin uptake in Escherichia coli is driven by either electrical potential or ATP; Fraimow HS et al.; Aminoglycoside antibiotics such as streptomycin and tobramycin must traverse the bacterial cytoplasmic membrane prior to initiating lethal effects . Previous data on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis have demonstrated that transport of aminoglycosides is regulated by delta psi, the electrical component of the proton motive force . However, several laboratories have observed that growth of bacterial cells can occur in the apparent absence of delta psi, and we wished to confirm these studies with E . coli and further investigate whether transport of aminoglycosides could occur in the absence of a membrane potential . Treatment of acrA strain CL2 with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissipated delta psi, decreased intracellular ATP levels, and resulted in cessation of growth; after a variable period of time (3 to 7 h), growth resumed, ultimately achieving growth rates comparable to those of untreated cells . Absence of delta psi in these cells was confirmed by absence of {3H}tetraphenyl phosphonium+ uptake as measured by membrane filtration, lack of flagellar motion, and inability of these cells to transport proline (but not methionine) . Regrowth was associated with restoration of normal intracellular ATP as measured by luciferin-luciferase bioluminescence assay . Unlike unacclimatized CL2 cells treated with CCCP, these cells transported {3H}tobramycin similarly to untreated cells; aminoglycoside-induced killing was seen in association with transport . These studies suggest that under certain circumstances aminoglycoside transport can be driven by ATP (or other high-energy activated phosphate donors) alone, in the absence of a measurable delta psi . delta uncBC mutants of CL2 incapable of interconverting delta psi and ATP were treated with CCCP, resulting in dissipation of delta psi but no alteration in ATP content . Despite maintenance of normal ATP, there was no transport of {3H} bramycin, confirming that under normal growth conditions ATP has no role in the transport of aminoglycosides. Mol Microbiol, 1991 May, 5(5), 1145 - 9 Chromosome strand segregation during sporulation in Bacillus subtilis; Errington J et al.; After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e . the prespore and the mother cell . The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties . We have used a procedure originally developed to label the terminus region of the B . subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation . We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated . The results show that the sister chromosomes segregate at random into the prespore . This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation. Plasmid, 1991 May, 25(3), 190 - 7 Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis; Boe L et al.; Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis . The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers . The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers . Treatment with rifampicin did not result in the accumulation of single-stranded DNA . However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers . From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation . Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis. Genetika, 1991 May, 27(5), 801 - 8 {Features of transformation of competent cells and Bacillus subtilis protoplasts by integrative vectors}; Nezametdinova VZ et al.; The effect of structural peculiarities of DNAs from integrative plasmids on the transformation activity was studied . Monomeric forms of the plasmids can only transform B . subtilis competent cells, when plasmid selective marker is inserted into chromosomal fragment within the plasmid . Polymeric forms are needed for efficient transformation . Both single- and double-stranded DNAs of integrative plasmids transform no B.subtilis protoplasts, this being irrespective of plasmid structure. J Appl Bacteriol, 1991 May, 70(5), 427 - 36 The effects of some halogen-containing compounds on Bacillus subtilis endospores; Williams ND et al.; Sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were more active against Bacillus subtilis 8236 spores in both viability and in germination and outgrowth studies than were polyvinylpyrrolidone-iodine (PVP-I) and Lugol's solution . Of the two chlorine compounds studied NaOCl proved to be the more active . The two iodine-containing compounds gave contrasting results with the Lugol's solution demonstrating increased antibacterial activity with increasing available iodine concentration . The antibacterial behaviour of PVP-I, however, reflected the more complex nature of aqueous iodine-surfactant mixtures . Initially, non-complexed iodine concentration (the active species) increased with increasing total available iodine concentration, resulting in increasing antibacterial activity . However, due to changes in the physical properties of the mixture, a maximum concentration of non-complexed iodine was reached so that a further increase in total available iodine resulted in a decrease in non-complexed iodine concentration and consequently a decrease in the antibacterial activity of the solution was observed . A greater inhibitory effect was observed in subsequent germination and outgrowth studies when spores were pre-treated with respective biocide than when untreated spores were added to germination media containing biocide at t = 0 . This may reflect a combination of different contact times plus the neutralizing effect of the germination media on such halogen compounds. J Gen Microbiol, 1991 May, 137 ( Pt 5), 1135 - 43 Identification of aecA mutations in Bacillus subtilis as nucleotide substitutions in the untranslated leader region of the aspartokinase II operon; Lu Y et al.; Recent genetic mapping of the aspartokinase II (lysC) operon of Bacillus subtilis {M . Petricek . L . Rutberg & L . Hederstedt (1989) FEMS Microbiology Letters 61, 85-88; N.Y . Chen . J . J . Zhang & H . Paulus (1989) Journal of General Microbiology 135, 2931-2940} has shown its chromosomal location to be close to the aecA locus, the mutation of which leads to highly increased levels of aspartokinase II . In order to examine the relationship between lysC and aecA, we have cloned the control regions of the lysC operon from several independent aecA mutants and determined their nucleotide sequences . The nucleotide sequences of the aecA mutants differed from the wild-type sequence by the substitution of one or two nucleotides at two widely separated sites in the transcribed leader region of the lysC operon . To confirm that the observed nucleotide changes are indeed responsible for the AecA phenotype and not simply the reflection of sequence polymorphisms in different B . subtilis strains, we introduced the same nucleotide substitutions as those observed in the aecA strains into the leader region of the wild-type lysC operon by oligonucleotide-directed mutagenesis . The expression of the mutagenized genes was analysed after transcriptional or translational fusion to lacZ in a single-copy integration vector . The levels of beta-galactosidase were greatly elevated by the nucleotide substitutions, with similar increases observed in transcriptional and translational fusions . The high level of expression of beta-galactosidase in the lysC'-lac'Z strains with nucleotide substitutions corresponding to the aecA mutations was resistant to repression by L-lysine but was completely abolished by the inactivation of the lysC promoter.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Microbiol, 1991 M