Bacillus subtilis

Genetics, 1995 Jul, 140(3), 917 - 32
The log-linear relationship between sexual isolation and sequence divergence in Bacillus transformation is robust; Zawadzki P et al.; The relationship between sexual isolation and sequence divergence in Bacillus transformation was previously shown to be log linear . In the present study, we have shown that this relationship is robust with respect to naturally occurring genetic variation among recipient strains of Bacillus subtilis and B . mojavensis . Naturally occurring restriction endonuclease activity was shown not to affect this relationship . Also, seven out of eight recombination mutants tested for their sensitivity to sequence divergence have shown the same relationship between sequence divergence and sexual isolation; a mutant for recH was more sensitive to sequence divergence, suggesting that the product of this gene may be involved in resolution of mismatches in heterogamic transformation . We have also shown that the relationship between sexual isolation and sequence divergence is robust with respect to variation in the conditions of transformation, including variation in the length of donor DNA, the concentration of donor DNA, and intracellular competition between donor-derived and recipient-derived DNA . The robustness of the relationship between sexual isolation and sequence divergence among naturally occurring strains and across transformation conditions allows us to predict the eventual outcome of sequence divergence among B . subtilis and its closest relatives.

Biotechnol Prog, 1995 Jul-Aug, 11(4), 475 - 8
Metabolic engineering of Escherichia coli to enhance recombinant protein production through acetate reduction; Aristidou AA et al.; Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities . In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumulation in Escherichia coli is presented . The alsS gene from Bacillus subtilis encoding the enzyme acetolactate synthase was introduced into E . coli cells using a multicopy plasmid . This newly introduced heterologous enzyme modifies the glycolytic fluxes by redirecting excess pyruvate away from acetate to acetolactate . Acetolactate is then converted to a nonacidic and less harmful byproduct acetoin, which appears in the broth . Furthermore, comparative fermentation studies show that the reduction in acetate accumulation leads to a significant improvement of recombinant protein production . The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the specific protein activity (to a level of 30% of total cellular protein) and 50% in terms of the volumetric activity in a batch fermenter . In fed-batch cultivation, the engineered strain achieved a volumetric recombinant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-galactosidase), which represented a 220% enhancement over the control strain . In the meantime, acetate excretion was maintained below 20 mM compared with 80 mM for the control, and the final cell density was improved by 35%.

Biotechnol Prog, 1995 Jul-Aug, 11(4), 380 - 5
Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures: emergence of pyruvate kinase as a potential metabolic engineering site; Goel A et al.; Microbial cultures typically produce acids when metabolizing the common carbon source, glucose . Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and culture stability, thereby reducing productivity . On the basis of prior work, acid production was attributed to be due to a mismatch between glycolytic and tricarboxylic acid (TCA) cycle capacities . To suppress acid production, a strategy entailing adding citrate to glucose minimal medium proved extremely effective . The effect of citrate on in-vivo flux distribution was quantified using a detailed flux-model . When the molar glucose-citrate ratio was varied between 3 and 6, a significant reduction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose . Adding other biosynthetic precursors such as glutamine did not invoke the same suppression, thus indicating that citrate's effect is at the regulatory level . We hypothesized that the reduction of glycolytic flux in the presence of citrate results from its transport being coupled with the uptake of divalent metal ions . Citrate transport alters the intracellular balance of metal ions which in turn could trigger a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), the regulatory enzymes of glycolysis . On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potential metabolic engineering site . It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a much lower tendency for making acid byproducts.

Eur J Biochem, 1995 Jul 1, 231(1), 236 - 41
Expression, purification and characterisation of the product from the Bacillus subtilis hemD gene, uroporphyrinogen III synthase; Stamford NP et al.; Uroporphyrinogen III synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane . The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage lambda PR and PL promoters in a pCE30-derived vector . Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of uroporphyrinogen III synthase . The protein produced was soluble and was readily purified . Pure uroporphyrinogen III synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3 . Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg-1 and the Km for this substrate is 330 +/- 30 nM . The N-terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Leu, in agreement with the gene-derived sequence . Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of uroporphyrinogen III synthase . Significantly, this synthase from B . subtilis is substantially more thermostable than the enzymes from previously studied sources.

Biochem J, 1995 Jul 1, 309 ( Pt 1), 279 - 83
High-yield purification of cytochrome aa3 and cytochrome caa3 oxidases from Bacillus subtilis plasma membranes; Henning W et al.; When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa3-quinol oxidase and cytochrome caa3 oxidase . This paper describes a high-yield conventional procedure for purifying the two haem A-containing oxidases from the same aerobic culture of Bacillus subtilis . Yields of close to 40% of the total haem A are achieved and about 6 mg of each of the purified oxidases is obtained from 4 litres of liquid culture . Both of the purified enzymes have two subunits, with apparent molecular masses of 71.6 kDa and 34.3 kDa for the cytochrome caa3 oxidase, and 67.6 kDa and 37.2 kDa for aa3-quinol oxidase . These features are in agreement with the sequence data for the corresponding structural genes in the aa3 and caa3 operons of B . subtilis . Some spectral and enzymic features of the two purified oxidases are reported that are consistent with the inclusion of both of these enzymes as members of the cytochrome oxidase superfamily.

Biochem J, 1995 Jul 1, 309 ( Pt 1), 113 - 8
Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane; Hernandez L et al.; Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10) . This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4 . The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5 . Its activity is optimal at pH 5.0 . It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis) . In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type . A . diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate . The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase . The kinetic parameters of the two enzymes are of the same order of magnitude . The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A . diazotrophicus levansucrase during sucrose transformation . We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.

Appl Environ Microbiol, 1995 Jul, 61(7), 2787 - 90
Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from killing by dry heat; Setlow B et al.; Dry Bacillus subtilis spores lacking their two major DNA-binding proteins (small, acid-soluble proteins {SASP} alpha and beta) were much more sensitive to dry heat than were wild-type spores . Survivors of dry heat treatment of both wild-type and mutant spores exhibited a high frequency of mutations, and the DNA from the heated spores had increased numbers of single-strand breaks . These data indicate that SASP alpha and beta provide significant protection to spore DNA against the damaging effects of dry heat . This DNA damage may be in part depurination, and a purified alpha/beta-type SASP gave significant protection against dry heat-induced DNA depurination in vitro.

J Bacteriol, 1995 Jul, 177(14), 4144 - 8
Translation of the open reading frame encoded by comS, a gene of the srf operon, is necessary for the development of genetic competence, but not surfactin biosynthesis, in Bacillus subtilis; D'Souza C et al.; A small open reading frame, comS of the srf operon, is the site of mutations that impair competence development in Bacillus subtilis . comS open reading frame translation was required for competence, as was confirmed by the suppression of a comS amber mutation {comS(Am)} by the nonsense suppressor sup-3 . comS(Am), when introduced into the srf operon, eliminated late competence gene expression but had no significant effect on surfactin production.

J Bacteriol, 1995 Jul, 177(14), 4105 - 12
The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift; Deuerling E et al.; The ftsH gene of Bacillus subtilis has been identified as a salt-sensitive insertion mutation in strain UG1 . Here, we show that UG1 has an insertion near the 3' end of ftsH . The salt sensitivity of this mutant was caused by reduction of ftsH mRNA levels by the synthesis of an artificial antisense RNA originating at a promoter located within the insertion and reading backwards into the ftsH gene . The salt-sensitive phenotype could be overcome by deleting the promoter from which the antisense RNA was transcribed . A physiological analysis of the isogenic wild-type strain in minimal medium revealed unimpaired growth at up to 1 M NaCl, and growth above 1.2 M NaCl was observed only after addition of the osmoprotectant proline or glycine betaine . In contrast, growth of strain UG1 was reduced at a salt concentration above 0.2 M, which could be rescued by the two compatible solutes already mentioned and also by trehalose . Primer extension revealed one potential transcription start site downstream of a putative vegetative promoter, which was activated after osmotic or temperature upshift . Northern (RNA blot) experiments led to the detection of a 2.1-kb transcript, suggesting that ftsH is monocistronic . A transcriptional fusion between ftsH and the gus reporter gene exhibited a twofold increase in beta-glucuronidase activity after osmotic upshift . To further confirm the need for an enhanced level of FtsH protein after osmotic upshift, the promoter was replaced by the sucrose-inducible promoter PsacB . Whereas this mutant strain could grow in the absence of inducer in LB medium, it stopped growth immediately after addition of 1.1 M NaCl . We conclude that an increased amount of FtsH protein is essential for B . subtilis to cope with an increase in osmolarity or temperature.

J Bacteriol, 1995 Jul, 177(14), 3923 - 31
Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis; Hauser PM et al.; To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation . Because the propensity of B . subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells . From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift . The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle . The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.

J Bacteriol, 1995 Jul, 177(14), 3904 - 10
Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated; Ahmed M et al.; The Bacillus subtilis genome encodes two multidrug efflux transporters sharing 51% sequence identity: Bmr, described previously, and Blt, described here . Overexpression of either transporter in B . subtilis leads to a similar increase in resistance to ethidium bromide, rhodamine and acridine dyes, tetraphenylphosphonium, doxorubicin, and fluoroquinolone antibiotics . However, Blt differs widely from Bmr in its expression pattern . Under standard cultivation conditions, B . subtilis expresses Bmr but Blt expression is undetectable . We have previously shown that Bmr expression is regulated by BmrR, a member of the family of MerR-like transcriptional activators . Here we show that blt transcription is regulated by another member of the same family, BltR . The DNA-binding domains of BmrR and BltR are related, but their putative inducer-binding domains are dissimilar, suggesting that Bmr and Blt are expressed in response to different inducers . Indeed, rhodamine, a substrate of Bmr and Blt and a known inducer of Bmr expression, does not induce Blt expression . Blt expression has been observed only in B . subtilis, carrying mutation acfA, which, as we show here, alters the sequence of the blt gene promoter . Unlike bmr, which is transcribed as a monocistronic mRNA, blt is cotranscribed with a downstream gene encoding a putative acetyltransferase . Overall, the differences in transcriptional control and operon organization between bmr and blt suggest that the transporters encoded by these genes have independent functions involving the transport of distinct physiological compounds.

J Bacteriol, 1995 Jul, 177(13), 3855 - 62
Characterization of the involvement of two compensatory autolysins in mother cell lysis during sporulation of Bacillus subtilis 168; Smith TJ et al.; The 30-kDa sporulation-specific peptidoglycan hydrolase CwlC of Bacillus subtilis 168 was purified and characterized . It is an N-acetylmuramoyl-L-alanine amidase (amidase) that is associated with the mother cell wall of sporulating cells, and although it is secreted, it undergoes no N-terminal processing except removal of the initial methionine . It was found that mother cells of a strain insertionally inactivated in cwlC and lytC (the major vegetative amidase gene) did not lyse at the end of sporulation . Mutants with single mutations in cwlC or lytC lysed, and so the two autolysins must have mutually compensatory roles in mother cell lysis . Active CwlC and LytC are present at the time of mother cell lysis; however, reporter gene analysis revealed that lytC transcription ceases early in sporulation, and therefore the function that LytC has in mother cell lysis is performed by material remaining from presporulation expression . Autolytic enzymes similar in molecular mass to CwlC were detected in two other Bacillus species by their cross-reactivity with anti-CwlC antiserum.

J Bacteriol, 1995 Jul, 177(13), 3771 - 80
Separate mechanisms activate sigma B of Bacillus subtilis in response to environmental and metabolic stresses; Voelker U et al.; sigma B is a secondary sigma factor that controls the general stress response of Bacillus subtilis . sigma B-dependent transcription is induced by the activation of sigma B itself, a process that involves release of sigma B from an inhibitory complex with its primary regulator, RsbW . sigma B becomes available to RNA polymerase when RsbW forms a complex with an additional regulatory protein (RsbV) and, because of this, fails to bind sigma B . Using Western blot (immunoblot) analyses, reporter gene fusion assays, and measurements of nucleotide pool sizes, we provide evidence for two independent processes that promote the binding of RsbW to RsbV . The first occurs during carbon limitation or entry into stationary phase . Activation of sigma B under these circumstances correlates with a drop in the intracellular levels of ATP and may be a direct consequence of ATP levels on RsbW's binding preference . The second activation process relies on the product of a third regulatory gene, rsbU . RsbU is dispensable for sigma B activation during carbon limitation or stationary phase but is needed for activation of sigma B in response to any of a number of different environmental insults (ethanol treatment, salt or acid shock, etc.) . RsbU, or a process dependent on it, alters RsbW binding without regard for intracellular levels of ATP . In at least some instances, the effects of multiple inducing stimuli are additive . The data are consistent with RsbW being a regulator at which distinct signals from separate effectors can be integrated to modulate sigma B activity.

J Bacteriol, 1995 Jul, 177(13), 3736 - 42
Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth; Hicks KA et al.; spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis . Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth . We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth . In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation . However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth . The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon . The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H . The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H . The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.

J Bacteriol, 1995 Jul, 177(13), 3687 - 94
A single amino acid substitution in sigma E affects its ability to bind core RNA polymerase; Shuler MF et al.; We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis . sigma E is required for the production of endospores in B . subtilis but not for vegetative growth . Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores . Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase . The substitution at position 68 did not affect the stability of the protein in B . subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor . The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.

Microbiology, 1995 Jul, 141 ( Pt 7), 1781 - 4
An operon encoding a novel ABC-type transport system in Bacillus subtilis; Rodriguez F et al.; Downstream from the surfactin synthetase operon in Bacillus subtilis a new operon-type structure has been localized which, on the basis of sequence determination, potentially encodes an ABC-type transport system . The 268 amino acid protein, the product of orf1, represents the solute-binding component of the system whereas the orf2 product, a 234 amino acid protein, is the transmembrane component . Finally orf3 potentially encodes a typical 241 amino acid ATP-binding protein involved in energy supply . Comparison of the three proteins with the subunits of other ABC-type systems suggests that this new system is involved in amino acid transport.

Microbiology, 1995 Jul, 141 ( Pt 7), 1771 - 9
Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure; Jacobs MF et al.; High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants . We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy . Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted . This led to the identification of a conditional B . subtilis secretion mutant after nitrosoguanidine mutagenesis . At 42 degrees C, but not at 30 degrees C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and and was also defective in the secretion of subtilisin Carlsberg . The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non-permissive temperature . The secretion defect was not linked to the secA/div locus.

Mol Microbiol, 1995 Jul, 17(2), 281 - 90
Evidence that the transcriptional activator Spo0A interacts with two sigma factors in Bacillus subtilis; Baldus JM et al.; The transcriptional regulator Spo0A activates transcription from two types of promoters . One type of promoter is used by RNA polymerase containing sigma A, whereas the other type is used by RNA polymerase containing sigma H . There are Spo0A-binding sites near the -35 region of both types of promoters . It has been reported that some transcriptional regulators that bind near the -35 regions of promoters directly interact with the sigma subunit of RNA polymerase . Therefore, we looked for evidence that Spo0A interacts with both sigma factors by searching for single amino acid substitutions in these factors that specifically prevent expression from Spo0A-dependent promoters, but that do not decrease activity of Spo0A-independent promoters . Two such amino acid substitutions were isolated in sigma A and one was isolated in sigma H . The amino acid substitutions in sigma A prevented expression from the Spo0A-activated promoters, spoIIG and spoIIE, but expression was not impaired from the Spo0A-independent, sigma A-dependent promoter tms or from the Spo0A-activated, sigma H-dependent promoter, spoIIA . The amino acid substitution in sigma H prevented expression from the spoIIA promoter but not from the Spo0A-independent promoter, citGp2, which is used by sigma H-RNA polymerase . All of these amino acid substitutions occur in the carboxyl terminus of the sigma factors . These amino acid substitutions may define the sites of contact between the sigma factors and Spo0A . The ability of response regulators such as Spo0A to interact with multiple sigma factors may increase the variety of responses made by bacteria using a limited number of transcription factors.

Mol Microbiol, 1995 Jul, 17(2), 271 - 9
The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli; Voskuil MI et al.; The promoter (amyP) of the Bacillus subtilis alpha-amylase gene, which is recognized by E sigma A, has a three out of six match to the consensus promoter in both the -35 and -10 hexamers . Oligonucleotide-directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers . Mutations in the sequence TGTG extending from positions -18 to -15 (the -16 region) caused a 5-94-fold decrease in alpha-amylase production . A G-C transversion at position -15 was the most detrimental mutation: it essentially eliminated amyP utilization in B . subtilis and in Escherichia coli . Mutating the -35 and -10 hexamers to the E sigma A consensus promoter increased alpha-amylase production 56-fold in B . subtilis and fivefold in E . coli . Introducing the -15 G to C transversion into the consensus promoter reduced alpha-amylase production threefold, in contrast to the 94-fold reduction for the wild-type promoter in B . subtilis . The -15 G to C transversion did not reduce alpha-amylase synthesis directed by the consensus promoter in E . coli . The alpha-amylase gene is subject to two forms of transcriptional regulation: catabolite repression and temporal regulation . None of the mutants constructed in this study had any effect on either type of regulation . The -16 region, especially the G at position -15, appears to be moderately conserved in B . subtilis and in other Gram-positive organisms and weakly conserved in E . coli . The evidence suggests that the -16 region is an additional region of E sigma A promoters in B . subtilis and E sigma 70 promoters in E . coli, essential in some weak promoters such as the alpha-amylase promoter but, of little benefit to very strong promoters.

Mol Microbiol, 1995 Jul, 17(1), 13 - 23
Identification and characterization of new DNA replication terminators in Bacillus subtilis; Franks AH et al.; A functional DNA replication terminator of Bacillus subtilis contains two overlapping binding sites, A and B, for the replication terminator protein (RTP) . A degenerate 17-mer oligonucleotide corresponding to the consensus B site has been used to detect four new terminators in the B . subtilis chromosome, in addition to the previously identified and closely spaced IRI and IRII . All the new terminators lie in the terminus region of the chromosome, on both sides of IRI and IRII, with their positions spanning < 10% of its length . Their DNA sequences are characterized by clearly identifiable A- and B-binding sites . They bind RTP in a manner indistinguishable from IRI, although precise affinities have not been compared . Each new terminator is functional in causing fork arrest when present in a plasmid replicating in B . subtilis . Three of the four were tested for polarity in fork-arrest activity and exhibited the polarity expected . The total of six terminators now identified in B . subtilis have been named TerI-TerVI . TerI and TerII correspond to the previously identified IRI and IRII, respectively . The chromosomal orientations of all but one of the terminators (TerIV) have been established and they conform to an arrangement similar to that in Escherichia coli in which two opposed groups of polar terminators provide a replication-fork trap ensuring that the approaching forks meet within a restricted region of the chromosome . The development of a strikingly similar arrangement of terminators in the two organisms, despite the lack of any detectable similarity in their respective DNA terminators and terminator proteins, emphasizes the importance of the replication-fork trap in each case.

J Mol Biol, 1995 Jun 30, 250(1), 11 - 23
Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis; Asayama M et al.; The Spo0A protein of Bacillus subtilis is a transcriptional regulator that shows extensive homology to the regulator proteins in bacterial two-component regulatory systems . Phosphorylation of Spo0A is absolutely necessary for the initiation of sporulation . We now show that phospho-Spo0A is a dimer, binds specifically to the spo0F promoter region, and stimulates the transcription from the P2 promoter recognized by sigma H-RNA polymerase . Biochemical and biological analyses suggest that phospho-Spo0A interacts directly with the "0A-like box" sequence (TGTCGTA) located in the spo0F promoter region . Phosphorylation of Spo0A enhanced its affinity to the 0A-like box . Evidence is also presented that the spo0F promoter region contains a static bend having two sets of oligo(dA-dT) tracts . It was demonstrated that the bending region overlaps with the recognition site for the phospho-Spo0A.

J Mol Biol, 1995 Jun 23, 249(5), 843 - 56
Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis; Gardan R et al.; Three genes called rocD, rocE and rocF were found near the rocR gene in B . subtilis . The product of rocD is similar to eukaryotic ornithine aminotransferases . The product of rocE shares similarity with the product of B . subtilis rocC and with the product of E . coli lysP . The rocE gene may encode an arginine permease . The rocF gene encodes a polypeptide similar to several arginases . Heterologous expression in E . coli indicated that rocD encodes an ornithine aminotransferase and that rocF encodes an arginase . Arginine utilization was abolished in both rocD and rocF mutants of B . subtilis confirming the role of these genes in arginine catabolism . The rocDEF genes form an operon transcribed from a -12, -24 promoter almost identical to the -12, -24 promoter of the rocABC operon . The expression of the rocDEF operon was induced by the presence of arginine, ornithine or proline in the growth medium and depended on the presence of the sigma factor SigL . Transcription of this operon was also abolished in a B . subtilis strain containing a null mutation in the regulatory gene rocR . Two tandemly repeated upstream activating sequences very similar to those previously identified in the rocABC system were found centered at positions -120 and -70, respectively, upstream from the transcription start site of rocDEF . Deletion analysis showed that at least one upstream activating sequence is involved in the expression of the rocDEF operon . These sequences are probably the target of RocR . Analysis of a rocR'-'lacZ fusion strain showed that the expression of rocR is not induced by arginine and is negatively autoregulated.

J Mol Biol, 1995 Jun 16, 249(4), 743 - 53
The Bacillus subtilis flagellar regulatory protein sigma D: overproduction, domain analysis and DNA-binding properties; Chen YF et al.; Flagellar biosynthesis requires an alternative sigma (sigma) subunit of RNA polymerase to allow recognition of the promoters for flagellin and other late genes of the flagellar regulon . We have now overproduced and characterized Bacillus subtilis sigma D: the prototype of the sigma 28 family of flagellar sigma factors . Limited protease digestion studies indicate that sigma D contains an amino-terminal domain, comprising conserved regions 1.2 and 2, and a carboxyl-terminal domain containing conserved regions 3.2 and 4 . The protease-sensitive region between these two domains correlates with a region of very low sequence conservation among bacterial sigma factors . Unlike the primary sigma factor, sigma D binds to DNA . In non-denaturing polyacrylamide gel electrophoresis the sigma D-DNA complex has an apparent equilibrium dissociation constant of 1 microM . Binding of sigma D to the promoter for flagellin, PD-6, appears to lead to an altered DNA structure near the -35 and -10 recognition elements as detected by DNase I footprinting and by the enhanced reactivity of several bases to dimethylsulfate.

EMBO J, 1995 Jun 15, 14(12), 2935 - 44
Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding; Hardt WD et al.; We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA . Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions . Partially modified E . coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation . Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354 . Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA . Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs . In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E . coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

J Mol Biol, 1995 Jun 2, 249(2), 319 - 31
Stabilization of a ribosomal RNA tertiary structure by ribosomal protein L11; Xing Y et al.; Interactions between ribosomal protein L11 and a domain of large subunit rRNA have been highly conserved and are essential for efficient protein synthesis . To study the effects of L11 on rRNA folding, a homolog of the Escherichia coli L11 gene has been amplified from Bacillus stearothermophilus DNA and cloned into a phage T7 polymerase-based expression system . The expressed protein is 93% homologous to the L11 homolog from Bacillus subtilis, denatures at temperatures above 72 degrees C, and has nearly identical rRNA binding properties as the Escherichia coli L11 in terms of RNA affinity constants and their dependences on temperature, Mg2+ concentration, monovalent cation, and RNA mutations . Mg2+ and NH4+ are specifically bound by the RNA-protein complex, with apparent ion-RNA affinities of 1.6 mM-1 and 19 M-1, respectively, at 0 degree C . The effect of the thermostable L11 on the unfolding of a 60 nucleotide rRNA fragment containing its binding domain has been examined in melting experiments . The lowest temperature RNA transition, which is attributed to tertiary structure unfolding, is stabilized by approximately 25 degrees C, and the interaction has an intrinsic enthalpy of approximately 13 kcal/mol . The thermal stability of the protein-RNA complex is enhanced by increasing Mg2+ concentration and by NH4+ relative to Na+ . Thus L11, NH4+, and Mg2+ all bind and stabilize the same rRNA tertiary interactions, which are conserved and presumably important for ribosome function.

Genes Dev, 1995 Jun 1, 9(11), 1316 - 26
A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis; Wu LJ et al.; Spore formation in Bacillus subtilis begins with an asymmetric cell division that superficially resembles the division of vegetative cells . Mutations in the spoIIIE gene of B . subtilis partially block partitioning of one chromosome into the smaller (prespore) compartment of the sporulating cell . Point mutations that specifically block prespore chromosome partitioning affect a carboxy-terminal domain of SpoIIIE that shows significant sequence similarity to the DNA transfer (Tra) proteins of several conjugative plasmids of Streptomyces . In wild-type sporulating cells, the prespore chromosome passes through an intermediate stage resembling the state in which spoIIIE mutant cells are blocked . The prespore chromosome is then transferred progressively through the newly formed spore septum . We propose that translocation of the prespore chromosome occurs by a mechanism that is functionally related to the conjugative transfer of plasmid DNA.

Appl Environ Microbiol, 1995 Jun, 61(6), 2224 - 9
Gene cloning, nucleotide sequence, and expression of a cephalosporin-C deacetylase from Bacillus subtilis; Mitsushima K et al.; The gene encoding a cephalosporin-C deacetylase (CAH) from Bacillus subtilis SHS 0133 was cloned and sequenced . The nucleotide sequence contained an open reading frame encoding a polypeptide consisting of 318 amino acids, the molecular weight of which was in good agreement with the value obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The deduced amino acid sequence contained the common sequence Gly-X-Ser-X-Gly found in many esterases, lipases, and serine proteases . This indicates that CAH is a serine enzyme . A possible promoter sequence which is very similar to the consensus sequences of -35 and -10 regions recognized by B . subtilis RNA polymerase utilizing sigma factor H was found in the 5'-flanking region of the CAH structural gene . Two repeated A+T-rich blocks consisting of 24 bp were also found in the upstream region of the initiation codon . We constructed a series of expression plasmids by inserting the CAH gene into Escherichia coli ATG vectors . The degree of CAH gene expression depended on promoters and vector plasmids, which have different replication origins . The expressed CAH protein was an active form in the soluble fraction obtained after cell disruption . The highest expression level was accomplished with an expression plasmid, pCAH400, which has the trp promoter and the replication origin derived from pAT153 . In the fermentation using a 30-liter jar fermentor, the transformant E . coli JM103(pCAH400) produced 440 U of CAH per ml of culture during a 24-h incubation . This value corresponded to 2.1 g of CAH protein in 1 liter of culture broth.

Curr Microbiol, 1995 Jun, 30(6), 357 - 66
A new alkaline serine protease from alkalophilic Bacillus sp.: cloning, sequencing, and characterization of an intracellular protease; Yamagata Y et al.; To obtain a new serine protease from alkalophilic Bacillus sp . NKS-21, shotgun cloning was carried out . As a result, a new protease gene was obtained . It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence . The molecular weight was 34,624 . The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B . polymyxa, and alkalophilic Bacillus sp . No . 221 . The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus) . The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out . The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants . The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.

J Bacteriol, 1995 Jun, 177(12), 3540 - 5
A gene at 333 degrees on the Bacillus subtilis chromosome encodes the newly identified sigma B-dependent general stress protein GspA; Antelmann H et al.; In Bacillus subtilis, general stress proteins (Gsps) are induced in response to different stresses (heat, salt, or ethanol) or after nutrient starvation . The majority of the genes for the Gsps are organized in a very large stationary-phase or stress regulon which is controlled by alternative sigma factor sigma B . The most striking spots on Coomassie-stained two-dimensional gels belong to GsiB and GspA, which are synthesized at extremely high levels in response to different stresses . Therefore, we determined the N-terminal protein sequence of GspA, which exhibited total identity to a hypothetical 33.5-kDa protein of B . subtilis encoded by open reading frame 2 (ipa-12d) in the sacY-tyrS1 intergenic region . The GspA-encoding gene gspA and the upstream and downstream regions were cloned with the aid of the PCR technique . By primer extension experiments, one sigma B-dependent promoter immediately upstream of the coding region was identified . A putative factor-independent terminator closely followed the coding region . By Northern (RNA) blot analysis, a 0.95-kb transcript was detected which indicates a monocistronic transcriptional unit . The gspA mRNA was strongly induced by different stimuli like heat or salt stress and starvation for glucose . Analysis of RNA isolated from a sigma B deletion mutant revealed that the transcription of gspA is sigma B dependent . Insertional inactivation of the B . subtilis chromosomal gspA gene confirmed that the gspA gene is not essential for either vegetative growth or growth under the influence of different stresses . In gspA mutant cells, the level of flagellin was increased severalfold over that in wild-type cells.

J Bacteriol, 1995 Jun, 177(12), 3451 - 4
DNA restriction-modification systems mediate plasmid maintenance; Kulakauskas S et al.; Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp . strain RFL6), enhance plasmid segregational stability in E . coli and Bacillus subtilis, respectively . Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems . We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells . Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease . Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome . Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells . Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.

J Bacteriol, 1995 Jun, 177(12), 3394 - 406
Characterization of cotJ, a sigma E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores; Henriques AO et al.; The outermost protective structure found in endospores of Bacillus subtilis is a thick protein shell known as the coat, which makes a key contribution to the resistance properties of the mature spore and also plays a role in its interaction with compounds able to trigger germination . The coat is organized as a lamellar inner layer and an electron-dense outer layer and has a complex polypeptide composition . Here we report the cloning and characterization of an operon, cotJ, located at about 62 degrees on the B . subtilis genetic map, whose inactivation results in the production of spores with an altered pattern of coat polypeptides . The cotJ operon was identified by screening a random library of lacZ transcriptional fusions for a conditional (inducer-dependent) Lac+ phenotype in cells of a strain in which the structural gene (spoIIGB) for the early-acting, mother-cell-specific transcriptional factor sigma E was placed under the control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible Pspac promoter . Sequence analysis of cloned DNA from the cotJ region complemented by genetic experiments revealed a tricistronic operon preceded by a strong sigma E-like promoter . Expression of an SP beta-borne cotJ-lacZ fusion commences at around h 2 of sporulation, as does expression of other sigma E-dependent genes, and shows an absolute requirement for sigma E . Studies with double-reporter strains bearing a cotJ-gusA fusion and lacZ fusions to other cot genes confirmed that expression of cotJ is initiated during sporulation prior to activation of genes known to encode coat structural proteins (with the sole exception of cotE) . An in vitro-constructed insertion-deletion mutation in cotJ resulted in the formation of spores with no detectable morphological or resistance deficiency . However, examination of the profile of electrophoretically separated spore coat proteins from the null mutant revealed a pattern that was essentially identical to that of a wild-type strain in the range of 12 to 65 kDa, except for polypeptides of 17 and 24 kDa, the putative products of the second (cotJB) and third (cotJC) cistrons of the operon, that were missing or reduced in amount in the coat of the mutant . Polypeptides of the same apparent sizes are detected in spores of a cotE null mutant, on which basis we infer that the products of the cotJ operon are required for the normal formation of the inner layers of the coat or are themselves structural components of the coat . Because the onset of cotJ transcription is temporally coincident with the appearance of active sigma E, we speculate that the cotJ-encoded products may be involved in an early state of coat assembly.

J Bacteriol, 1995 Jun, 177(12), 3386 - 93
Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis; Harry EJ et al.; We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation . Sporangia were doubly stained with propidium iodide to visualize the forespore and mother cell nucleoids and with fluorescein-conjugated antibodies to visualize the location of beta-galactosidase produced under the control of the sporulation RNA polymerase sigma factors sigma E and sigma F . In confirmation and extension of earlier reports, we found that expression of a lacZ fusion under the control of sigma E was confined to the mother cell compartment of sporangia at the septation (II) and engulfment (III) stages of morphogenesis . Conversely, sigma F-directed gene expression was confined to the forespore compartment of sporangia at postseptation stages of development . Little indication was found for sigma E- or sigma F-directed gene expression prior to septation or in both compartments of postseptation sporangia . Gene expression under the control of the forespore sigma factor sigma G also exhibited a high level of compartmentalization . A high proportion of sporangia exhibited fluorescence in our immunostaining protocol, which should be suitable for the subcellular localization of sporulation proteins for which specific antibodies are available.

J Bacteriol, 1995 Jun, 177(11), 3308 - 11
Possible role for the essential GTP-binding protein Obg in regulating the initiation of sporulation in Bacillus subtilis; Vidwans SJ et al.; We fused obg, encoding an essential GTP-binding protein in Bacillus subtilis, to the LacI-repressible, IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter Pspac . Depletion of Obg, following removal of IPTG, caused a defect in sporulation and in expression of sporulation genes that are activated by Spo0A approximately P . These defects were significantly relieved by a mutation in spo0A (rvtA11) that bypasses the normal phosphorylation pathway, indicating that Obg might normally be required, either directly or indirectly, to stimulate activity of the phosphorelay that activates Spo0A.

J Bacteriol, 1995 Jun, 177(11), 3045 - 51
ComEA, a Bacillus subtilis integral membrane protein required for genetic transformation, is needed for both DNA binding and transport; Inamine GS et al.; The competence-related phenotypes of mutations in each of the four open reading frames associated with the comE locus of Bacillus subtilis are described . comEA and comEC are required for transformability, whereas the products of comEB and of the overlapping comER, which is transcribed in the reverse direction, are dispensable . Loss of the comEA product decreases the binding of DNA to the competent cell surface and the internalization of DNA, in addition to exhibiting a profound effect on transformability . The comEC product is required for internalization but is dispensable for DNA binding . ComEA is shown to be an integral membrane protein, as predicted from hydropathy analysis, with its C-terminal domain outside the cytoplasmic membrane . This C-terminal domain possesses a sequence with similarity to those of several proteins known to be involved in nucleic acid transactions including UvrC and a human protein that binds to the replication origin of the Epstein-Barr virus.

Orig Life Evol Biosph, 1995 Jun, 25(1-3), 277 - 93
DNA stability and survival of Bacillus subtilis spores in extreme dryness; Dose K et al.; The inactivation of Bacillus subtilis spores during long-term exposure (up to several months) to extreme dryness (especially vacuum) is strain-dependent, through only to a small degree . During a first phase (lasting about four days) monolayers of spores lose about 20% of their viability, regardless of the strain studied . During this phase loss in viability can be equally attributed both to damages of hydrophobic structures (membranes and proteins) and DNA . During a second phase lasting for the remaining time of experimental observation (weeks, months and years) the loss in viability is slowed . A viability of 55% to 75% (depending on the strain) is attained after a total exposure of 36 days . The loss in viability during the second phase can be correlated with the occurrence of DNA double strand breaks . Also covalent DNA-protein cross-links are formed by vacuum exposure . If the protein moiety of these cross-links is degraded by proteinase K-treatment in vitro additional DNA double strand breaks result . The data are also discussed with respect to survival on Mars and in near Earth orbits.

J Mol Evol, 1995 Jun, 40(6), 585 - 93
Identification and chromosomal distribution of DNA sequence segments conserved since divergence of Escherichia coli and Bacillus subtilis; Kunisawa T; DNA sequence segments conserved since divergence of Escherichia coli and Bacillus subtilis were identified, using the GenBank sequence database . Chromosomal locations of the conserved segments were compared between the two bacteria, and the following three features were observed . (1) Although the two genomes are nearly identical in size, chromosomal arrangements of the conserved segments are considerably different from each other . (2) In many cases, chromosomal locations of a conserved segment in the two species have deviated from each other by a multiple of 60 degrees . (3) There are many instances in which a contiguous segment in one genome is split into two or more segments located at distinct positions in the other genome, and these split segments were found to tend to lie on the E . coli or B . subtilis genome separated by distances of multiples of 60 degrees . On the basis of these observations, genome organizations of the two bacteria were discussed in terms of genome doublings as well as random chromosomal rearrangements.

J Appl Bacteriol, 1995 Jun, 78(6), 669 - 76
Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils; Ababouch LH et al.; The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated . As expected, spores were more resistant to heat when suspended in oils than in buffer . This was ascribed to the low aw of oils and to their content of free fatty acids . Linear survivor curves were obtained for spores suspended in buffer at 105 degrees C or above and for B . subtilis A spores suspended in a vegetable oil . However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing . The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate . It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.

Eur J Biochem, 1995 Jun 1, 230(2), 760 - 5
Induction of terminal enzymes for heme biosynthesis during differentiation of mouse erythroleukemia cells; Taketani S et al.; To examine the induction of terminal enzymes of the heme-biosynthetic pathway during erythroid differentiation, mouse protoporphyrinogen oxidase (PPO) cDNA has been cloned . The deduced amino acid sequence derived from the nucleotide sequence revealed that mouse PPO consists of 477 amino acid residues, without the leader peptide, which is imported into mitochondria . Comparison of the amino terminus of the deduced amino acid sequence of mouse PPO cDNA with that of purified bovine PPO provided conclusive evidence for lack of the leader peptide in the former . The amino acid sequence has 86% and 28% identity with human PPO and Bacillus subtilis HemY, respectively . When mouse erythroleukemia (MEL) cells were induced with dimethylsulfoxide, PPO mRNA was induced within 12 h of treatment, and with further incubation, reached a plateau . mRNAs for coproporphyrinogen oxidase (CPO) and ferrochelatase (FEC) were induced within 12 h, and continued to increase with time up to 48 h . The activities of CPO and FEC markedly increased with time up to 72 h, while PPO activity increased 1.8-fold within 12 h and remained unchanged thereafter . Immunoblot analysis showed that levels of PPO, CPO and FEC paralleled their corresponding activities . The magnitude of PPO induction was less than that of CPO and FEC . Thus, induction of three terminal enzymes of the heme-biosynthetic pathway is an early event in MEL cell differentiation . The concomitant induction may play an important role in producing large amounts of heme during erythroid differentiation.

Trends Biotechnol, 1995 Jun, 13(6), 210 - 6
The Bacillus subtilis genome project: aims and progress; Devine KM; The members of the bacterial genus Bacillus are important organisms for both research and industrial purposes, and a major international effort is under way to sequence the complete genome of Bacillus subtilis, the type species for this genus . In this article the organization of the project is summarized; the strategies employed for cloning, sequencing and data handling; the progress to date, and the likely benefits which will accrue to basic research and to the biotechnology industry upon completion of the sequence.

Mutat Res, 1995 Jun, 347(1), 25 - 30
Thymine auxotrophy is associated with increased UV sensitivity in Escherichia coli and Bacillus subtilis; Lojo MM; Thymine auxotrophy was shown to be associated with an increase in UV sensitivity both in Bacillus subtilis and in Escherichia coli . This UV sensitization became clearly evident in polA5 mutants of Bacillus subtilis: at UV doses of 16 J/m2, a reduction of more than 10-fold in the survivor population is observed in thymine requiring spontaneous mutants (polA5 thyA thyB) compared to the parental strains (polA5) . Reversion of either thyA or thyB mutation led to a partial recovery in the UV resistance . This result suggests that DNA repair polymerization might be improved by the biosynthesis of thymidylate or some effect associated with such activity.

Arch Microbiol, 1995 Jun, 163(6), 432 - 8
Properties of the menaquinol oxidase (Qox) and of qox deletion mutants of Bacillus subtilis; Lemma E et al.; Menaquinol oxidase isolated from the membrane of Bacillus subtilis W23 was found to consist of four polypeptides (QoxA, B, C, and D) that were predicted by the sequence of the qox operon of B . subtilis 168 (Santana et al . 1992) . The preparation contained 7 mol cytochrome aa3 per g protein, which corresponds to 2 mol heme A per mol enzyme of 144 kDa molecular mass . Respiration with dimethylnaphthoquinol catalyzed by the enzyme was ten times faster than that with menadiol . Activities with more electropositive quinols were negligible . The activity of the enzyme was inhibited by equimolar amounts of HQNO, while antimycin, myxothiazol, and stigmatellin were more than tenfold less effective . When cells of both strains of B . subtilis (W23 and 168) were grown with glucose, quinol respiration was an order of magnitude more active than respiration with N,N,N',N'-tetramethyl-1,4-phenylenediamine plus ascorbate . Surprisingly, the same result was obtained with mutant strains lacking qoxB . As cytochromes a and d were virtually absent, a second quinol oxidase, possibly of the cytochrome o-type, was apparently formed by the mutants.

Microbiology, 1995 Jun, 141 ( Pt 6), 1433 - 42
A Bacillus subtilis spore coat polypeptide gene, cotS; Abe A et al.; A gene, cotS, encoding a spore coat polypeptide of Bacillus subtilis, was isolated from an EcoRI fragment (5.4 kb) of the chromosome by using synthetic oligonucleotide probes corresponding to the NH2-terminal amino acid sequence of Cot40-2 previously purified from the spore coat of B . subtilis . The nucleotide sequence (2603 bp) was determined and sequence analysis suggested the presence of two contiguous ORFs, ORF X and cotS, followed by the 5'-region of an additional ORF, ORF Y, downstream of cotS . The cotS gene is 1053 nucleotides long and encodes a polypeptide of 351 amino acids with a predicted molecular mass of 41083 Da . The predicted amino acid sequence was in complete agreement with the NH2-terminal amino acid sequence of Cot40-2 . The orfX gene is 1131 nucleotides long and encodes a polypeptide of 377 amino acids with a predicted molecular mass of 42911 Da . The gene product of cotS was confirmed to be identical to Cot40-2 by SDS-PAGE and immunoblotting from Escherichia coli transformed with a plasmid containing the cotS region . Northern hybridization analysis indicated that a transcript of cotS and orfX appeared at about 5 h after the onset of sporulation . The transcriptional start point determined by primer extension analysis suggested that -10 and -35 regions are present upstream of orfX and are very similar to the consensus sequence for the sigma k-dependent promoter . Terminator-like sequences were not found in the DNA fragment (2603 bp) sequenced in this paper, which suggested that the cotS locus may be part of a multicistronic operon . The cotS gene is located between dnaB and degQ at about 270-275 degrees on the genetic map . Insertional mutagenesis of the cotS gene by introducing an integrative plasmid resulted in no alteration of growth or sporulation, and had no effect on germination or resistance to chloroform.

J Bacteriol, 1995 Jun, 177(12), 3465 - 71
A polypurine sequence that acts as a 5' mRNA stabilizer in Bacillus subtilis; Hue KK et al.; A segment of early RNA from Bacillus subtilis bacteriophage SP82 was shown to function as a 5' stabilizer in B . subtilis . Several heterologous RNA sequences were stabilized by the presence of the SP82 sequence at the 5' end, and expression of downstream coding sequences was increased severalfold . The SP82 RNA segment encodes a B . subtilis RNase III cleavage site, but cleavage by B . subtilis RNase III was not required for stabilization . The sequence that specifies 5' stabilizer function was localized to a polypurine sequence that resembles a ribosome binding site . The ability of the SP82 sequence to stabilize downstream RNA was dependent on its position relative to the 5' end of the RNA . These results demonstrate the existence of a new type of 5' stabilizer in B . subtilis and indicate that attack at the 5' end is a principal mechanism for initiation of mRNA decay in B . subtilis.

Mol Microbiol, 1995 Jun, 16(5), 969 - 76
Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance; Podlesek Z et al.; The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined . The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed . The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins . The bcrB and bcrC genes were found to encode hydrophobic proteins, which may function as membrane components of the permease . Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli . The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target . In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane . The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the importance of various regions in transport.

J Biol Chem, 1995 May 26, 270(21), 12452 - 6
Structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis; Babitzke P et al.; A filter binding assay was used to determine the structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis . We examined the ability of L-tryptophan and 26 of its analogs to activate TRAP . Our findings show that TRAP activation by L-tryptophan is highly cooperative . We also observed that TRAP activation is stereospecific; D-tryptophan did not activate . Our results further indicate that the alpha-amino group and the carbonyl moiety of the alpha-carboxyl group of the ligand are required for TRAP activation and that the heterocyclic amino nitrogen of L-tryptophan greatly enhances TRAP activation . We also found that changes at several positions of the indole ring of L-tryptophan resulted in reduced TRAP activation . In addition, indole and 5-methylindole were shown to be effective competitors of L-tryptophan activation of TRAP.

Biochem Biophys Res Commun, 1995 May 16, 210(2), 317 - 23
Depletion of small cytoplasmic RNA confers fusidic-acid resistance on Bacillus subtilis; Shibata T et al.; Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of a signal recognition particle (SRP)-like RNA family . To analyze the function of scRNA in protein synthesis, a B . subtilis strain SC201NA was constructed in which the expression of intact scRNA is regulated by an IPTG-inducible promoter . In this strain, depletion of scRNA leads to deficient translation and sporulation as well as morphological changes . In addition, the growth of SC201NA in the absence of IPTG became fusidic-acid resistant . The acquisition of fusidic-acid resistant phenotype by depletion of scRNA suggested that scRNA is associated with elongation factor G (EF-G) in the translation process.

Mol Gen Genet, 1995 May 10, 247(3), 387 - 90
Carotenoid synthesis in Streptomyces setonii ISP5395 is induced by the gene crtS, whose product is similar to a sigma factor; Kato F et al.; In many species of actinomycetes, carotenogenesis can be photoinduced . The capacity to respond to photoinduction is, however unstable and, in various strains of Streptomyces, is lost at a relatively high frequency . In Streptomyces setonii ISP5395, which normally produces no carotenoids, carotenoid-producing mutants can be obtained following protoplast regeneration . We report here the characterization of a gene, crtS, which was isolated from one such mutant and can confer on wild-type S . setonii ISP5395 cells the capacity to synthesize carotenoids . Sequence analysis of crtS reveals an open reading frame, which shows homology to genes that encode alternative sigma factors in Bacillus subtilis . We propose that crtS encodes a sigma factor which is necessary for the expression of a cryptic gene(s) for carotenoid biosynthesis in S . setonii ISP5395.

Biochim Biophys Acta, 1995 May 10, 1229(3), 356 - 62
The trinuclear iron-sulfur cluster S3 in Bacillus subtilis succinate:menaquinone reductase; effects of a mutation in the putative cluster ligation motif on enzyme activity and EPR properties; Hagerhall C et al.; Succinate:quinone reductases (SQRs) and quinol:fumarate reductases (QFRs) each contain a bi-, a tri- and a tetra-nuclear iron-sulfur cluster . The C-terminal half of the iron-sulfur protein subunit of these enzymes shows two fully conserved motifs of cysteine residues, stereotypical for ligands of {3Fe-4S} and {4Fe-4S} clusters . To analyze the functional role of the trinuclear cluster S3 in Bacillus subtilis SQR, a fourth cysteine residue was introduced into the putative ligation motif to that cluster . A corresponding mutation in Escherichia coli QFR results in a tri- to tetranuclear conversion (Manodori et al . (1992) Biochemistry 31, 2703-2731) . We have found that presence of the extra cysteine in B . subtilis SQR does not result in cluster conversion . It does, however, affect the EPR properties of the cluster S3, whereas those of the other two clusters remain normal . The results strongly support the view that residues in the most C-terminal cysteine motif in the iron-sulfur protein subunit of SQRs and QFRs ligate the trinuclear cluster . Compared to wild-type SQR, S3 in the B . subtilis mutant enzyme is not sensitive to methanol and the midpoint redox potential is close to normal . The quinone reductase activity of the mutant enzyme is only 35% of normal . Thus, the architecture around cluster S3 plays a role in electron transfer to quinone or in the binding of quinone to the enzyme.

Biochim Biophys Acta, 1995 May 10, 1229(3), 347 - 55
Purification of three catalase isozymes from facultatively alkaliphilic Bacillus firmus OF4; Hicks DB; Cell extracts of facultatively alkaliphilic B . firmus OF4 were assayed for catalase activity and their catalase isozyme content was analyzed on native polyacrylamide gels stained for catalase activity . pH-10.5-grown cells had about twice the specific catalase activity of pH-7.5-grown cells . The higher activity, however, did not confer resistance to exogenous hydrogen peroxide challenge relative to pH-7.5-grown cells, and in fact, the pH-10.5-grown cells were much more sensitive to the challenge . Electrophoresis resolved three catalase isozymes in cell extracts . The isozymes, labeled I-III in order of decreasing electrophoretic mobility, were purified and their Nterminal amino acid sequences were obtained . Isozyme III corresponded to the product of a cloned gene fragment that had been shown to possess substantial sequence similarity to the KatE (HP-II) catalase of E . coli (Quirk, P.G., Krulwich, T.A . and Hicks, D.B . (1993) Biophys J . 64, 164A) and which had similar biochemical properties to HP-II, i.e., it was a chlorin-containing enzyme expressed only in stationary phase . Isozyme II, a protoheme enzyme, was responsible for the higher activity of alkaline-grown cells and was induced in cells treated with hydrogen peroxide or ascorbate . It showed sequence similarity to katA of Bacillus subtilis (Bol, D . and Yasbin, R . (1991) Gene 109, 31-37) . Isozyme I was the only isozyme that exhibited detectable levels of peroxidase activity in addition to catalase activity, resembling a catalase enzyme purified from a different alkaliphile, Bacillus YN-2000 (Yumoto, I., Fukumori, Y . and Yamanaka, T . (1990) J . Biochem . 108, 583-587), to which it showed some sequence similarity.

FEBS Lett, 1995 May 8, 364(2), 157 - 60
Overexpression of phosphatidylglycerophosphate synthase restores protein translocation in a secG deletion mutant of Escherichia coli at low temperature; Kontinen VP et al.; The E . coli secG deletion mutant is unable to grow and is defective in protein translocation at low temperature . A gene of Bacillus subtilis, which is able to restore the growth of the deletion mutant at low temperature, was found as a multi-copy suppressor . Sequencing of this gene revealed significant homology to E . coli pgsA, which encodes phosphatidylglycerophosphate synthase, an enzyme involved in acidic phospholipid synthesis . A plasmid carrying E . coli pgsA also restored the growth of the deletion mutant . Furthermore, protein translocation in the deletion mutant was stimulated when it harbored a plasmid carrying pgsA . A possible mechanism underlying the pgsA-dependent suppression of the secG deletion mutation is discussed.

Mol Biol (Mosk), 1995 May-Jun, 29(3), 507 - 11
{Cloning and expression of the Bacillus stearothermophilus neutral proteinase gene in Bacillus subtilis cells}; Sidorenkov IN et al.; Gene of thermostable Bacillus stearothermophilus metalloprotease was cloned and expressed in mesophilic Bacillus subtilis cells . It was demonstrated that this gene is closely related by its restriction map to B . stearothermophilus T metalloprotease gene cloned earlier . Thermostability level and thermal optimum of activity of metalloprotease, the product of cloned gene expression, were estimated.

Antibiot Khimioter, 1995 May, 40(5), 3 - 7
{The characteristics of alirin B1--the basic component of a fungicidal preparation produced by the Bacillus subtilis 10-VIZR strain}; Shenin IuD et al.; Alirin B1, the major active component of a fungicidal complex produced by Bacillus subtilis 10-VIZR was separated . Its physico-chemical and biological properties as well as the amino acid composition were investigated . Alirin B1 was shown to differ from the polypeptide substances described in the literature . The antibiotic was classified as belonging to the bacteriocins.

Biosci Biotechnol Biochem, 1995 May, 59(5), 915 - 6
A 3-deazauracil-resistant mutant of Bacillus subtilis with increased production of cytidine; Asahi S et al.; Bacillus subtilis No . 344 is a cytidine-producing mutant strain derived from wild type strain No . 122 . When 3-deazauracil-resistant mutants were derived from strain No . 344, some of the mutants had higher productivities of cytidine . Among them, strain No . 428 accumulated 14.2 mg/ml cytidine in the culture . Cytidine 5'-triphosphate (CTP) synthetase from strain No . 428 changed to be free from feedback inhibition by CTP, compared with the enzyme from strain No . 344.

Biosci Biotechnol Biochem, 1995 May, 59(5), 864 - 8
Comparison of o-phthalaldehyde modification of alpha-amylases from porcine pancreas and Bacillus subtilis with Taka-amylase A; Ueyama H et al.; A fluorescent reagent, o-phthalaldehyde (OPA), competitively inhibited porcine pancreatic alpha-amylase (PPA) with Ki values of 0.7-0.9 mM, while alpha-amylase from Bacillus subtilis (BS) was uncompetitively inhibited, with Ki values of 5.8-7.6 mM . In both cases, OPA gave a time-dependent irreversible inactivation, where the amylase activity was lost faster than the maltosidase activity . Zymograms of the course of OPA modification showed that PPA was converted into at least six, faster moving components and BS gave two components . The OPA modification was retarded by the addition of the substrate analog, cyclodextrins, and the OPA modified enzymes decreased in affinity for the substrate soluble starch . Stoichiometric measurement showed that both PPA and BS was inactivated by the incorporation of 1 mol of OPA per mol of enzyme . The role of OPA modification of alpha-amylases was discussed in relation to the regulation of catalytic activity of enzymes.

Microbiology, 1995 May, 141 ( Pt 5), 1199 - 200
The Bacillus subtilis dnaI gene is part of the dnaB operon; Bruand C et al.; The dnaI gene of Bacillus subtilis, previously identified through the isolation of the dnaI2 mutant, was found to be the second gene of the dnaB operon . The nucleotide substitution in the dnaI2 mutant gene was determined.

Microbiology, 1995 May, 141 ( Pt 5), 1193 - 8
Mutations in the glycerol kinase gene restore the ability of a ptsGHI mutant of Bacillus subtilis to grow on glycerol; Wehtje C et al.; Although glycerol is not taken up via the phosphotransferase system (PTS) in Bacillus subtilis, some mutations that affect the general components of the PTS impair the ability of cells to grow on glycerol . Five revertants of a pts deletion mutant that grow on glycerol were analysed . They were shown to carry mutations in the glycerol kinase gene . These are missense mutations located in parts of the glpK gene that could encode regions important for the activity of glycerol kinase . The results strongly suggest that the main effect of the PTS on glycerol utilization in B . subtilis is mediated via glycerol kinase.

J Bacteriol, 1995 May, 177(10), 2933 - 7
Genes that protect against the host-killing activity of the E3 protein of Bacillus subtilis bacteriophage SPO1; Wei P et al.; A cloned rpoB gene, specifying an apparently mutant RNA polymerase beta subunit, protected Escherichia coli against the cytocidal effects of the E3 protein of bacteriophage SPO1, suggesting that RNA polymerase is the primary cellular target of the E3 protein . Two segments of the wild-type E . coli genome, one of which specifies a suppressor of dnaK mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpoB did not.

J Bacteriol, 1995 May, 177(10), 2912 - 3
Site of phosphorylation of SpoIIAA, the anti-anti-sigma factor for sporulation-specific sigma F of Bacillus subtilis; Najafi SM et al.; Sigma F is regulated by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA . SpoIIAB also functions as a phosphokinase which transfers phosphate from ATP to SpoIIAA; this phosphorylation is thought to be involved in the regulatory mechanism . By using {gamma-32P}ATP to phosphorylate SpoIIAA, cleaving the protein proteolytically, and analyzing the one resulting radiolabelled peptide by the Edman degradation procedure, we show that the site of phosphorylation in SpoIIAA is Ser-58.

J Bacteriol, 1995 May, 177(10), 2863 - 9
Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coli; Tolner B et al.; Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons . This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidation of ascorbate-phenazine methosulfate or by artificial ion gradients . An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake . The transporter is specific for L-glutamate and L-aspartate . L-Glutamate transport is inhibited by beta-hydroxyaspartate and cysteic acid but not by alpha-methyl-glutamate . The gene encoding the L-glutamate transport protein of B . subtilis (gltPBsu) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was determined . Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions . UUG is most likely the start codon . gltPBsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate . Both sodium- and proton-coupled transporters belong to this family of dicarboxylate transporters . Hydropathy profiling and multiple alignment of the family of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.

J Bacteriol, 1995 May, 177(10), 2721 - 6
Purification and characterization of the phospho-alpha(1,1)glucosidase (TreA) of Bacillus subtilis 168; Gotsche S et al.; The intracellular phospho-alpha(1,1)glucosidase TreA from Bacillus subtilis has been overproduced in Escherichia coli and purified by ion-exchange chromatography and gel filtration . The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 64 kDa . Isoelectric focusing indicated homogeneity of the protein, and its pI was determined to be 4.3 . Characterization of the enzyme showed a protein which is stable up to 44 degrees C after temperature treatment for 15 min . The temperature optimum was found to be 37 degrees C, and the pH optimum was 4.5 . TreA activity is stimulated by high salt concentrations with different efficiencies depending on the kind of salt . When increasing amounts of ammonium sulfate are used, the increase of TreA activity is correlated with a conformational change of the protein or dimerization . The substrate specificity of the purified enzyme was characterized, showing additionally that trehalose is also hydrolyzed, but to a much smaller extent than trehalose-6-phosphate . In vitro, the presence of glucose reduces TreA activity, indicating product inhibition of the enzyme.

J Bacteriol, 1995 May, 177(9), 2594 - 601
Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis; Gu L et al.; We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis . This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins . The incorporation of chlamydial alpha subunit protein into the E . coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.

J Bacteriol, 1995 May, 177(9), 2572 - 5
Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon; Bower S et al.; The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized . The birA gene maps at 202 degrees on the B . subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein . Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein . The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E . coli BirA protein . B . subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E . coli.

J Bacteriol, 1995 May, 177(9), 2560 - 3
FtsZ and nucleoid segregation during outgrowth of Bacillus subtilis spores; Partridge SR et al.; Spores of a strain of Bacillus subtilis in which ftsZ was under the control of the spac promoter were allowed to germinate and grow out in the presence of increasing concentrations of isopropyl-beta-D-thiogalactopyranoside (IPTG) . Over the IPTG concentration range of 0 to 10(-3) M, the level of FtsZ from the time when the first nucleoid segregations were occurring, measured in Western blot (immunoblot) transfer experiments, varied between 15 and 100% of that in the wild type . Septation was completely blocked (for at least several hours) when the amount of FtsZ was < 30% of the wild-type level . At all levels of ftsZ induction, the timing and rate of segregation of nucleoids following the first round of replication were unaltered . It is concluded that FtsZ has no direct role in nucleoid segregation in this situation.

J Bacteriol, 1995 May, 177(9), 2403 - 7
Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis; Kunst F et al.; Growth under conditions of salt stress has important effects on the synthesis of degradative enzymes in Bacillus subtilis . Salt stress strongly stimulates the expression of sacB, encoding levansucrase (about ninefold), and downregulates the expression of aprE, encoding alkaline protease (about sixfold) . It is suggested that the DegS-DegU two-component system is involved in sensing salt stress . Moreover, it has been shown that the level of sacB expression strongly depends on the growth conditions; its expression level is about eightfold higher in cells grown on agar plates than in cells grown in liquid medium.

J Bacteriol, 1995 May, 177(9), 2283 - 91
Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp . israelensis; Dervyn E et al.; The CryIVD protein is involved in the overall toxicity of the Bacillus thuringiensis subsp . israelensis parasporal inclusions and is one of the four major components of the crystals . Determination of the DNA sequence indicated that the cryIVD gene is the second gene of an operon which includes three genes . The first one encodes a 19-kDa polypeptide and has sequence homology with the orf1 gene of the Bacillus thuringiensis cryIIA and cryIIC operons . The second and third genes have already been identified and encode the CryIVD crystal protein and the P20 polypeptide, respectively . The promoter region was located by deletion analysis, and the 5' end of the mRNA was determined by primer extension mapping . Transcription of the cryIVD gene in B . thuringiensis subsp . israelensis strains is induced 9 h after the beginning of sporulation . Sequence analysis indicated two potential promoters, a strong one and a weak one, recognized respectively by the RNA polymerase associated with the sigma 35 or the sigma 28 factor of B . thuringiensis (sigma E and sigma K of Bacillus subtilis, respectively) . Transcriptional lacZ fusion integrated in single copy into the chromosome of various B . subtilis sporulation mutants confirmed the sigma E dependence of cryIVD gene transcription.

Mutagenesis, 1995 May, 10(3), 165 - 70
Bacterial ribonuclease: mutagenic effect in microbial test-systems; Ilinskaya O et al.; Pure enzyme samples of ribonuclease from Bacillus intermedius 7P (known commercially as 'binase') were investigated for genotoxicity in four microbial tests: the Ames plate incorporation method, AraR-assay; the prophage induction test; and the DNA-repair test . The weak mutagenic effect of binase at high concentrations (0.1 mg/plate, 1 mg/plate) was established by induction of forward AraR-mutations and histidine-reverse mutations (both frameshift mutations and base pair substitution) . Metabolic activation with rat or chicken liver, human placenta or plant (from tulip bulbs) microsomal fractions in vitro was seen to abolish the binase mutagenicity . Bacillus intermedius 7P ribonuclease appears to possess DNA damaging activity in uvrA- and polA- mutants, but not in the recA-deficient Escherichia coli strain, and exhibits an induction of recA-dependent mutagenesis detected by the 8-fold increase of the prophage-induction level in lysogenic Bacillus subtilis culture and by the 5-fold increase of this level in the Streptomyces lavendulae 3 lysogenic strain . The importance of the roles of both of enzyme catalytic activity and native structure is emphasized . A proposed mechanism for exogenous ribonuclease action is discussed . Bacillus intermedius 7P ribonuclease probably does not act as a direct genotoxic agent interacting with DNA, but could provoke nucleotide imbalance through its catalytic action on membrane-associated RNAs, which results in alteration of DNA replication and, as a consequence, in recA-dependent mutagenesis.

Mol Plant Microbe Interact, 1995 May-Jun, 8(3), 454 - 64
A new Bradyrhizobium japonicum gene required for free-living growth and bacteroid development is conserved in other bacteria and in plants; Weidenhaupt M et al.; In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, a new DNA region, orf74, was discovered which is required for optimal free-living growth and, by consequence, also necessary for the formation of an effective symbiosis . A Tn5-233 insertion of orf14 resulted in a mutant, strain 74, that has a reduced growth rate in free-living cultures under all conditions tested and less than 1% residual symbiotic nitrogen fixation activity as compared with the wild type . Nodule distribution and nodule morphology are severely affected similarly as in a previously characterized B . japonicum nifA mutant . Protein databank searches revealed that the 142-amino-acid protein encoded by orf74 is homologous to a 161-amino-acid protein encoded by orf17 of Bacillus subtilis (approximately 46% identity; J . C . R . Struck, R . Kretschmer-Kazemi Far, W . Schroder, F . Hucho, H . Y . Toschka, and V . A . Erdmann, Biochim . Biophys . Acta, 1050:80-83, 1990) as well as to a 178-amino-acid protein encoded by orf178 of Escherichia coli (approximately 45% identity; K . L . Poulsen, N . W . Larsen, S . Molin, and P . Andersson, Mol . Microbiol., 6:895-905, 1992) . Significant similarity was also found with unknown ORFs of two plant species . When expressed from a strong constitutive promoter, orf17 of B . subtilis could partially complement B . japonicum mutant 74 . By contrast, orf74 of B . japonicum was unable to functionally complement an E . coli orf178 mutant . The conservation of this DNA region in gram-negative and gram-positive bacteria suggests that the gene is essential for a fundamental cellular process which is required in B . japonicum for both free-living and symbiotic growth.

Appl Environ Microbiol, 1995 May, 61(5), 1953 - 8
Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli; Wanker E et al.; The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter . The enzyme was purified from crude E . coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE-Sepharose CL-6B, S-Sepharose, and MonoQ-Sepharose . The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence . Levanase was active on levan, inulin, and sucrose with Km values of 1.2 microM, 6.8 mM, and 65 mM, respectively . The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55 degrees C . Levanase was rapidly inactivated at 60 degrees C, but activity could be retained for longer times by adding fructose or glycerol . The enzyme activity was completely inactivated by Ag+ and Hg2+ ions, indicating that a sulfhydryl group is involved . A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml . The mechanism of enzyme action was investigated . No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography-low-angle laser light scattering-interferometric differential refractive index techniques . This indicates that levanase is an exoenzyme acting by the single-chain mode.

Biophys J, 1995 May, 68(5), 1937 - 43
Interactions of surfactin with membrane models; Maget-Dana R et al.; Surfactin, an acidic cyclic lipopeptide produced by strains of Bacillus subtilis, is a powerful biosurfactant possessing biological activities . Interactions of ionized surfactin (two negative charges) with lecithin vesicles have been monitored by changes in its CD spectra . These changes are more important in the presence of Ca2+ ions . We have studied the penetration of ionized surfactin into lipid monolayers . Using dimyristoyl phospholipids, the surfactin penetration is more important in DMPC than in DMPE monolayers and is greatly reduced in DMPA monolayers because of electrostatic repulsion . The surfactin penetration is lowered when the acyl chain length of the phospholipids increases . The exclusion pressure varies from 40 mN m-1 for DMPC to 30 mN m-1 for DPPC and 18 mN m-1 for egg lecithin . The presence of Ca2+ ions, which neutralize the charges of both surfactin and lipids in the subphase, leads to an important change of the penetration process that is enhanced in the case of acidic, but also of long chain (higher than C14) zwitterionic phospholipids (DPPC and lecithin) . From compression isotherms of mixed surfactin/phospholipid monolayers, it appears that surfactin is completely miscible with phospholipids . The present study shows that surfactin penetrates spontaneously into lipid membranes by means of hydrophobic interactions . The insertion in the lipid membrane is accompanied by a conformation change of the peptide cycle.

Appl Microbiol Biotechnol, 1995 May-Jun, 43(2), 310 - 4
Regulation of mosquitocidal toxin synthesis in Bacillus sphaericus; Ahmed HK et al.; Translational lacZ fusions to the promoters of the parasporal, crystal protein (binary toxin) and 100-kDa ADP-ribosylating mosquitocidal toxin genes of Bacillus sphaericus were prepared and expression of the toxin genes monitored as beta-galactosidase activity . Transcription of the crystal protein gene fusion began immediately before the end of exponential growth and continued into stationary phase in both B . sphaericus and Bacillus subtilis but accompanied exponential-phase growth in Escherichia coli . Expression of this fusion was severely delayed in a B . subtilis spo0A mutant and decreased relative to the wild type in a B . subtilis spoIIAC background . beta-Galactosidase activity from the 100-kDa toxin gene fusion was restricted to early exponential phase in B . sphaericus, but in B.subtilis it continued into late exponential phase . Expression was about eightfold lower in B . sphaericus than B . subtilis suggesting an element of negative control in the native host.

Biofactors, 1995 May, 5(1), 29 - 37
Physiological mechanisms regulating the conversion of selenite to elemental selenium by Bacillus subtilis; Garbisu C et al.; We have demonstrated that the common soil bacterium, Bacillus subtilis, reduces selenite to an insoluble and much less toxic product--the red form of elemental selenium . Reduction was effected by an inducible system that appears to deposit elemental selenium between the cell wall and the plasma membrane . Glucose and sucrose supported selenite reduction . Although malate and citrate supported growth, no significant reduction of selenite occurred, indicating the importance of the redox state of the culture substrate . Selenite reduction in the millimolar concentration range (i.e., cultures supplemented with 1 mM selenite) was not affected by a ten-fold excess of nitrate or sulfate--compounds that serve as alternate electron acceptors and antagonize selenite reduction by anaerobic bacteria . Similarly, nitrite and sulfite did not significantly affect the rate or extent of selenite reduction . B.subtilis was able to grow and produce selenium (Se degree) at selenite concentrations ranging from 0.6 microM to 5 mM (50 ppb to 395 ppm selenium) . At the lowest selenite concentration tested, 50 ppb selenium, B.subtilis removed 95% of the selenite from the liquid phase . The results suggest that selenite is reduced via an inducible detoxification system rather than dissimilatory electron transport . The findings establish the potential utility of B.subtilis for the bioremediation of selenite-polluted sites.

Biochem Mol Biol Int, 1995 May, 35(6), 1245 - 51
Comparison between thymidylate synthase B of Bacillus subtilis ATCC6633 and 168; Montorsi M et al.; Bacillus subtilis 168 possesses two thymidylate synthase genes: thyA and thyB, encoding for a thermolabile and a thermostable enzyme respectively . B . subtilis ATCC6633 also possesses two thy genes, both producing thermostable enzymes . A comparison of the thymidylate synthase B amino acid sequences from the two strains shows, among others, a Pro to Ala mutation which may affect the dUMP binding site . The apparent Km and Vmax values for dUMP and tetrahydrofolate were determined at a permissive temperature for both enzymes . The kinetic data showed a significant difference in the Km, but not in the Vmax, for dUMP between the two enzymes . The Km and Vmax for tetrahydrofolate were very similar.

Mol Microbiol, 1995 May, 16(4), 709 - 18
Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis: induction, repression and growth-rate regulation; Putzer H et al.; The thrS gene in Bacillus subtilis is specifically induced by starvation for threonine and is, in addition, autorepressed by the overproduction of its own gene product, the threonyl-tRNA synthetase . Both methods of regulation employ an antitermination mechanism at a factor-independent transcription terminator that occurs just upstream of the start codon . The effector of the induction mechanism is thought to be the uncharged tRNA(Thr), which has been proposed to base pair in two places with the leader mRNA to induce antitermination . Here we show that the autoregulation by synthetase overproduction is likely to utilize a mechanism similar to that characterized for induction by amino acid starvation, that is by altering the levels of tRNA charging in the cell . We also demonstrate that the base pairing interaction at the two proposed contact points between the tRNA and the leader are necessary but not always sufficient for either form of regulation . Finally, we present evidence that the thrS gene is expressed in direct proportion to the growth rate . This method of regulation is also at the level of antitermination but is independent of the interaction of the tRNA with the leader region.

Biochim Biophys Acta, 1995 Apr 13, 1243(3), 552 - 4
Cloning and sequencing of beta-mannanase gene from Bacillus subtilis NM-39; Mendoza NS et al.; A gene encoding beta-mannanase from Bacillus subtilis NM-39 was cloned into Escherichia coli DH5 alpha by using pUC 18 and its nucleotide sequence was determined . The beta-mannanase gene was 1080 base pairs long and encoded a mature protein of 336 amino acids and a signal peptide of 24 amino acids . The deduced amino acid sequence of the cloned mannanase showed sequence homology with mannanase from alkalophilic Bacillus sp . strain AM-001 (about 50%).

FEBS Lett, 1995 Apr 10, 362(3), 257 - 60
An estimation of minimal genome size required for life; Itaya M; The number of indispensable chromosomal loci for a bacterium, Bacillus subtilis was estimated . Seventy-nine randomly selected chromosomal loci were investigated by mutagenesis . Mutation at only six loci rendered B . subtilis unable to form colonies . In contrast, mutants for the rest of the 73 loci retained the ability to form colonies . Mutant B . subtilis with multiple-fold mutations of those dispensable loci (7-, 12- or 33-fold) were not impaired in their ability to form colonies on nutritionally adequate medium, indicating that up to 33 dispensable loci were simultaneously abolished . Given the statistical analyses for the frequency of indispensable loci (6 out of 79), total indispensable genetic material would be included within about 562 kbp . The hypothetical minimum genome size lies in the range of those currently determined smallest genomes for bacteria.

J Biol Chem, 1995 Apr 7, 270(14), 8076 - 80
Cloning of a human cDNA for protoporphyrinogen oxidase by complementation in vivo of a hemG mutant of Escherichia coli; Nishimura K et al.; Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway . Hemes are essential components of redox enzymes, such as cytochromes . Thus, a hemG mutant strain of Escherichia coli deficient in PPO is defective in aerobic respiration and grows poorly even in rich medium . By complementation with a human placental cDNA library, we were able to isolate a clone that enhanced the poor growth of such a hemG mutant strain . The clone encoded the gene for human PPO . Sequence analysis revealed that PPO consists of 477 amino acids with a calculated molecular mass of 50.8 kilodaltons . The deduced protein exhibited a high degree of homology over its entire length to the amino acid sequence of PPO encoded by the hemY gene of Bacillus subtilis . The NH2-terminal amino acid sequence of the deduced PPO contains a conserved amino acid sequence that forms the dinucleotide-binding site in many flavin-containing proteins . Northern blot analysis revealed the synthesis of a 1.8-kilobase pair mRNA for PPO . A homogenate of the monkey kidney COS-1 cells that had been transfected with the cDNA had much higher PPO activity than an extract of control cells, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO . Furthermore, the cDNA was expressed in vitro as 51-kilodalton protein, and after incubation with isolated mitochondria the protein was found to be located in the mitochondria, having just the same size as before, an indication that PPO is a mitochondrial enzyme and has no apparent transport-specific leader sequence.

EMBO J, 1995 Apr 3, 14(7), 1439 - 45
Cell-type specificity during development in Bacillus subtilis: the molecular and morphological requirements for sigma E activation; Shazand K et al.; Development in Bacillus subtilis involves the formation of two cell types with activation of the transcription factors sigma F in the forespore and sigma E in the mother cell . Activation of sigma E is due to the processing of the inactive precursor pro-sigma E, which requires the putative protease SpoIIGA and the presence of active sigma F . We have introduced missense mutations altering the promoter recognition properties of sigma F . These mutations abolish pro-sigma E processing, suggesting that sigma F is involved through its transcriptional activity and that the processing machinery responds to a signal generated by the product(s) of some unidentified gene(s) transcribed in the forespore . The role of the septum in transducing this signal was investigated . Induction of sigma F during exponential growth in cells producing SpoIIGA and pro-sigma E led to a high level of processing and sigma E activity . Moreover, pro-sigma E was efficiently processed in a mutant strain blocked prior to septation and synthesizing sigma F in active form at the onset of sporulation . Therefore, the sporulation septum is not required for induction of pro-sigma E processing and pro-sigma E can be processed in the same cell in which sigma F is active . These results suggest that some unknown mechanism must exist to prevent sigma E from becoming active in the forespore.

J Bacteriol, 1995 Apr, 177(7), 1888 - 91
Effects of Bacillus subtilis sporulation regulatory protein SpoIIID on transcription by sigma K RNA polymerase in vivo and in vitro; Halberg R et al.; SpoIIID is a sequence-specific, DNA-binding protein that activates or represses transcription of different genes by sigma K RNA polymerase in vitro . A Bacillus subtilis strain engineered to produce both sigma K and SpoIIID during growth showed effects of SpoIIID on expression of sigma K-dependent genes that were consistent with the effects of a small amount of SpoIIID on transcription of these genes in vitro, indicating that the strain provides a simple, in vivo method to screen for effects of SpoIIID on transcription of sigma K-dependent genes.

J Bacteriol, 1995 Apr, 177(7), 1727 - 33
Isolation and sequence analysis of the Pseudomonas syringae pv . tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase; Morris VL et al.; Pseudomonas syringae pv . tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings . It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes . We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism . Uptake studies have shown that DC3481 is not deficient in transport . A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced . The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases . However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively) . PGMs not requiring the cofactor DPG are usually found in plants and algae . Enzyme assays confirmed that P . syringae PGM activity required an intact ORF1 . Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P . syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM . PGM activity appears essential for the growth and pathogenicity of P . syringae pv . tomato on its host plant.

Mol Cell Biol, 1995 Apr, 15(4), 2101 - 8
A Saccharomyces cerevisiae mitochondrial transcription factor, sc-mtTFB, shares features with sigma factors but is functionally distinct; Shadel GS et al.; In Saccharomyces cerevisiae mitochondria, sc-mtTFB is a 341-amino-acid transcription factor required for initiation of transcription from mitochondrial DNA promoters . Specific transcription in vitro requires only sc-mtTFB and the bacteriophage-related core sc-mtRNA polymerase . Mutational analysis of sc-mtTFB has defined two regions of the protein that are important for normal function both in vivo and in vitro . These regions overlap portions of the protein that exhibit similarity to conserved region 2 of bacterial sigma factors . One mutation in this region of sc-mtTFB (tyrosine 108 to arginine {Y108R}) has a defective phenotype that matches that observed for mutations in the corresponding residue of Bacillus subtilis sigma A and sigma E proteins . However, mutations in the sigma 2.4-like region, including a 5-amino-acid deletion corresponding to crucial promoter-contacting amino acids of sigma factors, did not eliminate the ability of sc-mtTFB to initiate transcription specifically in vitro . This suggests a mechanism of promoter recognition for sc-mtRNA polymerase different from that used by bacterial RNA polymerases . Two mutations in a basic region of sc-mtTFB resulted in defective proteins that were virtually dependent on supercoiled DNA templates in vitro . These mutations may have disrupted a DNA-unwinding function of sc-mtTFB that is only manifested in vitro and is partially rescued by DNA supercoiling.

Curr Microbiol, 1995 Apr, 30(4), 193 - 9
Induction of cold shock proteins in Bacillus subtilis; Lottering EA et al.; The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described . Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37 degrees C . The B . subtilis cells were cold shocked at 25 degrees C, 20 degrees C, 15 degrees C, and 10 degrees C . A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis . General stress proteins were identified by a comparative analysis with the heat shock response of B . subtilis . Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced . Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.

Eur J Biochem, 1995 Apr 1, 229(1), 99 - 106
5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis is an allosteric enzyme . Analysis of Arg24-->Asp, Pro105-->Ser and His385-->Lys mutations suggests a hidden phosphoenolpyruvate-binding site; Majumder K et al.; 5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis has been cloned, expressed and purified to near homogeneity . Clustal alignment of the amino acid sequences from different bacteria revealed several conserved residues located in the N-terminal, middle and C-terminal domains . The role of conserved Arg24, Pro105, and His385 residues has been examined by site-directed mutagenesis . Steady-state kinetic analysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5-enolpyruvylshikimate-3-phosphate synthase enzymes investigated so far . Both substrates, phosphoenolpyruvate (P-pyruvate) and shikimate 3-phosphate have multiple interaction sites . There are two sites for P-pyruvate binding, catalytic and non-catalytic . Glyphosate (N-phosphonomethyl glycine) competes for binding at the catalytic site and does not interact at the secondary site . Glyphosate in the absence of ammonium ions increases cooperativity of P-pyruvate binding and favors dimerization of the enzyme through an interaction between P-pyruvate-binding sites . The ammonium-ion-activated 5-enolpyruvylshikimate-3-phosphate synthase displays no cooperativity with respect to P-pyruvate . Absence of ammonium ions decreases affinity for substrates and introduces cooperativity . Cooperativity was also introduced in the enzyme by point mutations, Arg24-->Asp and His385-->Lys . The latter mutant of the native enzyme exists as a dimer and aggregates to a tetrameric form in the presence of glyphosate . The occurrence of multimeric forms of the synthase has been demonstrated by staining for the enzyme activity on the native gel and by resolving purified enzyme preparations on a sucrose density gradient . A model describing the alteration in the aggregation status of the enzyme by the inhibitor, activator and the substrates has been proposed.

Mol Microbiol, 1995 Apr, 16(1), 111 - 120
Cleavage of trehalose-phosphate in Bacillus subtilis is catalysed by a phospho-alpha-(1-1)-glucosidase encoded by the treA gene; Helfert C et al.; A 2.5 kb DNA fragment contain a gene encoding a phospho-alpha-(1-1)-glucosidase (phosphotrehalase), designated treA, was isolated from a Bacillus subtilis chromosomal library by complementation of the tre-12 mutation . The major TreA activity was found in the cytoplasm . TreA exhibits high sequence similarity to thermostable oligo 1,6 beta-glucosidases of several species and the trehalose-6-phosphate hydrolase TreC of Escherichia coli . TreA activity is induced by trehalose and repressed by glucose, fructose or mannitol . Induction by trehalose and repression by glucose are concentration dependent . The highest activity of TreA occurs 90 min before the end of the exponential growth phase in crude cell extracts . The enzyme is able to cleave para-nitrophenyl-glucopyranoside and trehalose-6-phosphate but not trehalose . These results indicate that treA encodes a specific phospho-alpha-(1-1)-glucosidase which cleaves trehalose-6-phosphate in the cytoplasm after transport and phosphorylation of trehalose . The 5' flanking region of treA contains an open reading frame which was partially sequenced, whose product shows about 40% identity to sucrose Enzyme II of the phosphotransferase transport system from several organisms.

Zoolog Sci, 1995 Apr, 12(2), 225 - 30
Humoral defense of the nematode Ascaris suum: antibacterial, bacteriolytic and agglutinating activities in the body fluid; Kato Y; Three humoral defense activities (antibacterial, bacteriolytic and agglutinating) were detected in the body fluid of the nematode Ascaris suum . Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) were more sensitive to the antibacterial activity than the Gram-negative bacteria (Escherichia coli) . The antibacterial activity was heat stable and was lost by trypsin digestion . The molecular mass of the factor responsible for antibacterial activity was estimated as 6 kDa . The bacteriolytic activity against dried Micrococcus luteus was also detected . The bacteriolytic factor was 6-9 kDa in molecular mass, heat sensitive and trypsin sensitive . Both E . coli and glutaraldehyde-fixed trypsin-treated human A-type red blood cells were agglutinated in the body fluid . An analytical gel permeation HPLC revealed the agglutinating activity consists of at least two factors . Activities of both agglutinating factors were lost by heat treatment or trypsin digestion . The molecular masses estimated for the two agglutinating factors were 500 kDa and 25 kDa . Under experimental conditions, microbe-injection was not a prerequisite for the appearance of these defense activities.

Mol Microbiol, 1995 Apr, 16(2), 345 - 55
The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids; Lazarevic V et al.; We report the nucleotide sequence and the characterization of the Bacillus subtilis tagGH operon . The latter is controlled by a sigma A-dependent promoter and situated in the 308 degrees chromosomal region which contains genes involved in teichoic acid biosynthesis . TagG is a hydrophobic 32.2 kDa protein which resembles integral membrane proteins belonging to polymer-export systems of Gram-negative bacteria . Gene tagH encodes a 59.9 kDa protein whose N-moiety contains the ATP-binding motif and shares extensive homology with a number of ATP-binding proteins, particularly with those associated with the transport of capsular polysaccharides and O-antigens . That the tagGH operon is essential for cell growth was established by the failure to inactivate tagG and the 5'-moiety of tagH by insertional mutagenesis . During limited tagGH expression, cells exhibited a cocoid morphology while their walls contained reduced amounts of phosphate as well as galactosamine . These observations, revealing impaired metabolism of both wall teichoic acids of B . subtilis 168, i.e . poly(glycerol phosphate), and poly(glucose galactosamine phosphate), combined with sequence homologies, suggest that TagG and TagH are involved in the translocation through the cytoplasmic membrane of the latter teichoic acids or their precursors.

J Biol Chem, 1995 Mar 31, 270(13), 7594 - 600
Protease evolution in Streptomyces griseus . Discovery of a novel dimeric enzymes; Sidhu SS et al.; This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus . Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product . The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S . griseus protease D (SGPD) . A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60 . The sprD gene was subcloned into an Escherichia coli/B . subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin . The gene fusion was subsequently expressed in B . subtilis DB104, and active protease was purified . SGPD has a high degree of sequence homology to previously described S . griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer . The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S . griseus proteases A, B, and C . The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids . Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S . griseus.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2845 - 9
Krebs cycle function is required for activation of the Spo0A transcription factor in Bacillus subtilis; Ireton K et al.; Expression of genes early during sporulation in Bacillus subtilis requires the activity of the transcription factor encoded by spo0A . The active, phosphorylated form of Spo0A is produced through the action of a multicomponent pathway, the phosphorelay . A mutant defective in the first three enzymes of the Krebs citric acid cycle was unable to express early sporulation genes, apparently because of a failure to activate the phosphorelay . Cells that produce an altered Spo0A protein that can be phosphorylated by an alternative pathway were not dependent on Krebs cycle function for early sporulation gene expression . These findings suggest that Krebs cycle enzymes transmit a signal to activate the phosphorelay and that B . subtilis monitors its metabolic potential before committing itself to spore formation.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2582 - 6
Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element; Fredrick K et al.; Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28) . We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region {upstream promoter (UP) element} both in vivo and in vitro . This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase . The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding . Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

Biochemistry, 1995 Mar 21, 34(11), 3823 - 31
Chemotactic methylation and behavior in Bacillus subtilis: role of two unique proteins, CheC and CheD; Rosario MM et al.; We characterized mutants in two novel genes of Bacillus subtilis, cheC and cheD . Mutants in CheC had a high smooth swimming bias and exhibited poor adaptation to positive stimuli . Analysis of tethered cells revealed two distinct subpopulations which differ in their prestimulus bias and extent of adaptation . The receptors, the methyl-accepting chemotaxis proteins (MCPs), of this mutant strain were overmethylated, as a result of an increase in CheR activity . We speculate that CheC helps to control tumbling frequency by regulating CheR, perhaps by a feedback mechanism through the MCPs . In contrast, a cheD mutant exhibited very tumbly behavior, and many of the MCPs were unmethylated . It seems that some B . subtilis MCPs require the presence of CheD for CheR to methylate them, a unique feature of B . subtilis chemotaxis . It is hypothesized that CheD is part of a complex that facilitates methylation of some of the MCPs, and dissociation of CheD from this complex affects CheA activity and may help bring about adaptation.

Mol Gen Genet, 1995 Mar 20, 246(6), 756 - 60
Bent DNA is found in some, but not all, regions recognized by the Bacillus subtilis AbrB protein; Strauch MA et al.; The AbrB protein of Bacillus subtilis is a transcriptional regulator of numerous genes in diverse metabolic pathways that commence expression at the onset of stationary phase . AbrB is a DNA-binding protein with specific affinity for defined DNA regions of its target genes . Analysis of these regions has failed to detect a readily apparent sequence determinant for AbrB binding and it is believed that AbrB recognizes a specific DNA structure formed by a finite subset of base sequences . We have examined six AbrB binding regions for intrinsic bending and found that three do not contain a detectable intrinsic bend, whereas three do . AbrB binding to these regions resulted in electrophoretic mobility patterns that were consistent with, but did not prove, that the DNA moieties were present in a bent configuration.

Mol Gen Genet, 1995 Mar 20, 246(6), 680 - 6
Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants; Alonso JC et al.; We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome . The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria . S . aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa) . We have shown that the S . aureus recF gene partially complements the defect of a B . subtilis recF mutant, but does not complement an E . coli recF strain . The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions (alpha to theta) and to predict five new conserved regions within the gram-positive group (a to f) . We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.

J Biol Chem, 1995 Mar 17, 270(11), 6181 - 5
The outB gene of Bacillus subtilis codes for NAD synthetase; Nessi C et al.; The outB gene of Bacillus subtilis is involved in spore germination and outgrowth and is essential for growth . The OutB protein was obtained by expression in Escherichia coli and purified to apparent homogeneity . Here we report experiments showing that OutB is a NH3-dependent NAD synthetase, the enzyme that catalyzes the final reaction in the biosynthesis of NAD . The enzyme is composed of two identical subunits of 30,240 Da and is NH3-dependent, whereas glutamine is inefficient as an amide donor . The NAD synthetase is highly resistant to heat, with a half-time of inactivation at 100 degrees C of 13 min . A mutant NAD synthetase was purified from a B . subtilis strain temperature-sensitive during spore germination and outgrowth . The mutant enzyme was 200 times less active than the wild-type one, with a lower temperature optimum and a non-hyperbolic kinetic versus NH4+ . The time course of synthesis of OutB showed that synthesis of the enzyme started during germination and outgrowth, and reached the highest level at the end of exponential growth . The enzyme could be recovered from dormant spores.

Biochem Biophys Res Commun, 1995 Mar 17, 208(2), 610 - 6
Purification and characterization of DNA dependent RNA polymerase from Staphylococcus aureus; Deora R et al.; DNA dependent RNA polymerase from exponentially growing Staphylococcus aureus cells was purified . An SDS-polyacrylamide gel analysis of the most purified preparation revealed that it consists of beta, beta', alpha, and sigma with apparent molecular masses of 151, 147, 42, and 55 kDa, respectively . The sigma subunit cross reacted with a polyclonal antibody against Bacillus subtilis sigma 43 . The cross reacting peptide co-migrated with the B . subtilis sigma 43 subunit . The implications of these results are discussed . Promoter specific in vitro run-off transcripts were obtained using the purified enzyme preparation . Specific conditions for the polymerization reaction are defined.

FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 65 - 72
Expression of the genes for insecticidal crystal proteins in Bacillus thuringiensis: cryIVA, not cryIVB, is transcribed by RNA polymerase containing sigma H and that containing sigma E; Yoshisue H et al.; To investigate the mechanism of transcriptional regulation of cryIVA and cryIVB, encoding 130-kDa dipteran-active crystal proteins, in Bacillus thuringiensis subsp . israelensis, we introduced each gene into several sporulation mutants of Bacillus subtilis . A spoIIG mutation, the wild-type gene of which encodes sigma E precursor, completely blocked the cryIVB transcription . In contrast, low but detectable transcription of cryIVA was observed in the spoIIG mutant . In the wild-type B . subtilis, no transcription of cryIVB was detected before T2 (2 h after the onset of stationary phase), while the cryIVA transcription started at the late exponential phase at low levels . Furthermore, in a wild-type strain of B . thuringiensis subsp . israelensis, transcription of cryIVA began earlier than that of genes encoding other crystal components, cryIVB and cytA . A consensus sequence recognized by an RNA polymerase containing sigma H of B . subtilis was found upstream of the transcription start point of cryIVA, which overlapped with that recognized by sigma E.

FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 121 - 6
Cloning, sequencing and production of the lantibiotic mersacidin; Bierbaum G et al.; Mersacidin is a lanthionine-containing peptide antibiotic that shows a good in vivo efficiency against methicillin-resistant Staphylococcus aureus . It is excreted during early stationary phase and could be purified from culture supernatant in a one-step procedure by reversed phase HPLC . Its structural gene was cloned from chromosomal DNA of the producer strain Bacillus subtilis HIL Y-85,54728 . Sequencing revealed that pre-mersacidin consists of an unusually long 48 amino acid leader sequence and a 20 amino acid propeptide part which is modified during biosynthesis to the mature lantibiotic . The comparison of the mersacidin prepeptide with those of hitherto known lantibiotics demonstrates that mersacidin is more closely related to type B lantibiotic cinnamycin than to type A lantibiotics.

FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 11 - 5
Studies on lipopeptide biosynthesis by Bacillus subtilis: isolation and characterization of iturin-, surfactin+ mutants; Feignier C et al.; Two mutant strains, M35 and M89, were obtained by UV irradiation from a wild-type Bacillus subtilis producing iturin and surfactin . Sporulation and surfactin production were similar in both mutants and in the parent strain, while the iturin production of M35 was 300-fold less than that of the wild-type strain; M89 did not produce any iturin . The analysis of the incorporation of sodium {1-14C}acetate into cellular lipids and lipopeptides showed that M89 still synthesized beta-amino fatty acids, the lipid moiety of iturin.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2012 - 6
Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis; Karow ML et al.; Sporulation of Bacillus subtilis requires the coordinated expression of two separate developmental programs in the mother cell and forespore compartments by sigma E and sigma F, respectively . This coordination is maintained through the action of cross-regulatory factors that control the activities of the various sporulation-specific sigma factors . We present here the isolation and characterization of one such cross-regulatory factor, the spoIIR gene . Using a genetic screen, we have isolated four mutant alleles of spoIIR . These mutants were isolated as expressing sigma F-directed genes but not sigma E-directed genes . The block in sigma E-directed gene expression in spoIIR mutants was caused by an inability to process pro-sigma E to its active form . Cloning and characterization of the spoIIR gene determined that its transcription is directed by sigma F . Thus, SpoIIR is required for linking the activation of sigma E to the activation of sigma F and coordinating the initiation of the two developmental programs required to form a spore.

Mol Gen Genet, 1995 Mar 10, 246(5), 610 - 8
Molecular analysis of the phosphoenolpyruvate-dependent L-sorbose: phosphotransferase system from Klebsiella pneumoniae and of its multidomain structure; Wehmeier UF et al.; We have cloned a 3.4 kb DNA fragment from the chromosome of Klebsiella pneumoniae that codes for a phosphoenolpyruvate-dependent L-sorbose: phosphotransferase system (PTS) . The cloned fragment was sequenced and four open reading frames coding for 135 (sorF), 164 (sorB), 266 (sorA) and 274 (sorM) amino acids, respectively, were found . The corresponding proteins could be detected in a T7 overexpression system, which yielded molecular masses of about 14,000 for SorF, 19,000 for SorB, 25,000 for SorA and 27,000 for SorM . SorF and SorB have all the characteristics of soluble and intracellular proteins in accordance with their functions as EIIASor and EIIBSor domains of the L-sorbose PTS . SorA and SorM, by contrast, are strongly hydrophobic, membrane-bound proteins with two to five putative transmembrane helices that alternate with a series of hydrophilic loops . They correspond to domains EIICSor and EIIDSor . The four proteins of the L-sorbose PTS resemble closely (27%-60%) the four subunits of a D-fructose PTS (EIIALev, EIIBLev, EIICLev, and EIIDLev) from Bacillus subtilis and the three subunits of the D-mannose PTS (EIIA,BMan, EIICMan, and EIIDMan) from Escherichia coli K-12 . The three systems constitute a new PTS family, and sequence comparisons revealed highly conserved structures for the membrane-bound proteins . A consensus sequence for the membrane proteins was used to postulate a model for their integration into the membrane.

Biochemistry, 1995 Mar 7, 34(9), 2883 - 92
Biosynthesis of riboflavin . Studies on the reaction mechanism of 6,7-dimethyl-8-ribityllumazine synthase; Kis K et al.; The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of 3 alpha subunits . The beta subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with (3S)-3,4-dihydroxy-2-butanone under formation of 6,7-dimethyl-8-ribityllumazine . This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation yielding 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione as second product . (3R)- and (3S)-3,4-dihydroxy-2-butanone 4-phosphate were synthesized . Both enantiomers can serve as substrate for 6,7-dimethyl-8-ribityllumazine synthase . The reaction rate of the natural S-enantiomer is about 6-fold higher than that of the R-enantiomer . The Km value for (3S)-3,4-dihydroxy-2-butanone 4-phosphate is 130 microM, and the Km value for the pyrimidine substrate is 5 microM . Diacetyl and 3,4-dihydroxy-2-butanone 3-phosphate do not serve as substrates for lumazine synthase . The enzyme-catalyzed condensation of the carbohydrate with the pyrimidine is strictly regiospecific . The enzyme does not catalyze the exchange of protons between (3S)-3,4-dihydroxy-2-butanone 4-phosphate and solvent water in the absence of the pyrimidine cosubstrate . A reaction mechanism starting with the formation of a Schiff base followed by elimination of phosphate and cyclization is proposed . The lumazine synthase activities of the native enzyme complex and of reconstituted, hollow beta 60 capsids are virtually identical (about 12,000 nmol mg-1 h-1).

FEBS Lett, 1995 Mar 6, 360(3), 307 - 9
Kinetics of the unfolding-folding transition of Bacillus subtilis levansucrase precursor; Scotti PA et al.; The reversible folding-unfolding transition of mature and precursor forms of Bacillus subtilis levansucrase were compared under physiological conditions of pH and temperature . The time constant of the folding reaction was not modified by the presence of the signal sequence and the precursor in the native form was slightly more resistant to the denaturing action of urea . However, the folding pathway could be different for each protein since a domain of the mature levansucrase underwent an independent transition which is not observed during the renaturation process of prelevansucrase.

Biochem J, 1995 Mar 1, 306 ( Pt 2), 371 - 7
Enzymic characterization of Bacillus subtilis GTP cyclohydrolase I . Evidence for a chemical dephosphorylation of dihydroneopterin triphosphate; De Saizieu A et al.; GTP cyclohydrolase I catalyses the first committing step in the biosynthesis of the pterin moiety of folic acid: conversion of GTP to dihydroneopterin triphosphate . GTP cyclohydrolase I of Bacillus subtilis was purified to homogeneity and shown to have a homo-octameric structure . The enzyme had an apparent Km for GTP of 4 microM and, in the absence of cations, a Vmax . of 80 nmol/min per mg of protein . K+ ions moderately increased its Vmax., whereas UTP and Ca2+ and Mg2+ ions drastically increased its Km for GTP . Dihydrofolate and other products of the folate and tetrahydrobiopterin pathways did not inhibit GTP cyclohydrolase I . In addition to their effect on the enzyme activity, Ca2+ and Mg2+ ions catalysed the chemical dephosphorylation of dihydroneopterin triphosphate to non-cyclic dihydroneopterin monophosphate, the substrate for the phosphomonoesterase reaction in folate biosynthesis . This dephosphorylation was specific and did not require the action of a phosphatase . We suggest a physiological role for Ca2+ ions and UTP in regulation of folate biosynthesis at the levels of GTP cyclohydrolase I and dephosphorylation of dihydroneopterin triphosphate.

J Bacteriol, 1995 Mar, 177(6), 1630 - 3
Identification of a Bacillus subtilis spo0H allele that is necessary for suppression of the sporulation-defective phenotype of a spo0A mutation; Bramucci MG et al.; A mutation in Bacillus subtilis spo0A codon 97 suppressed the sporulation defect caused by the spo0A9V mutation . The suppressor activity of the codon 97 mutation was evident only in the presence of a novel spo0H allele . Our results suggest that the spo0A gene product interacts with the sigma factor subunit of RNA polymerase.

J Bacteriol, 1995 Mar, 177(6), 1527 - 35
New beta-glucoside (bgl) genes in Bacillus subtilis: the bglP gene product has both transport and regulatory functions similar to those of BglF, its Escherichia coli homolog; Le Coq D et al.; The Bacillus subtilis sacY and sacT genes encode antiterminator proteins, similar to the Escherichia coli bglG gene product and required for transcription of sucrose metabolism genes . A Tn10 insertion into bglP (formerly sytA) has been previously identified as restoring sucrose utilization to a strain with deletions of both sacY and sacT . The nucleotide sequence of bglP showed a high degree of homology with the E . coli bglF gene (BglF is a beta-glucoside permease of the phosphotransferase system and also acts as a negative regulator of the BglG antiterminator) . Complementation studies of an E . coli strain with a deletion of the bgl operon showed that BglP was a functional beta-glucoside permease . In B . subtilis, bglP complemented in trans both the bglP::Tn10 original insertion and a phenotypically similar bglP deletion . Disruption of licT abolished the suppressor phenotype in a bglP mutant . LicT is a recently identified third B . subtilis antiterminator of the BglG/SacY family . These observations indicated that BglP was also a negative regulator of LicT . Both LicT and BglP seem to be involved in the induction by beta-glucosides of an operon containing at least two genes, bglP itself and bglH, encoding a phospho-beta-glucosidase . Other beta-glucoside genes homologous to bglP and bglH have been recently described in B . subtilis . Thus, B . subtilis possesses several sets of beta-glucoside genes, like E . coli, but these genes do not appear to be cryptic.

J Bacteriol, 1995 Mar, 177(5), 1409 - 13
The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis; Ogawa K et al.; Bacillus subtilis can use either nitrate or nitrite as a sole source of nitrogen . The isolation of the nasABCDEF genes of B . subtilis, which are required for nitrate/nitrite assimilation, is reported . The probable gene products include subunits of nitrate/nitrite reductases and an enzyme involved in the synthesis of siroheme, a cofactor for nitrite reductase.

J Bacteriol, 1995 Mar, 177(5), 1315 - 25
Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression; Lu Y et al.; Expression of the Bacillus subtilis pyr operon is regulated by exogenous pyrimidines and the protein product of the first gene of the operon, PyrR . It has been proposed that PyrR mediates transcriptional attenuation at three untranslated segments of the operon (R.J . Turner, Y . Lu, and R.L . Switzer, J . Bacteriol., 176:3708-3722, 1994) . In this study, transcriptional fusions of the pyr promoter followed by the pyr attenuation sequences, either individually or in tandem to a lacZ reporter gene, were used to examine the physiological functions of all three attenuators through their ability to affect beta-galactosidase expression . These fusions were studied as chromosomal integrants in various B . subtilis strains to examine the entire range of control by pyrimidines, PyrR dependence, amd developmental control of pyr gene expression . The nutritional regulation of each attenuator separately was roughly equivalent to that of the other two and was totally dependent upon PyrR, and that of tandem attenuators was cumulative . The regulation of a fusion of the spac promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the corresponding fusion containing the pyr promoter, demonstrating that attenuator-dependent regulation is independent of the promoter . Extreme pyrimidine starvation gave rise to two- to threefold-higher levels of expression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved . Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent regulation is not a specific property of the pyr operon . Conversion of the initiator AUG codon in a small open reading frame in the pyrP:pyrB intercistronic region to UAG reduced expression by about half but did not alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism . Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR, and the participation of SpoOA was excluded.

Microbiology, 1995 Mar, 141 ( Pt 3), 645 - 8
A putative new peptide synthase operon in Bacillus subtilis: partial characterization; Tognoni A et al.; A large operon-type structure has been located between the gltA and citB loci on the Bacillus subtilis chromosome . On the basis of the analysis of the 25 kb sequenced so far, it potentially encodes at least three large proteins which contain structural motifs associated with the subunits of all characterized peptide synthases . The amino acid recognition specificity of this new peptide synthase is discussed in the light of sequence homology with other synthases.

Microbiology, 1995 Mar, 141 ( Pt 3), 641 - 4
The Bacillus subtilis dnaC gene encodes a protein homologous to the DnaB helicase of Escherichia coli; Sakamoto Y et al.; Within the region of the Bacillus subtilis chromosome assigned to us in the genome sequencing project, we found a gene, the product of which is similar to the DnaB protein (replicative DNA helicase) of Escherichia coli . Three B . subtilis dna gene mutations, dnaC30 and ts56 causing defects in elongation and ts199 causing a defect in the initiation of replication, were mapped in the gene by transformation and DNA sequencing . Both dnaC30 and ts56 have been located near the amino-terminal end of the B . subtilis DnaC protein . In contrast, ts199 has been located near the carboxy-terminal of the protein . Our results indicate that the B . subtilis dnaC gene encodes a counterpart of the E . coli dnaB helicase.

Microbiol Rev, 1995 Mar, 59(1), 1 - 30
The sigma factors of Bacillus subtilis; Haldenwang WG; The specificity of DNA-dependent RNA polymerase for target promotes is largely due to the replaceable sigma subunit that it carries . Multiple sigma proteins, each conferring a unique promoter preference on RNA polymerase, are likely to be present in all bacteria; however, their abundance and diversity have been best characterized in Bacillus subtilis, the bacterium in which multiple sigma factors were first discovered . The 10 sigma factors thus far identified in B . subtilis directly contribute to the bacterium's ability to control gene expression . These proteins are not merely necessary for the expression of those operons whose promoters they recognize; in many instances, their appearance within the cell is sufficient to activate these operons . This review describes the discovery of each of the known B . subtilis sigma factors, their characteristics, the regulons they direct, and the complex restrictions placed on their synthesis and activities . These controls include the anticipated transcriptional regulation that modulates the expression of the sigma factor structural genes but, in the case of several of the B . subtilis sigma factors, go beyond this, adding novel posttranslational restraints on sigma factor activity . Two of the sigma factors (sigma E and sigma K) are, for example, synthesized as inactive precursor proteins . Their activities are kept in check by "pro-protein" sequences which are cleaved from the precursor molecules in response to intercellular cues . Other sigma factors (sigma B, sigma F, and sigma G) are inhibited by "anti-sigma factor" proteins that sequester them into complexes which block their ability to form RNA polymerase holoenzymes . The anti-sigma factors are, in turn, opposed by additional proteins which participate in the sigma factors' release . The devices used to control sigma factor activity in B, subtilis may prove to be as widespread as multiple sigma factors themselves, providing ways of coupling sigma factor activation to environmental or physiological signals that cannot be readily joined to other regulatory mechanisms.

Bull Math Biol, 1995 Mar, 57(2), 299 - 344
The development of concentration gradients in a suspension of chemotactic bacteria; Hillesdon AJ et al.; When a suspension of bacterial cells of the species Bacillus subtilis is placed in a chamber with its upper surface open to the atmosphere complex bioconvection patterns are observed . These arise because the cells: (1) are denser than water; and (2) usually swim upwards, so that the density of an initially uniform suspension becomes greater at the top than the bottom . When the vertical density gradient becomes large enough, an overturning instability occurs which ultimately evolves into the observed patterns . The reason that the cells swim upwards is that they are aerotactic, i.e., they swim up gradients of oxygen, and they consume oxygen . These properties are incorporated in conservation equations for the cell (N) and oxygen (C) concentrations, and these are solved in the pre-instability phase of development when N and C depend only on the vertical coordinate and time . Numerical results are obtained for both shallow- and deep-layer chambers, which are intrinsically different and require different mathematical and numerical treatments . It is found that, for both shallow and deep chambers, a thin boundary layer, densely packed with cells, forms near the surface . Beneath this layer the suspension becomes severely depleted of cells . Furthermore, in the deep chamber cases, a discontinuity in the cell concentration arises between this cell-depleted region and a cell-rich region further below, where no significant oxygen concentration gradients develop before the oxygen is fully consumed . The results obtained from the model are in good qualitative agreement with the experimental observations.

Genes Dev, 1995 Mar 1, 9(5), 547 - 58
Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis; Solomon JM et al.; Development of genetic competence in Bacillus subtilis is regulated by extracellular signaling molecules, including the ComX pheromone, a modified 9- or 10-amino-acid peptide . Here, we present characterization of a second extracellular competence stimulating factor (CSF) . CSF appears to be, at least in part, a small peptide of between 520 and 720 daltons . Production of CSF requires several genes that are needed both for initiation of sporulation and development of competence (spo0H, spo0A, spo0B, and spo0F) . Although both peptide factors regulate competence, two different sensing pathways mediate the response to the ComX pheromone and CSF . Analysis of double mutants indicated that ComX pheromone is on the same genetic pathway as the membrane-bound histidine protein kinase encoded by comP and that CSF is on the same genetic pathway as the oligopeptide permease encoded by spo0K . Furthermore, the cellular response to partly purified ComX pheromone requires the ComP histidine protein kinase, whereas the response to partly purified CSF requires the Spo0K oligopeptide permease . These two sensing pathways converge to activate competence genes . Both factors and their convergent sensing pathways are required for normal development of competence and might function to integrate different physiological signals.

J Ind Microbiol, 1995 Mar-Apr, 14(3-4), 218 - 25
The influence of pH and external K+ concentration on caesium toxicity and accumulation in Escherichia coli and Bacillus subtilis; Perkins J et al.; Toxicity screening of Escherichia coli NCIB 9484 and Bacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs+ to be the most toxic Group 1 metal cation . However, toxicity and accumulation of Cs+ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K+ . Over the pH range 6-9 both E . coli and B . subtilis showed increasing sensitivity towards caesium as the pH was raised . The presence of K+ and Na+ in the laboratory media used lowered caesium toxicity and lowered accumulation of the metal . In order to assess accurately Cs+ toxicity towards the bacterial strains it was therefore necessary to define the K+:Cs+ ratio in the external medium . The minimum inhibitory K+:Cs+ concentration ratio for the Bacillus strains tested was in the range 1:2-1:3 while E . coli had a minimum inhibitory K+:Cs+ concentration ratio of 1:6.

RNA, 1995 Mar, 1(1), 102 - 12
Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli; Nurse K et al.; tRNA pseudouridine 55 (psi 55) synthase, the enzyme that is specific for the conversion of U55 to psi 55 in the m5U psi CG loop in most tRNAs, has been purified from Escherichia coli and cloned . On SDS gels, a single polypeptide chain with a mass of 39.7 kDa was found . The gene is a previously described open reading frame, p35, located at 68.86 min on the E . coli chromosome between the infB and rpsO genes . The proposed name for this gene is truB . There is very little protein sequence homology between the truB gene product and the hisT (truA) product, which forms psi in the anticodon arm of tRNAs . However, there was high homology with a fragment of a Bacillus subtilis gene that may produce the analogous enzyme in that species . The cloned gene was fused to a 5'-leader coding for a (His)6 tract, and the protein was overexpressed > 400-fold in E . coli . The recombinant protein was purified to homogeneity in one step from a crude cell extract by affinity chromatography using a Ni(2+)-containing matrix . The SDS mass of the recombinant protein was 41.5 kDa, whereas that calculated from the gene was 37.3 . The recombinant protein was specific for U55 in tRNA transcripts and reacted neither at other sites for psi in such transcripts nor with transcripts of 16S or 23S ribosomal RNA or subfragments . The enzyme did not require either a renatured RNA structure or Mg2+, and prior formation of m5U was not required . Stoichiometric formation of psi occurred with no requirement for an external source of energy, indicating that psi synthesis is thermodynamically favored.

Protein Eng, 1995 Mar, 8(3), 211 - 5
The 2 A crystal structure of subtilisin E with PMSF inhibitor; Chu NM et al.; Using enzyme prepared by the DNA recombination technique, subtilisin E from Bacillus subtilis was crystallized in space group P2(1)2(1)2(1) with two molecules in an asymmetric unit . The crystal structure of PMSF-inhibited subtilisin E was solved by molecular replacement followed by refinement with the X-PLOR program . This resulted in the 2.0 A structure of subtilisin E with an R-factor of 0.191 for 8-2 A data and r.m.s . deviations from ideal values of 0.021 A and 2.294 for bond lengths and bond angles respectively . The PMSF group covalently bound to Ser221 appeared very clearly in the electron density map . Except for the active site disturbed by PMSF binding, the structural features of subtilisin E are almost the same as in other subtilisins . The calcium-binding sites are different in detail in the two independent molecules of subtilisin E . Based on the structure, the remarkably enhanced heat stability of mutant N118S of subtilisin E is discussed . It is very likely that there is an additional water molecule in the mutant structure, which is hydrogen bonded to side chains of Ser118 and its neighbouring residues Lys27 and Asp120.

Gene, 1995 Feb 27, 154(1), 23 - 9
Cloning and characterization of a Bacillus thuringiensis homolog of the spoIIID gene from Bacillus subtilis; Yoshisue H et al.; The SpoIIID protein of Bacillus subtilis (Bs) is a small DNA-binding protein that is essential for gene expression of the mother cell compartment during sporulation . We have cloned a DNA fragment from Bacillus thuringiensis (Bt) that showed a specific hybridization with the Bs spoIIID gene . Sequence analysis found an open reading frame encoding 90 amino acids (aa), which are 89% identical to the deduced aa sequence of Bs spoIIID . Upstream from the transcription start point (tsp), a nucleotide sequence highly homologous to the consensus sequence motif for the sigma 35-recognized promoters was found . Northern blot analysis has indicated that the expression of the gene is induced only at the midsporulation stage, and that the gene constitutes an operon with a downstream gene, mreB . The Bs strain carrying the spoIIID delta erm or spoIIID83 mutation completely restored sporulation ability upon introduction of the spoIIID homologous gene from Bt . These results strongly suggest that the gene we have cloned is a Bt homolog of spoIIID.

Gene, 1995 Feb 27, 154(1), 1 - 6
Characterization of an insertion in the phage phi 105 genome that blocks host Bacillus subtilis lysis and provides strong expression of heterologous genes; Leung YC et al.; A defective prophage vector, phi 105MU331, for high-level protein overproduction in Bacillus subtilis, was derived by random insertion of a lacZ reporter gene . The site of insertion not only provided efficient inducible transcription of heterologous genes, but also prevented lysis of the host cell . The region of the insertion in phi 105MU331 lies close to the right cohesive end of phi 105 . DNA sequence analysis revealed that this region of phi 105 somewhat resembles the lysis cassette of various phages, including lambda . The site of insertion lies in a possible 'holin' gene, which could explain the block in host cell lysis . Dual promoters apparently responsible for the strong inducible transcription lie in an untranslated region just upstream from the putative holin gene . This region is probably equivalent to the site of the major late promoter and antiterminator of the lambdoid phages . The sequence features could, thus, account for the useful properties of the phi 105MU331 vector system.

Nucleic Acids Res, 1995 Feb 25, 23(4), 612 - 9
Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis; Meijer WJ et al.; In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication . The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060 . The SSO of pTA1015 was isolated by shotgun cloning in a specially designed vector, pWM100, which has no SSO of its own . Sequence analysis revealed that the SSO of pTA1015 is almost identical to formerly described palT type SSOs . Also pTA1020 and pTA1060 were shown to contain SSOs highly homologous to palT . Using Southern hybridization with the palT of pTA1015 as a probe, the SSO of pTA1040 was cloned . Sequence analysis revealed a region of 200 bp which is 77% identical to the palT of pTA1015 . The plasmids pTA1030 and pTA1050 contain an SSO which is highly homologous to the SSO of pTA1040 . The majority of the SSOs of rolling-circle plasmids from B.subtilis seem to belong to two related families which we denote as palT1 (present on pTA1015, pTA1020 and pTA1060) and palT2 (present on pTA1030, pTA1040 and pTA1050) . Both families of SSOs are highly efficient single-strand-conversion signals in B.subtilis.

Virology, 1995 Feb 20, 207(1), 23 - 31
Transcription regulation in Bacillus subtilis phage phi 29: expression of the viral promoters throughout the infection cycle; Monsalve M et al.; Transcription of the genome of Bacillus subtilis phage phi 29 is tightly controlled, taking place in two stages, early and late . We have analyzed the abundance of the transcripts produced from each viral promoter throughout the infection cycle . We compare the relative strength of each promoter, as well as get a better understanding of the regulatory events, finding a new promoter regulated by the viral protein p4 . The two strong early promoters, A2b and A2c, responsible for the expression of genes 6 to 1, are coordinately repressed by the viral protein p4, although repression is not complete: both promoters are still active at late times of infection . Since repression by protein p4 was very efficient in vitro, and affects its own synthesis, it is likely that this protein is produced in limiting amounts, not being bound to all viral DNA molecules present in the cell at a given time . Protein p4, also known to activate the late promoter responsible for the expression of all the structural and morphogenetic genes, is the key regulator of phage phi 29 development.

J Biol Chem, 1995 Feb 17, 270(7), 2938 - 45
The Bacillus subtilis histone-like protein Hbsu is required for DNA resolution and DNA inversion mediated by the beta recombinase of plasmid pSM19035; Alonso JC et al.; The beta recombinase, encoded by the Gram-positive bacterial plasmid pSM19035, is unable to mediate DNA recombination in vitro unless a host factor is provided . The factor has now been identified as the Bacillus subtilis Hbsu protein . Hbsu is a nonspecific DNA-binding and DNA-bending protein . The beta recombinase, in the presence of highly purified Hbsu protein, is able to catalyze in vitro intramolecular recombination between two specific recombination sites on a supercoiled DNA molecule . DNA resolution was obtained when the two crossing over sites (six sites) were directly oriented, whereas DNA inversion was the product when the six sites were in inverse orientation . The ability of the Escherichia coli chromatin-associated proteins HU, IHF, Fis, and H-NS to substitute for Hbsu was investigated . HU efficiently stimulated beta-mediated recombination, while the effect of IHF was partial and that of Fis and H-NS was undetectable . In addition, the beta protein was able to mediate DNA recombination in both wild-type and IHF-deficient E . coli cells, but failed to do so in an HU-deficient strain . The data presented provide direct evidence that a chromatin-associated protein is strictly required for beta-mediated recombination.

Genes Dev, 1995 Feb 15, 9(4), 503 - 8
Cell-cell signaling pathway activating a developmental transcription factor in Bacillus subtilis; Londono-Vallejo JA et al.; Transcription in the mother cell at early stages of sporulation in Bacillus subtilis is controlled by sigma E, a sigma factor that is synthesized in the predivisional cell as an inactive larger precursor, pro-sigma E . Activation of sigma E depends on sigma F, the factor that governs transcription in the forespore . Genetic experiments have indicated that transduction of the activation signal from the forespore to the mother cell requires the products of some genes belonging to the sigma F-controlled regulon . We have identified and characterized a sigma F-dependent gene, csfX, encoding a protein necessary and sufficient for triggering processing of pro-sigma E . The CsfX protein contains a typical amino-terminal signal sequence suggesting that, although synthesized in the forespore, it may act across the septum to activate the membrane-bound enzyme that is responsible for pro-sigma E processing in the mother cell.

FEBS Lett, 1995 Feb 6, 359(1), 23 - 6
HOQNO interaction with cytochrome b in succinate:menaquinone oxidoreductase from Bacillus subtilis; Smirnova IA et al.; 2-n-Heptyl 4-hydroxyquinoline-N-oxide (HOQNO) inhibits the succinate:quinone oxidoreductase activity of isolated and membrane-bound succinate:menaquinone oxidoreductase of B . subtilis . The inhibition pattern resembles closely that observed for alpha-thenoyltrifluoroacetone and carboxins in the mitochondrial succinate:ubiquinone oxidoreductase: ca . 90% of the activity is highly sensitive to HOQNO (Ki ca . 0.2 microM for the isolated enzyme) whereas the rest 10% proves to be resistant to the inhibitor . HOQNO binding is shown to perturb the absorption spectrum of the ferrous di-heme cytochrome b of the B . subtilis succinate:quinone oxidoreductase both in the alpha and Soret bands . In addition, the inhibitor is shown to bring about a negative shift of Em of the low-potential heme b . It is suggested that HOQNO interacts with a menasemiquinone binding site near the low-potential heme and suppresses the MQ.(-)-to-MQH2 step of the quinone reductase reaction but allows partly for the MQ-to-MQ.- transition to occur; dismutation of MQ . formed in the latter reaction to MQ and MQH2 may account for the 10% of the enzyme activity insensitive to HOQNO.

J Chromatogr B Biomed Appl, 1995 Feb 3, 664(1), 219 - 23
Characterization of weak hydrophobic composite sorbents and their application to the isolation of bacterial lectin; Ivanov AE et al.; Composite sorbents based on wide-pore glass and silica coated with N-butyl polyacrylamide (butyl-PA-glass and butyl-PA-silica) were studied . The surface tension of butyl-PA-silica is 50-55 mJ/m2 as evaluated by the sedimentation volume technique . The linear dependences of log k' on ammonium sulphate concentration were determined by isocratic chromatography of the dipetide Trp-Trp and lysozyme on butyl-PA-glass . Both solutes were shown to have a weaker retention on butyl-PA-glass than on butyl-Toyopearl 650C . This weaker retention is beneficial in the purification of sialic acid-binding lectin from Bacillus subtilis.

PCR Methods Appl, 1995 Feb, 4(4), 212 - 8
Background-minimized cassette mutagenesis by PCR using cassette-specific selection markers: a useful general approach for studying structure-function relationships of multisubstrate enzymes; Majumder K et al.; An efficient protocol, termed background-minimized cassette mutagenesis (BMCM) by PCR, has been developed for multiple mutagenesis of DNA . This method uses suitable extension primers for incorporating various mutation(s) and is not limited by either the nature of the mutation or the size and spatial location of mutational loci . Minimization of the wild type background clone and mutant selection at very high frequency were easily achieved through a two-step process . First, a deletion of a unique restriction site within the cassette was introduced through additional silent mutation(s) . Then, the recombinant clones were digested with the corresponding enzyme followed by transformation when selective linearization of wild-type clone led to its near total removal leaving the mutant clones as the only practicable transformants . Because it is generally possible to design several such cassette-specific unique background minimization markers for any gene, for multiple mutagenesis involving distally located portions of the gene the present protocol is superior to other currently available methods . The efficiency of BMCM-PCR has been demonstrated here by using the multisubstrate enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) of Bacillus subtilis as a model system . Three different sets of cassettes of varying sizes were generated to encompass the three putative active/binding regions in the beginning, middle, and the end of the gene encoding EPSPs . Very high efficiency of mutation incorporation and selection were obtained in all cases . Furthermore, by taking advantage of the unique cassette-specific background elimination markers, it was possible to generate a nested set of double and/or triple mutants . The mutant enzymes were overexpressed in Escherichia coli and purified to near homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutat Res, 1995 Feb, 316(3), 123 - 31
Restriction, methylation and ligation of 5-hydroxymethyluracil-containing DNA; Vilpo JA et al.; Oxidation of DNA and its components can cause genetic mutations and chromosomal instability . These changes have generally been implicated in aging . Oxidation of the methyl group of thymidine residues in DNA is known to result in the formation 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) . We have utilized Bacillus subtilis phage SPO1 DNA as a model of oxidatively damaged DNA . In this phage, all thymine (Thy) residues are replaced by 5-hydroxymethyluracil (5HmUra), but the species is naturally devoid of other oxidatively-induced DNA lesions . Particular attention was paid to the behavior of 5HmUra-containing DNA as a target for several enzymes employing DNA as substrate; restriction endonucleases, dam DNA methylase and T4 DNA ligase . We noticed that susceptibility of SPO1 DNA varied when different restriction endonucleases having 5HmUra in the restriction sites were tested . Endonucleolytic cleavage brought about Sau3A proceeded as effectively with SPO1 DNA as with conventional DNA (lambda phage) . The same was true when the ligation of Sau3A sites was performed with T4 DNA ligase . In contrast, both endonucleolytic cleavage and ligation were slower in SPO1 DNA, compared with lambda phage, when Taq I and T4 DNA ligase were used for restriction and ligation, respectively . We also noticed that SPO1 phage does not naturally contain N6-methyladenine (N6MeAde) opposite 5HmUra, i.e., no hydrolysis of SPO1 DNA was observed when assessed with methylation-dependent restriction endonuclease DpnI . Our results show that the presence of 5HmUra in the respective site of DNA does not, per se, prevent the activity of restriction endonucleases, ligases or DNA methylases . These data support the view that oxidation of Thy to 5HmUra in target DNA does not necessarily result in substantial deterioration in the functions of DNA processing enzymes.

J Bacteriol, 1995 Feb, 177(4), 1112 - 5
Identification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis; Glaser P et al.; The Bacillus subtilis narA locus was shown to include narQ and narA . The putative product of narQ is similar to FdhD, which is required for formate dehydrogenase activity in Escherichia coli . NarA showed homology to MoaA, a protein involved in biosynthesis of the molybdenum cofactor for nitrate reductase and formate dehydrogenase . Analysis of mutants showed that narA but not narQ is required for both nitrate assimilation and respiration.

J Bacteriol, 1995 Feb, 177(4), 1082 - 5
Sporulation protein SpoIVFB from Bacillus subtilis enhances processing of the sigma factor precursor Pro-sigma K in the absence of other sporulation gene products; Lu S et al.; Processing of inactive pro-sigma K to active sigma K in the mother cell compartment of sporulating Bacillus subtilis is governed by a signal transduction pathway emanating from the forespore and involving SpoIVFB in the mother cell . Coexpression of spoIVFB and sigK (encoding pro-sigma K) genes in growing B . subtilis or Escherichia coli enhanced pro-sigma K processing in the absence of other sporulation-specific gene products . The simplest explanation of these results is that SpoIVFB is a protease that processes pro-sigma K.

EMBO J, 1995 Feb 1, 14(3), 619 - 28
Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance; Sahoo T et al.; The termination of DNA replication at a sequence-specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication terminator protein (RTP) of subunit mol . wt 14,500 . RTP has become an attractive protein with which to study the molecular mechanism of termination because its crystal structure has now been solved and the previous lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well-studied Gram-negative Escherichia coli system . We have exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E . coli only when two RTP dimers are bound co-operatively to overlapping core and auxiliary sequences comprising the terminus . A core sequence by itself binds one dimer of RTP, but elicits no contrahelicase activity . Binding of two RTP dimers to a tandem head-to-tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for termination . RTP blocks unwinding of DNA substrates containing heteroduplex regions that include the terminus and are in the size range of approximately 50 to > 1000 bp in length . Thus, the protein blocks authentic helicase-catalyzed unwinding rather than just the translocation of the helicase on DNA.

J Bacteriol, 1995 Feb, 177(3), 861 - 3
Different roles for KinA, KinB, and KinC in the initiation of sporulation in Bacillus subtilis; LeDeaux JR et al.; Activation (phosphorylation) of the transcription factor encoded by spo0A is essential for the initiation of sporulation in Bacillus subtilis . At least three histidine protein kinases are involved in the phosphorylation of Spo0A . Under some growth conditions, KinA was the primary kinase, but under other conditions, KinB had the more critical role . KinC was required for the initial activation of Spo0A, even in the presence of KinA and KinB.

J Bacteriol, 1995 Feb, 177(3), 839 - 42
The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis; Hoffman RJ et al.; The mtrB gene from Bacillus pumilus encodes a 76-amino-acid polypeptide with 77% identity to the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis . B . pumilus TRAP binds trp leader RNA from either B . subtilis or B . pumilus in a tryptophan-dependent manner . Altering threonine 52 to alanine eliminated RNA-binding activity of B . pumilus TRAP.

J Bacteriol, 1995 Feb, 177(3), 765 - 73
Bacillus subtilis possesses a second determinant with extensive sequence similarity to the Escherichia coli mreB morphogene; Abhayawardhane Y et al.; A gene with substantial sequence similarity to the mreB morphogene of Bacillus subtilis has been identified at 302 degrees on the chromosomal map by A . Decatur, B . Kunkel, and R . Losick (Harvard University; personal communication) . Our characterization has revealed that the protein product of this determinant (termed mbl for mreB-like) is 55 and 53% identical in sequence to the MreB proteins of B . subtilis and Escherichia coli, respectively . The protein is 86% identical to a protein identified as MreB from Bacillus cereus, suggesting that the B . cereus protein is actually Mbl . Insertional inactivation of mbl indicated that this gene is not essential for cell viability or sporulation . Cells bearing mutant mbl alleles display a decreased growth rate and an altered cellular morphology . The cells appear bloated and are frequently twisted . Intergenic suppressor mutations which restore the growth rate to an approximately normal level arise within the mutant population . A second site mutation, designated som-1, was mapped to the hisA-mbl region of the chromosome by transduction.

J Bacteriol, 1995 Feb, 177(3), 716 - 22
Identification and characterization of the Bacillus subtilis spoIIP locus; Frandsen N et al.; We have identified an additional sporulation gene, named spoIIP, in the region of the Bacillus subtilis chromosome located immediately downstream of the gpr gene (227 degrees on the genetic map) . A null mutation of spoIIP arrests sporulation at an early stage of engulfment (stage IIii), a phenotype similar to that already described for spoIID and spoIIM mutants . This gene encodes a 401-residue polypeptide, which is predicted to be anchored in the membrane, most of the protein being localized outside the cytoplasm . The spoIIP gene is transcribed from a promoter located in the interval between the gpr and the spoIIP reading frames . This promoter has the structural and genetic characteristics of a sigma E-dependent promoter . Transcription of spoIIP is abolished by a mutation in spoIIGB, the gene encoding sigma E, and can be induced during exponential growth in cells engineered to produce an active form of sigma E . Plasmid integration-excision experiments leading to the formation of genetic mosaics during sporulation indicate that as with SpoIID and SpoIIM, SpoIIP is required only in the mother cell . Disruption of spoIIP had little or no effect on the expression of sigma F- and sigma E-controlled regulons but inhibited transcription from sigma G-dependent promoters and abolished transcription from promoters under the control of sigma K . We propose that, together with SpoIID and SpoIIM, the SpoIIP protein is involved in the dissolution of the peptidoglycan located in the sporulation septum.

J Bacteriol, 1995 Feb, 177(3), 573 - 9
Nitrogen regulation of nasA and the nasB operon, which encode genes required for nitrate assimilation in Bacillus subtilis; Nakano MM et al.; The divergently transcribed nasA gene and nasB operon are required for nitrate and nitrite assimilation in Bacillus subtilis . The beta-galactosidase activity of transcriptional lacZ fusions from the nasA and nasB promoters was high when cells were grown in minimal glucose medium containing poor nitrogen sources such as nitrate, proline, or glutamate . The expression was very low when ammonium or glutamine was used as the sole nitrogen source . The repression of the genes during growth on good sources of nitrogen required wild-type glutamine synthetase (GlnA), but not GlnR, the repressor of the glnRA operon . Primer extension analysis showed that the -10 region of each promoter resembles those of sigma A-recognized promoters . Between the divergently oriented nasA and nasB promoters is a region of dyad symmetry . Mutational analysis led to the conclusion that this sequence is required in cis for the activation of both nasA and nasB . The derepression of these genes in a glnA mutant also required this sequence . These results suggest that an unidentified transcriptional activator and glutamine synthetase function in the regulation of nasA and the nasB operon.

Mol Microbiol, 1995 Feb, 15(3), 455 - 62
comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis; van Sinderen D et al.; comK is a positive autoregulatory gene occupying a central position in the competence-signal-transduction network . All regulatory routes identified in this network converge at the level of comK expression . The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA-binding and uptake apparatus . In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK) . Using portions of the region upstream of comC we show that the ComK-binding sequences are essential for the expression of competence . Moreover, we demonstrate that the presence of ComK stimulates the expression of comF-lacZ and comG-lacZ translational fusions in vivo in Escherichia coli . These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF).

Biosci Biotechnol Biochem, 1995 Feb, 59(2), 321 - 2
TmrB protein, which confers resistance to tunicamycin on Bacillus subtilis, binds tunicamycin; Noda Y et al.; Overproduction of TmrB protein, a 22.5-kDa protein with an N-terminal ATP-binding region and a C-terminal amphiphilic alpha-helix, confers resistance to tunicamycin on Bacillus subtilis . TmrB protein was found to bind Sepharose 6B to which tunicamycin was covalently linked . Experiments with mutant proteins found that the C-terminal region of TmrB protein might be involved in the binding to tunicamycin.

Microbiology, 1995 Feb, 141 ( Pt 2), 345 - 50
Analysis of errors in finished DNA sequences: the surfactin operon of Bacillus subtilis as an example; Fabret C et al.; Increased productivity in DNA sequencing would not be valid without a straightforward detection and estimation of errors in finished sequences . The sequence of the surfactin operon from Bacillus subtilis was obtained by two different groups and by chance we were also working on the same chromosome region . Taking advantage of this situation we report in this paper, the number and nature of errors found in the overlapping part of the DNA sequences obtained by the three laboratories . The coincidence of some of the errors with compression in sequence ladders and with secondary DNA structures as well as the detection of frameshift errors using computer programs, are demonstrated . Finally we discuss the definition of a new sequencing strategy that might minimize both the error rate and the cost of sequencing.

Microbiology, 1995 Feb, 141 ( Pt 2), 329 - 35
Sequence analysis of the 308 degrees to 311 degrees segment of the Bacillus subtilis 168 chromosome, a region devoted to cell wall metabolism, containing non-coding grey holes which reveal chromosomal rearrangements; Lazarevic V et al.; The 29.71 kb chromosomal region of Bacillus subtilis 168 extending from 308 degrees to 311 degrees contains 18 ORFs . Functions of most of these ORFs were identified and associated with cell wall metabolism . Sequences of two non-coding regions of 0.7 and 2.2 kb flanking the ggaAB operon involved in the synthesis of poly(3-O-beta-D-glucopyranosyl N-acetylgalactosamine 1-phosphate), a minor teichoic acid, correspond to five degenerate segments of neighbouring protein-coding regions . We discuss the possibility that such grey holes are indicative of a chromosomal rearrangement which could have arisen from horizontal gene transfer.

Microbiology, 1995 Feb, 141 ( Pt 2), 323 - 7
Complete nucleotide sequence of a skin element excised by DNA rearrangement during sporulation in Bacillus subtilis; Takemaru K et al.; As part of the Bacillus subtilis genome sequencing project, we have determined the complete nucleotide sequence of a skin element which is located between spoIVCB and spoIIIC . The entire sequence of this element is 48,032 bp in length, and contains 57 ORFs with putative ribosome-binding sites . Two of them correspond to previously sequenced and characterized genes, cwIA and spoIVCA . Furthermore, seven ORF products identified in this element show interesting similarities with known proteins present in data banks, including the phi 105 immunity repressor, the phi 105 Cro-like protein and the SPP1 terminase . These results indicate the possibility that the skin element is a cryptic remnant of an ancestral temperate phage.

Microbiology, 1995 Feb, 141 ( Pt 2), 321 - 2
Nucleotide sequence of the Bacillus subtilis dnaD gene; Bruand C et al.; The dnaD gene of Bacillus subtilis was identified within a 104 kb DNA segment cloned into a yeast artificial chromosome . The nucleotide sequence of the wild type and dnaD23 mutant genes were determined . dnaD is predicted to encode a protein of 232 amino acids with no similarity to proteins in the data banks.

Microbiology, 1995 Feb, 141 ( Pt 2), 311 - 9
The Bacillus subtilis chromosome region encoding homologues of the Escherichia coli mssA and rpsA gene products; Sorokin A et al.; A gene was found in Bacillus subtilis which encodes a protein highly homologous to the Escherichia coli rpsA gene product, the S1 ribosomal protein . The B . subtilis protein contains the domain responsible for binding to ribosomes and two S1 motifs, instead of four as found in the E . coli protein . The B . subtilis protein is similar in this way to the equivalent protein of plant chloroplast ribosomes, supposed to be the counterpart of E . coli S1 . The gene is expressed during vegetative growth in B . subtilis at the transcriptional and translational levels, as judged by Northern hybridization and expression in a translational fusion with a reporter gene . In contrast to the E . coli situation, it can be inactivated without dramatic effects on cell viability . Southern hybridization of the B . subtilis DNA fragment encoding this gene revealed specific homologous fragments in all other Gram-positive bacteria tested . The hybridization pattern with B . stearothermophilus suggests the presence of at least two homologous genes in this bacterium . We show that in B . subtilis the ORF preceding the rpsA homologue encodes a protein which is highly similar to the product of the E . coli mssA gene which is located upstream of rpsA . Again, in contrast to the E . coli situation, where these genes are co-transcribed, in B . subtilis they are separated by a transcription terminator and the mssA homologue is transcribed during sporulation . We suggest that during the evolution very similar structures and genetic organization of these two genes were conserved but acquired different functions in Gram-negative and Gram-positive bacteria.

Microbiology, 1995 Feb, 141 ( Pt 2), 299 - 309
Sequence around the 159 degree region of the Bacillus subtilis genome: the pksX locus spans 33.6 kb; Albertini AM et al.; The nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined . Six ORFs were recognized, one of which extended for 13,341 nucleotides . Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism . At the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans wA gene, responsible for the synthesis of a polyketide pigment, were recognized . The observed similarities suggest that the 20 kb region and the previously reported 13.6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.

Microbiology, 1995 Feb, 141 ( Pt 2), 291 - 7
The thyA gene from Bacillus subtilis exhibits similarity with the phage phi 3T thymidylate synthase gene; Tam NH et al.; The gene encoding thymidylate synthase A (thyA) was cloned from a genomic library of Bacillus subtilis 168 . The sequence of thyA was found to be highly similar to that of the phage phi 3T thymidylate synthase gene . This similarity is, however, limited to about 862 nucleotides, spanning the coding region and the adjacent 3' region . The flanking sequences are not related . An integrative plasmid containing a DNA fragment with a deletion within the coding region of thyA was constructed and used to replace the chromosomal thyA gene . Transformants unable to grow without thymidine at 47 degrees C were obtained . Genetic and physical mapping techniques were used to show that the cloned DNA fragment harbouring delta thyA was integrated at 168 degrees on the B . subtilis chromosome.

Microbiology, 1995 Feb, 141 ( Pt 2), 281 - 90
Genes encoding xylan and beta-glucan hydrolysing enzymes in Bacillus subtilis: characterization, mapping and construction of strains deficient in lichenase, cellulase and xylanase; Wolf M et al.; The gene encoding extracellular xylanase (xynA) was amplified as a 770 bp DNA fragment from Bacillus subtilis 168 chromosomal DNA by PCR . The genes encoding endo-beta-1,4-glucanase (eglS) and endo-beta-1,3-1,4-glucanase (bglS) were isolated from a genomic library of B . subtilis 168 . The sequences of xynA and eglS were identical to those of the xylanase and cellulase genes from B . subtilis PAP115 . Integrative plasmids containing DNA fragments with deletions in the coding region of the genes were constructed and used to replace the chromosomal eglS, bglS and xynA genes of B . subtilis 168 . Strains without any detectable activity against xylan (Xyn-), carboxymethylcellulose (Egl-) or mixed linked beta-1,3-1,4-glucan (Egl- Bgl-) were obtained . The genes were mapped at 170 degrees (eglS), 175 degrees (xynA) and 340 degrees (bglS) on the B . subtilis chromosome.

Microbiology, 1995 Feb, 141 ( Pt 2), 277 - 9
A 10 kb nucleotide sequence at the 5' flanking region (32 degrees) of srfAA of the Bacillus subtilis chromosome; Fujishima Y et al.; The nucleotide sequence of approximately 10 kb at the 5' flanking region (32 degrees) of srfAA of the Bacillus subtilis chromosome was determined . Eleven putative ORFs were identified . Three of them (orf6, orf7 and orf8) coincided with known B . subtilis genes (comJ, comI and tlpC) encoding a competence-specific protein, a DNA-entry nuclease and a transducer-like protein, respectively . The products of two other ORFs showed similarity to GlnP of Escherichia coli (orf1) and beta-glucosidase A of B . polymyxa (orf5).

Microbiology, 1995 Feb, 141 ( Pt 2), 269 - 75
Determination of a 21548 bp nucleotide sequence around the 24 degrees region of the Bacillus subtilis chromosome; Ogawa K et al.; A 21548 bp nucleotide sequence around the 24 degrees region of the Bacillus subtilis chromosome, located 306 kb downstream from the zero point of the physical map, was determined . Twenty-one putative ORFs were identified: two ORFs (Orf1 and Orf20) were consistent with the nucleotide and amino acid sequence of B . subtilis OrfJ, whose function is not known, and Pcp, respectively; four were found to display significant similarities to known proteins in data banks, i.e . 5-keto-3-deoxyglucarate dehydratase (Orf2), aldehyde dehydrogenase (Orf3) and two glucarate dehydratases (Orf4 and 5); three had considerable similarity to the sensor-regulator proteins of bacterial two-component signal transduction systems (Orf1, 11 and 12); two had considerably high levels of similarity to parts of known proteins (Orf13 and 19); and five showed low levels of similarity to known proteins in the data banks . The remaining six ORFs showed no similarity to known proteins.

Microbiology, 1995 Feb, 141 ( Pt 2), 261 - 8
SubtiList: a relational database for the Bacillus subtilis genome; Moszer I et al.; In the framework of the international collaborative project aiming to sequence the whole Bacillus subtilis chromosome, we have created a relational database for managing and analysing information associated with the molecular genetics of this bacterium: SubtiList . It allows recovery of non-redundant DNA sequences of the B . subtilis genome, as well as related information, i.e . genes, proteins, etc . A logical structure has been designed with appropriate links between the different objects, and a set of procedures has been implemented for data updating and management . The database is organized around a core constituted by all known contigs of B . subtilis, i.e . sets of non-redundant sequences created from original entries in the EMBL data library . A user-friendly interface has been developed to make the database easy to consult . Sequence analysis tools have been integrated into the database, such as a program for rapid similarity searching of protein data banks, and a powerful DNA pattern searching program . Thanks to the consistency of SubtiList, we have performed a codon usage analysis by Factorial Correspondence Analysis, and a study of the distribution of the isoelectric points of known proteins of B . subtilis . The SubtiList database is available through anonymous ftp (address 'ftp.pasteur.fr' or IP number 157.99.64.12, directory '/pub/GenomeDB/SubtiList').

J Appl Bacteriol, 1995 Feb, 78(2), 97 - 108
Antibiotic production and biocontrol activity by Bacillus subtilis CL27 and Bacillus pumilus CL45; Leifert C et al.; Bacillus subtilis CL27 and B . pumilus CL45 showed similar activity against Botrytis cinerea in in vitro plate assays . In a seedling bioassay, however, B . subtilis CL27 had activity similar to a commercial fungicide while B . pumilus CL45 failed completely to prevent seedling damping-off caused by Bot . cinerea . Antibiotic production by the two Bacillus strains was found to depend on the growth substrate and highest antibiotic production was found on media based on homogenized cabbage tissue . Antibiotic activity was found to depend on the pH and nutrient concentration in the assay medium . Antifungal antibiotics produced by B . subtilis CL27 and B . pumilus CL45 in different fermentation media were separated by thin layer chromatography . As suspected from the activity spectrum, three antibiotics (one with activity against Alternaria brassicicola, one with activity against Botrytis cinerea and one with activity against both fungi) could be detected in the fermentation broth of CL27, but only one in the fermentation broth of CL45 . The two antibiotics produced by strain CL27 with activity against A . brassicicola were identified as peptides since their bands on the TLC plates developed a green to blue/green colour after treatment with 4,4'-tetramethyldiamino-diphenylmethane (TDM) reagent . The third antibiotics produced by strain CL27 and antibiotic produced by CL45 had a similar Rf-value and appeared not to be peptides based on the reaction with TDM . However, they showed a slightly different activity spectrum when tested against a range of different fungi . Antibiotic production was clearly indicated as the mode of action of in vivo biocontrol by strain CL27 against damping off caused by Bot . cinerea of Astilbe micro-plants, because a u.v.-induced antibiotic negative mutant strain CL27b showed no activity in seedling bioassays in vivo . Also the mutant strain CL27a which produced the two peptide antibiotics but had lost the ability to produce the non-peptide antibiotic, showed greatly reduced in vivo activity.

Antibiot Khimioter, 1995 Feb, 40(2), 19 - 21
{A novel fungicide of the iturin group, obtained from a marine isolate of Bacillus subtilis . Isolation, physico-chemical and biochemical properties, identification}; Oleinikova GK et al.; Strain KMM 457 of Bacillus subtilis was isolated from a frozen sample of the soft coral Sarcophyton sp . The samples were collected in 1989 in the South China Sea near the Vietnam Shore during an expedition on the board of R/V "Akademik Oparin" . Metabolites of the isolate were investigated and it was found to produce a number of physiologically active compounds . One of them designated as OGA showed fungicidal activity . By the physicochemical properties it was referred to the group of iturins . The comparison with the described antibiotics of the iturin group suggested that it was a new representative of the iturin group.

Dtsch Tierarztl Wochenschr, 1995 Feb, 102(2), 101 - 2
{Preliminary results of microbiologic studies of the processing of contaminated plastics (recycling)}; Agthe O et al.; Spores of Bacillus subtilis were mixed with 200 g of polyethylene-granulate . The result was an initial concentration of about 10(5) colony-forming-units per g polyethylene . This mixture passed an injection-moulding machine . The formed material was collected under sterile conditions, cut into pieces of about 1 cm and analysed for bacteria . In 4 of 23 cases Bacillus subtilis was present after passing the injection-moulding machine . A quantitative determination had the result, that about 3% of the spores could be found after the process of passing the injection-moulding machine.

Appl Environ Microbiol, 1995 Feb, 61(2), 487 - 94
Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis; Walter S et al.; Various streptomyces strains {Streptomyces lividans 66, Streptomyces vinaceus, and Strepotmyces coelicolor A3 (2)} acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli . The expression level in these hosts was two to three times lower than in S . reticuli, indicating the absence of positive regulatory elements . Like S . reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part . The cel-1 gene with its original upstream region was not expressed within Escherichia coli . When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E . coli . However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms . As the cellulase (Avicelase) synthesized by S . reticuli is not cleaved by the E . coli proteases, its posttranslational modification is proposed . With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter.

Mol Microbiol, 1995 Feb, 15(3), 421 - 9
Isolation and analysis of mutants of the dnaK operon of Bacillus subtilis; Schulz A et al.; Bacillus subtilis contains at least three classes of heat-shock genes regulated by different mechanisms . We are studying class I heat-shock genes encoded by the operons dnaK and groE . These two operons are both expressed from a vegetative promoter, and their regulation involves a novel heat-shock element designated CIRCE . Here we show that induction of both operons results from enhanced synthesis of mRNA and is independent of de novo protein synthesis . To answer the question of whether dnaK is involved in the deregulation of the heat-shock response as reported for Escherichia coli, two different insertion mutations were isolated within the tetracistronic dnaK operon (orf39-grpE-dnaK-dnaJ) . In one mutant a cat cassette was inserted at the beginning of orf39 . Transcriptional analysis revealed that this mutation abolished expression of the whole operon . In contrast, the basal level of groE mRNA was significantly increased at 37 degrees C, followed by a prolonged delay in the shut off after temperature upshift . These data point to a crucial role for the orf39 gene in the regulation of class I heat-shock genes . In the other mutant an internal 0.8 kb Bg/II fragment of dnaK was replaced by the cat cassette . In contrast to E . coli dnaK null mutants, the two B . subtilis dnaK operon mutants could grow within a temperature range from 16-52 degrees C . At temperatures above 52 degrees C, they failed to form colonies on agar plates, started to filament, and lost motility . Furthermore, the induction profile of the groE and dnaK operons was not impaired in the dnaK::cat mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1995 Feb, 15(3), 395 - 401
Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria?
Hueck CJ, Hillen W.
Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified . The cis-acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram-positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus . CR of most genes regulated via CRE is also affected by the trans-acting factors CcpA and HPr . Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE . HPr, a component of the phosphoenolpyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fructose-1,6-diphosphate-activated kinase . A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities . This mutant phenotype is similar to the one exhibited by a ccpA mutant . Direct protein-protein interaction between CcpA and HPr(Ser-P) was recently demonstrated and constitutes a link between metabolic activity and CR.

Biochemistry, 1995 Jan 24, 34(3), 902 - 9
Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P; Pan T; Folding of the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA) has been examined by Fe(II)-EDTA protection and bimolecular association . Fe(II)-EDTA results show that, in the presence of Mg2+, P RNA is folded into a core structure which includes most of the phylogenetically conserved nucleotides . Folding is cooperative and is complete at 5-6 mM Mg2+ with the {Mg2+}1/2 at 2-3 mM . The Hill constant indicates that this folding transition requires binding of at least three additional Mg2+ ions . Two RNA molecules consisting of nucleotides 62-239 {p(62-239)} and 240-401 + 1-61 {p(240-61)} of the B . subtilis P RNA have been constructed . These RNAs can in principle form the catalytically active structure primarily, if not solely, through tertiary interactions . Although either molecule by itself is inactive, the bimolecular complex is as active as the circularly permuted P RNAs from which it is derived . The binding constant of the complex can be as low as 0.1 microM and is strongly dependent on Mg2+ and K+ concentrations . Association of these molecules also induces a Mg2+ dependent cleavage at nucleotide 103 in p(62-239) . p(62-239) gives a Fe(II)-EDTA protection pattern very similar to the wild-type P RNA at identical Mg2+ concentrations . However, Fe(II)-EDTA protection in p(240-61) is completely lost, even though it contains many nucleotides that are conserved among all P RNAs . These results suggest that, like other RNAs, P RNA contains domains that can fold in the absence of the rest of the molecule . The implications of these results are discussed in the context of the P RNA structure and catalysis.

Eur J Biochem, 1995 Jan 15, 227(1-2), 510 - 5
A reactive, surface cysteine residue of the class-II fructose-1,6-bisphosphate aldolase of Escherichia coli revealed by electrospray ionisation mass spectrometry; Packman LC et al.; The state of post-translational modification of the class-II fructose-1,6-bisphosphate aldolase (FBP-aldolase) purified from Escherichia coli was examined by electrospray ionisation mass spectrometry (ESI-MS) . The mass was larger than that expected from the known DNA sequence by approximately 80 +/- 6 Da, suggesting the presence of a covalent modification on the protein . Phosphorylation (+ 80 Da), a known modification in an FBP-aldolase from Bacillus subtilis and a suspected modification in this E . coli aldolase, was ruled out as the extra mass was readily removed by treatment with dithiothreitol . Purification of aldolase by a protocol which omitted 2-mercaptoethanol from all buffers resulted in the purified protein having the expected mass (39016 Da) . The extra mass was therefore established as a covalent adduct of the protein with 2-mercaptoethanol (+ 76 Da) . Reduction and alkylation studies, followed by isolation of tryptic peptides, established that the site of attachment was Cys36 . Although no significant effect of the modification on the activity of the protein was observed, the study underlines the ease with which a protein can be modified covalently by a simple and mild purification procedure; such labelling, which may not always be benign, would be undetectable without the routine use of mass spectrometric analysis.

Gene, 1995 Jan 11, 152(1), 69 - 74
Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis; Jacobs MF; The cloning and sequence of the 5' untranslated region (5'-UTR) of the Bacillus licheniformis (Bl) 6816 subtilisin Carlsberg gene (subC) are reported here . The 5' and 3' ends of subC transcripts were characterized, and the promoter identified . Expression was studied using a fused lacZ reporter gene integrated into the chromosome of heterologous host Bacillus subtilis (Bs) . beta Gal activities of mutants deleted within the promoter region identified a region which is required for stimulation by the transcriptional activator proteins, DegU and DegQ . This region is close to the transcription start point (tsp), and is adjacent to a sequence homologous to that involved in DegU/Q stimulation of the Bs subtilisin gene, aprE . Expression of subC in Bs was optimized by the use of heterologous promoter and by the deletion of UTR sequences predicted to be involved in secondary structures in the native subC mRNA . Sequence comparison with other subtilisin Carlsberg-type-encoding genes revealed a high degree of conservation of the entire 5'-UTR, including regulatory sequences and promoter, as well as part of the structural gene.

Arch Biochem Biophys, 1995 Jan 10, 316(1), 260 - 6
Evidence for substrate stabilization in regulation of the degradation of Bacillus subtilis aspartate transcarbamylase in vivo; Hu P et al.; Aspartate transcarbamylase (ATCase) is rapidly degraded in Bacillus subtilis cells that are starved for a carbon or nitrogen source or a required amino acid . The hypothesis that ATCase degradation may be regulated in vivo by protection of the enzyme by substrate binding was tested by studies of a mutant ATCase (Arg99 to Ala, R99A), which binds substrate so poorly that it fails to support pyrimidine-independent growth in a pyrB strain, but still has 10% of normal activity when saturated with substrates . Unlike normal ATCase, R99A ATCase was degraded rapidly in exponentially growing cells . Degradation of the mutant enzyme was two-fold slower in a relA strain, as was degradation of the normal ATCase . The stability of purified R99A ATCase to denaturation by heat or guanidine hydrochloride was identical to that of wild-type ATCase, as was its circular dichroic spectrum . The wild-type and R99A ATCase were degraded identically in vitro by subtilisin, except that the mutant enzyme was much less effectively protected against cleavage by carbamyl phosphate, as expected . The carbamyl phosphate pool in glucose-limited B . subtilis cells was only one-third of the pool in exponentially growing cells . These results indicate that protection of ATCase by carbamyl phosphate binding could be one of the elements that regulate ATCase stability in vivo . However, carbamyl phosphate pools were the same in cells grown with ammonium ions and with a mixture of 20 common amino acids, conditions under which ATCase stability in vivo differs . Thus, other means of regulating ATCase degradation must also exist.

J Biol Chem, 1995 Jan 6, 270(1), 296 - 303
Secondary structure of uracil-DNA glycosylase inhibitor protein; Balasubramanian S et al.; The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that inactivates uracil-DNA glycosylase from diverse organisms (Wang, Z., and Mosbaugh, D . W . (1989) J . Biol . Chem . 264, 1163-1171) . The secondary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance . The protein adopts a single well defined structure consisting of five anti-parallel beta-strands and two alpha-helices . Six loop or turn regions were identified that contain approximately one half of the acidic amino acid residues and connect the beta-strands sequentially to one another . The secondary structure suggests which regions of Ugi may be involved in interactions with uracil-DNA glycosylase.

Yi Chuan Xue Bao, 1995, 22(6), 478 - 86
{Cloning and analysis of prophage PBSX repressor gene from Bacillus subtilis}; Li N et al.; The repressor gene and its allele--a temperature sensitive mutant have been cloned from the defective prophage PBSX of Bacillus subtilis by means of PCR technique . The characterization of these sequences suggest that wild-type and mutant gene have the same open reading frame, which probably encode a repressor protein of 113 amino acids, and the putative promoter region and ribosome binding sites within the sequences have also been identified . The results from complementation experiments demonstrate that repressor encoded by wild-type gene is capable of blocking the temperature-shift to induce PBSX prophage in Bacillus subtilis.

Nucleic Acids Symp Ser, 1995, (34), 243 - 4
How to alter the bacterial genome structure; Itaya M et al.; Bacterial chromosomes, mostly of circular form, have an unique primary structure that are stably maintained . We initiated a systematic study to induce changes of the structure of the Bacillus subtilis chromosome . There are two main goals: (i) to obtain general concepts for possible plasticity of the bacterial genome and (ii) to apply the proposed genome technology to bacteria.

Annu Rev Genet, 1995, 29, 477 - 508
Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis; Grossman AD; Interconnected regulatory networks control the initiation of sporulation and the development of genetic competence in Bacillus subtilis . These two developmental pathways have both common and distinct elements and employ similar regulatory strategies . Activation of the ComK transcription factor serves to integrate many of the physiological signals that control competence development, including cell density signals . The cell density signals for competence are mediated by two different peptide pheromones, the ComX pheromone, a 9 or 10 amino acid peptide with a modified tryptophan residue, and the competence stimulating factor, CSF, which is at least in part a peptide . Activation of the Spo0A transcription factor by phosphorylation serves as a developmental checkpoint and to integrate several physiological signals that control entry into the sporulation pathway . The physiological signals are generated by conditions of nutrient deprivation, high cell density, the Krebs cycle, DNA replication, DNA damage, and some aspect of the chromosome partitioning machinery . Both the ComK and Spo0A transcription factors are part of autogenous regulatory loops that control entry into competence or sporulation.

Annu Rev Genet, 1995, 29, 41 - 67
Chromosome partitioning in bacteria; Wake RG et al.; This review addresses chromosome partitioning in Escherichia coli and Bacillus subtilis . The first part deals with events associated with completion of a round of replication to an extent that yields separable chromosomes . Events more directly involved in chromosome movement are covered in the second part . In the final section, a model for chromosome partitioning based on the information presented in the first two parts is presented.

Nucleic Acids Symp Ser, 1995, (33), 92 - 4
Generation and characterization of circular Bacillus subtilis RNase P RNA; activation by RNase P protein; Puttaraju M et al.; A circular form of Bacillus subtilis ribonuclease P RNA (C-P RNA) was generated in vitro by splicing permuted intron-exon (PIE) sequences containing the P RNA sequence . Steady-state cleavage of pre-tRNA(Asp) catalyzed by circular P RNA is slightly faster than the linear form . Furthermore, steady-state turnover catalyzed by circular RNase P RNA is activated by the addition of the Bacillus subtilis protein component of RNase P, to a rate constant equal to the linear holoenzyme under identical conditions . Also, the circles are resistant to nuclease degradation, have less sequence heterogeneity, and may enhance the formation of a unique structure . Therefore, circular forms of RNase P RNA should prove useful for mutagenesis and structural studies.

Dermatology, 1995, 191(1), 6 - 8
May Helicobacter pylori be important for dermatologists?
Rebora A, Drago F, Parodi A.
Helicobacter pylori, a microaerophilic gram-negative bacterium, is the major cause of gastritis, plays a key role in the etiology of peptic ulcer and is a risk factor for gastric cancer . Although 50% of the population is affected, dermatologist seem to be unaware of the impact H . pylori may have on cutaneous pathology . Among skin diseases, H . pylori has been related so far only with chronic urticaria and rosacea . In rosacea, histology of the stomach mucosa revealed tht 84% of 31 patients were H . pylori positive . Twenty percent of them were serologically negative, but, overall, 100% of the 20 patients with both histology and serology were H . pylori positive with either test . The consistency between clinical success with metronidazole and abatement of H . pylori isolates and serology after treatment was an additional evidence suggesting an etiologic relationship between rosacea and H . pylori infection . Rosacea has often been linked with gastrointestinal disturbances . H . pylori, therefore, may link them to the well-known beneficial activity of metronidazole on rosacea lesions . The role of H . pylori is more probable in erythrotic rosacea than in its papulopustular and granulomatous stages . As in Bacillus subtilis intoxication, a flush-inducing toxin cannot be excluded . Despite the difficulty to find patients accepting bioptic gastroscopies, large case-control studies should be done before a causal relationship with urticaria and rosacea is firmly established.

Symp Soc Exp Biol, 1995, 49, 91 - 107
Paths and patterns: the biology and physics of swimming bacterial populations; Kessler JO et al.; The velocity distribution of swimming micro-organisms depends on directional cues supplied by the environment . Directional swimming within a bounded space results in the accumulation of organisms near one or more surfaces . Gravity, gradients of chemical concentration and illumination affect the motile behaviour of individual swimmers . Concentrated populations of organisms scatter and absorb light or consume molecules, such as oxygen . When supply is one-sided, consumption creates gradients; the presence of the population alters the intensity and the symmetry of the environmental cues . Patterns of cues interact dynamically with patterns of the consumer population . In suspensions, spatial variations in the concentration of organisms are equivalent to variations of mean mass density of the fluid . When organisms accumulate in one region whilst moving away from another region, the force of gravity causes convection that translocates both organisms and dissolved substances . The geometry of the resulting concentration-convection patterns has features that are remarkably reproducible . Of interest for biology are (1) the long-range organisation achieved by organisms that do not communicate, and (2) that the entire system, consisting of fluid, cells, directional supply of consumables, boundaries and gravity, generates a dynamic that improves the organisms' habitat by enhancing transport and mixing . Velocity distributions of the bacterium Bacillus subtilis have been measured within the milieu of the spatially and temporally varying oxygen concentration which they themselves create . These distributions of swimming speed and direction are the fundamental ingredients required for a quantitative mathematical treatment of the patterns . The quantitative measurement of swimming behaviour also contributes to our understanding of aerotaxis of individual cells.

Annu Rev Microbiol, 1995, 49, 29 - 54
Mechanisms for the prevention of damage to DNA in spores of Bacillus species; Setlow P; The DNA in dormant spores of Bacillus subtilis as well as other Bacillus species is extremely well protected against damage resulting from treatments such as desiccation, heat, oxidizing agents, and UV and gamma radiation . This high degree of DNA protection is a major factor in the survival of spores of these species, not only when subjected to the treatments noted above, but also when incubated under common environmental conditions for many years . Factors that play major roles in overall spore resistance include the low permeability of spores to toxic chemicals and the decreased spore-core water content . However, although decreased spore permeability and water content appear to at least partially protect spore DNA from oxidative damage, these factors seem to play little or no role in protecting spore DNA from heat damage . The major factor preventing damage to spore DNA is the saturation of this DNA with a novel group of small, acid-soluble proteins of the alpha/beta-type whose binding greatly alters DNA's chemical and enzymatic reactivity as well as its UV photochemistry . Binding of these proteins is also a key factor in spore DNA resistance to desiccation, heat, oxidizing agents, and UV radiation.

Rocz Panstw Zakl Hig, 1995, 46(3), 299 - 304
{The influence of load size on the result of ethylene oxide gas sterilization}; Jakimiak B; The purpose of the study was establishing whether the size of the load--that is the degree of filling of sterilizer chamber with medical instruments--could have an effect on the time needed for eradication of all microorganisms subjected to the action of ethylene oxide . The test organism used was the strain of Bacillus subtilis var . niger ATTC 9372 . Tests were prepared with about 3.6 x 10(6) spores per one test . The tested samples were exposed to ethylene oxide at concentration of 750 mg/l, at 50-80 degrees C, at 40% humidity during 30, 60, 120, 180, 240 and 300 minutes in three variants: in empty sterilizer chamber im chamber loaded with plastic objects (polyethylene and polypropylene)--600 g weight in chamber with similar load weighing 1200 g . The obtained results showed that the degree of filling of sterilizer chamber influenced the efficiency of gas sterilization with ethylene oxide . The effectiveness of each sterilization process with ethylene oxide should be controlled using biological indicators.

J Comput Biol, 1995 Fall, 2(3), 417 - 37
Exceptional motifs in different Markov chain models for a statistical analysis of DNA sequences; Schbath S et al.; Identifying exceptional motifs is often used for extracting information from long DNA sequences . The two difficulties of the method are the choice of the model that defines the expected frequencies of words and the approximation of the variance of the difference T(W) between the number of occurrences of a word W and its estimation . We consider here different Markov chain models, either with stationary or periodic transition probabilities . We estimate the variance of the difference T(W) by the conditional variance of the number of occurrences of W given the oligonucleotides counts that define the model . Two applications show how to use asymptotically standard normal statistics associated with the counts to describe a given sequence in terms of its outlying words . Sequences of Escherichia coli and of Bacillus subtilis are compared with respect to their exceptional tri- and tetranucleotides . For both bacteria, exceptional 3-words are mainly found in the coding frame . E . coli palindrome counts are analyzed in different models, showing that many overabundant words are one-letter mutations of avoided palindromes.

J Bacteriol, 1995 Jan, 177(1), 176 - 82
Analysis of a suppressor mutation ssb (kinC) of sur0B20 (spo0A) mutation in Bacillus subtilis reveals that kinC encodes a histidine protein kinase; Kobayashi K et al.; sur0B20 is a mutation that suppresses the effects of spo0B delta B or spo0F221 mutations in Bacillus subtilis, sur0B20 is an allele of the spo0A gene (Glu-14 to Val-14 conversion) and restores the sporulation of spo0B or spo0F mutants to the wild-type level . Here, we report the isolation of suppressor mutations of sur0B20 (ssb) . One of these mutations, ssb-12, severely impairs the suppressor activity of sur0B20 . A 2.5-kbp MboI fragment which complements the ssb-12 mutation was cloned by the prophage transformation method using phi CM as a vector . Nucleotide sequencing of the fragment revealed two open reading frames (orf1 and orf2) . Gene disruption and complementation experiments showed that orf2 is the ssb gene . ssb was shown to encode a protein with a molecular weight of 48,846 (428 amino acid residues) showing strong similarity to transmitter kinases, especially KinA, of two-component regulatory systems . Therefore, ssb was renamed kinC . Deletion of kinC had no observable effect on sporulation . kinC transcription was induced at the onset of sporulation, probably from a sigma A-dependent promoter, and its expression was shut off at T3 . DNase I protection experiments showed that the Spo0A protein binds to two adjoining sites in the kinC promoter region with different affinities . These results suggest that kinC expression might be regulated by Spo0A.

J Bacteriol, 1995 Jan, 177(1), 166 - 75
Isolation and characterization of kinC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinases KinA and KinB in Bacillus subtilis; LeDeaux JR et al.; Phosphorylation of the transcription factor encoded by spo0A is required for the initiation of sporulation in Bacillus subtilis . Production and accumulation of Spo0A-P is controlled by histidine protein kinases and the spo0 gene products . To identify additional genes that might be involved in the initiation of sporulation and production of Spo0A-P, we isolated genes which when present on a multicopy plasmid could suppress the sporulation defect of a spo0K mutant . kinC was one gene isolated in this way . A multicopy plasmid containing kinC completely or partially suppressed the sporulation defect caused by mutations in spo0K, kinA, spo0F, and spo0B, indicating that at least when overexpressed, KinC is capable of stimulating phosphorylation of Spo0A independently of the normal phosphorylation pathway . The predicted product of kinC is 428 amino acids long and is most similar to KinA and KinB, the histidine protein kinases involved in the initiation of sporulation . In otherwise wild-type strains, kinC null mutations caused little or no defect in sporulation under the conditions tested . However, in the absence of a functional phosphorelay (spo0F or spo0B), KinC appears to be the kinase responsible for phosphorylation of the sof-1 and rvtA11 forms of Spo0A.

J Bacteriol, 1995 Jan, 177(1), 123 - 33
Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals; Wise AA et al.; sigma B of the gram-positive bacterium Bacillus subtilis is an alternative transcription factor activated by a variety of environmental stresses, including the stress imposed upon entry into the stationary growth phase . Previous reports have shown that this stationary-phase activation is enhanced when cells are grown in rich medium containing glucose and glutamine . The sigma B structural gene, sigB, lies in an operon with three other genes whose products have been shown to control sigma B activity in response to environmental stress . However, none of these is sufficient to explain the enhanced stationary-phase activation of sigma B in response to glucose . We show here that the four genes previously identified in the sigB operon constitute the downstream half of an eight-gene operon . The complete sigB operon is preceded by a sigma A-like promoter (PA) and has the order PA-orfR-orfS-orfT-orfU-PB-rsbV-rsbW-sig B-rsbX, where rsb stands for regulator of sigma-B and the previously identified sigma B-dependent promoter (PB) is an internal promoter preceding the downstream four-gene cluster . Although the genes downstream of PB were also transcribed by polymerase activity originating at PA, this transcription into the downstream cluster was not essential for normal induction of a sigma B-dependent ctc-lacZ fusion . However, deletion of all four upstream open reading frames was found to interfere with induction of the ctc-lacZ fusion in response to glucose . Additional deletion analysis and complementation studies showed that orfU was required for full glucose induction of sigma B-dependent genes . orfU encodes a trans-acting, positive factor with significant sequence identity to the RsbX negative regulator of sigma B . On the basis of these results, we rename orfU as rsbU to symbolize the regulatory role of its product.

J Bacteriol, 1995 Jan, 177(1), 114 - 22
The Bacillus subtilis rsbU gene product is necessary for RsbX-dependent regulation of sigma B; Voelker U et al.; sigma B is a secondary sigma factor of Bacillus subtilis . sigma B-dependent transcription is induced when B . subtilis enters the stationary phase of growth or is exposed to any of a number of different environmental stresses . Three genes (rsbV, rsbW, and rsbX), which are cotranscribed with the sigma B structural gene (sigB), encode regulators of sigma B-dependent gene expression . RsbW and RsbV have been shown to control sigma B activity, functioning as an inhibitory sigma B binding protein and its antagonist, respectively . Using the SPAC promoter (PSPAC) to control the expression of the sigB operon, a ctc::lacZ reporter system to monitor sigma B activity, and monoclonal antibodies to determine the levels of sigB operon products, we have now obtained evidence that RsbX is an indirect regulator of sigma B activity . Genetic data and in vivo measurements argue that RsbX negatively regulates an extension of the RsbV-RsbW pathway that requires the product of an additional regulatory gene (rsbU) which lies immediately upstream of the sigB operon . The results are consistent with RsbU, or a process dependent on RsbU, being able to facilitate the RsbV-dependent release of sigma B from RsbW but normally prevented from doing this by RsbX.

Microbiology, 1995 Jan, 141 ( Pt 1), 113 - 21
Acquisition of azide-resistance by elevated SecA ATPase activity confers azide-resistance upon cell growth and protein translocation in Bacillus subtilis; Nakane A et al.; We isolated four azide-resistant secA mutants of Bacillus subtilis and found that all of them were the result of a single amino acid replacement of threonine 128 of SecA by alanine or isoleucine . In the presence of 1.5 mM sodium azide, cell growth and protein translocation of the wild-type strain were completely inhibited, but those of the azide-resistant mutant strains were not . Wild-type and two mutant SecA proteins were purified . Both the basal level and the elevated ATPase activity of the mutant SecA proteins were threefold higher than those of the wild-type SecA . The elevated ATPase activity of the SecA mutants was reduced upon the addition of 1.5 mM sodium azide by only 5-10% as compared with 40% for that of the wild-type . These results indicate that the elevated ATPase activity of the SecA mutants is resistant to sodium azide and that is also required for the protein translocation process of B . subtilis.

Plant J, 1995 Jan, 7(1), 77 - 86
Control of de novo purine biosynthesis genes in ureide-producing legumes: induction of glutamine phosphoribosylpyrophosphate amidotransferase gene and characterization of its cDNA from soybean and Vigna; Kim JH et al.; Soybean (Glycine max) and mothbean (Vigna aconitifolia) cDNA clones encoding glutamine phosphoribosylpyrophosphate amidotransferase (PRAT), the first enzyme of the de novo purine biosynthesis pathway, have been isolated from nodule cDNA libraries . The amino acid sequence deduced from soybean clone showed > 85% homology to the PRAT sequence of mothbean and 33-47% homology to those of bacteria, yeast, chicken, rat and human . The soybean clone encodes a protein with an N-terminal sequence resembling a plastid-targeting peptide . Downstream from this peptide is a sequence similar to the 11 amino acid propeptide found in the Bacillus subtilis, chicken, rat and human PRAT proteins . The mothbean cDNA, although lacking most of the plastid presequence, encodes the putative propeptide and efficiently complements purine auxotrophy in an Escherichia coli purF mutant . Both the soybean and mothbean clones encode characteristic cysteine residues that are known to be involved in the assembly of a {Fe-S} cluster near the C-terminus of this protein . Levels of PRAT mRNA in mothbean nodules were found to increase steadily as the nodules matured from 13 days to 23 days . PRAT mRNA was not detectable in uninfected root tissue but a low level of transcript was detected in leaves . Treatment of uninfected root with L-glutamine induced the PRAT mRNA transcript suggesting that glutamine produced as a result of assimilation of fixed nitrogen is funnelled into the de novo purine biosynthesis and controls the expression of this pathway in root nodules.

J Bacteriol, 1995 Jan, 177(2), 372 - 7
An additional GerE-controlled gene encoding an abundant spore coat protein from Bacillus subtilis; Sacco M et al.; We describe the identification and characterization of a gene, herein designated cotG, encoding an abundant coat protein from the spores of Bacillus subtilis . The cotG open reading frame is 195 codons in length and is capable of encoding a polypeptide of 24 kDa that contains nine tandem copies of the 13-amino-acid long, approximately repeated sequence H/Y-K-K-S-Y-R/C-S/T-H/Y-K-K-S-R-S . cotG is located at 300 degrees on the genetic map close to another coat protein gene, cotB . The cotG and cotB genes are in divergent orientation and are separated by 1.3 kb . Like the promoter for cotB, the cotG promoter is induced at a late stage of sporulation under the control of the RNA polymerase sigma factor sigma K and the DNA-binding protein GerE . The -10 and -35 nucleotide sequences of the cotG promoter resemble those of other promoters recognized by sigma K-containing RNA polymerase, and centered 70 bp upstream of the apparent start site is a sequence that matches the consensus binding site for GerE . Spore coat proteins from a newly constructed cotG null mutant lack not only CotG but also CotB, a finding that suggests that CotG may be a morphogenetic protein that is required for the incorporation of CotB into the coat.

J Bacteriol, 1995 Jan, 177(2), 326 - 35
Cloning, nucleotide sequence, and mutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor; Popham DL et al.; An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight penicillin-binding proteins (PBPs) was used to identify the ponA gene encoding PBP1a and -1b (PBP1) of Bacillus subtilis . The identity of the ponA product was established by (i) the presence of a sequence coding for a peptide generated from PBP1 and (ii) the disappearance of PBP1 in a ponA mutant . DNA sequence analysis revealed that the amino acid sequence of PBP1 was similar to those of other class A high-molecular-weight PBPs and that ponA appeared to be cotranscribed with an upstream gene (termed prfA) of unknown function . Null mutations in ponA resulted in a slight decrease in growth rate and a change in colony morphology but had no significant effect on cell morphology, cell division, sporulation, spore heat resistance, or spore germination . Mutations in prfA which did not effect ponA expression produced a more significant decrease in growth rate but had no other significant phenotypic effects . Deletion of both prfA and ponA resulted in extremely slow growth and a reduction in sporulation efficiency . Studies of expression of transcriptional fusions of ponA and prfA to lacZ demonstrated that these two genes constitute an operon . Expression of these genes was relatively constant during growth, decreased during sporulation, and was induced approximately 15 min into spore germination . The ponA locus was mapped to the 200 degrees region of the chromosomal physical map.

J Bacteriol, 1995 Jan, 177(1), 271 - 4
Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis; Martinussen J et al.; Uracil phosphoribosyltransferase (UPRTase) catalyzes the key reaction in the salvage of uracil in many microorganisms . Surprisingly, two genes encoding UPRTase activity were cloned from Bacillus subtilis by complementation of an Escherichia coli mutant . The genes were sequenced, and the putative amino acid sequences were deduced . One gene showed a high level of homology to UPRTases from other organisms, whereas the other gene with a low level of homology to other UPRTases turned out to be the pyrR gene--the repressor of the pyr operon . The role of these genes in uracil metabolism was established by an analysis of the phenotypes of upp and pyrR mutants.

Rev Latinoam Microbiol, 1995 Jan-Mar, 37(1), 43 - 53
{Biological flow tracers: growth and survival of Bacillus subtilis 65-8 under environmental stress}; Hinojosa-Rebollar RE et al.; Microbial flow tracers are presently limited to a strain of Bacillus globigii and a few highly specific bacteriophages . Bacillus subtilis 65-8 produces a black pigment as part of the primary metabolism under minimal nutritional conditions, with glucose as the sole carbon and energy source . This work shows that Bacillus subtilis 65-8 spores are thermostable (55 degrees C during 150 dias), halotolerant (they germinate and grow in an enriched medium with up to 12% NaCl), persistent in a system of sand-soil and sewage, even in the presence of added commercial oil derivatives (kerosene, leaded gasoline and unleaded diesel), they are capable to move through porous systems even as the liquids, viscous as they may be, move through . Moreover, spores were resistant to the presence of autochtonous microorganisms in sewage, where we did not detect any other organism with differential characteristics like our strain (black pigment production in minimal medium) which could interfere with the identification of our biological flow tracer . The characteristics of Bacillus subtilis 65-8 make it a suitable biological flow tracer.

Protein Sci, 1995 Jan, 4(1), 35 - 43
Conformational stability of HPr: the histidine-containing phosphocarrier protein from Bacillus subtilis; Scholtz JM; The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments . The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea . These data were analyzed in several different ways to afford a measure of the cardinal parameters (delta Hg, Tg, delta Sg, and delta Cp) that describe the thermodynamics of folding for HPr . The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520-2525) was used to estimate delta Cp as was a global analysis of the thermal- and urea-induced unfolding data . Each method used to analyze the data gives a similar value for delta Cp (1,170 +/- 50 cal mol-1K-1) . Despite the high melting temperature for HPr (Tg = 73.5 degrees C), the maximum stability of the protein, which occurs at 26 degrees C, is quite modest (delta Gs = 4.2 kcal mol-1) . In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of delta Cp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685-691) . A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed . The methods presented will be of general utility in the characterization of the stability curve for many small proteins.

Biosci Biotechnol Biochem, 1995 Jan, 59(1), 126 - 9
Cloning of the gene encoding DNA binding protein HU from Bacillus stearothermophilus and its expression in Escherichia coli; Kawamura S et al.; The gene (hbst) encoding the DNA binding protein HU from Bacillus stearothermophilus was cloned, with the Bacillus subtilis HU gene (hbsu) as a hybridization probe . The nucleotide sequence, which contains a ribosome binding site, a transcriptional termination signal, as well as the coding region, was analyzed by the dideoxy chain-termination method . The deduced amino acid sequence of an open reading frame was perfectly matched with that of B . stearothermophilus HU (BstHU) determined by the protein chemical methods {M . Kimura and K . S . Wilson, J . Biol . Chem., 258, 4007-4011 (1983)} . The gene, hbst, was overexpressed using the expression vector pET-5a in Escherichia coli, and the recombinant HU protein (r-BstHU) was purified to be homogeneity by heparin-agarose column chromatography followed by ion-exchange column chromatography on S-Sepharose . The recombinant protein thus obtained had a circular dichroism spectrum identical to that of the authentic protein and bound to DNA to the same extent as the authentic protein.

Food Addit Contam, 1995 Jan-Feb, 12(1), 77 - 82
A one-plate microbiological screening test for antibiotic residue testing in kidney tissue and meat: an alternative to the EEC four-plate method?
Koenen-Dierick K, Okerman L, de Zutter L, Degroodt JM, van Hoof J, Srebrnik S.
A one-plate screening method is described for the microbiological detection of antibiotic residues by growth inhibition of Bacillus subtilis in agar medium, at pH 7, which contains trimethoprim for better detection of sulphonamides . beta-Glucuronidase is added to the samples to enable the detection of chloramphenicol residues . The sensitivity was determined for 16 antimicrobial substances frequently used in animal husbandry . A comparison was made with the sensitivity of the EEC four-plate test and existing maximum residue levels . The method has been used in Belgium for 5 years for the monitoring of antimicrobial residues in kidney tissue and meat of slaughter animals.

Mol Microbiol, 1995 Jan, 15(2), 287 - 95
A checkpoint involving RTP, the replication terminator protein, arrests replication downstream of the origin during the Stringent Response in Bacillus subtilis; Levine A et al.; Regulation of DNA replication in Bacillus subtilis involves a post-initiation mechanism which is subject to control by the Stringent System, an essential regulatory network, mediated by the alarmone, ppGpp . In detailed studies using DNA-DNA hybridization procedures, we have now shown that, following the induction of the Stringent Response, replication is blocked downstream of the origin, on the left, close to the hut marker (-175 kb) and on the right, beyond the soft10 marker (+199 kb) . In addition, we provide evidence that inhibition of replication under these conditions requires the replication terminator protein (RTP) . In a mutant lacking RTP, a protein normally involved in termination of chromosomal replication through recognition of specific terminator sequences, replication continues past the sites normally blocked by the Stringent Response . These data strengthen the argument that this second level of control of DNA replication occurs at specific sites, the Strigent Terminus (STer) sites, either side of orlC . Such sites are presumably related to the sequence involved in RTP recognition at the terminus, terC . We propose that the binding of RTP must be modulated, perhaps through the action of ppGpp, to recognize post-initiation control sequences during the Stringent Response, in order to block replisome movement . This, therefore, acts as a checkpoint in chromosome elongation.

Mol Microbiol, 1995 Jan, 15(2), 213 - 23
Differential expression of two closely related deoxyribonuclease genes, nucA and nucB, in Bacillus subtilis; van Sinderen D et al.; Despite the lack of involvement of the competence-specific, membrane-associated deoxyribonuclease (DNase) in competence development, the expression of the gene encoding this protein, nucA, was shown to be dependent on the competence signal transduction pathway, and in particular on ComK, the competence transcription factor, which was shown to bind to the DNA region upstream of nucA . The expression of nucB, specifying an extracellular DNase, which was cloned on the basis of its homology to nucA, was shown to be sporulation-specific and dependent on the gene products of spo0A and spoIIG, the latter constituting an operon responsible for the synthesis of the mother-cell-specific sigma factor sigma E . The observed differential expression of nucA and nucB demarcates the appearance of DNase activities which are either associated with the cytoplasmic membrane or secreted into the medium during different post-exponential growth-phase processes.

Mol Microbiol, 1995 Jan, 15(2), 203 - 11
Expression of the ATP-dependent deoxyribonuclease of Bacillus subtilis is under competence-mediated control; Haijema BJ et al.; Transcription of the ATP-dependent deoxynuclease operon (addAB), as monitored by means of an addAB-lacZ transcriptional fusion, has a low, constitutive level and is initiated from a sigma A type promoter . Transcription of addAB is independent of DNA-damaging agents known to induce the SOS response in Bacillus subtilis . However, addAB transcription increased significantly during competence development . This competence-specific induction was dependent on the gene products of srfA, degU and comK, but not on that of recA . Deletion analysis of the addAB promoter region demonstrated that the competence-specific transcription induction requires DNA sequences located upstream of the addAB promoter that associated with ComK, the competence transcription factor . The latter finding indicates that a direct regulatory link exists between the establishment of the competent state and the synthesis of AddAB, required for recombination of internalized donor DNA.

Mikrobiologiia, 1995 Jan-Feb, 64(1), 44 - 50
{The role of amino acids in intensification of Bacillus subtilis exopolysaccharide biosynthesis in deep growth conditions}; Osadchaia AI et al.; The influence of various combinations of amino acids on the Bacillus subtilis growth activity and exopolysaccharide biosynthesis has been studied . The effects of amino acid additions on these two processes have appeared to be different . The strains reveal the specificity in amino acids requirements while cultivation in liquid media . To judge by the average indirect contributions, the growth as well as the exopolysaccharide biosynthesis of B . subtilis strain 39 have been significantly affected by introduction of alanine, valine, phenylalanine, tryptophane and glycine in triple combinations into the culture medium . The exopolysaccharide production and growth of B . subtilis strain 51 have been influenced markedly by methionine, leucine, isoleucine, cystine, glycine, tryptophane and alanine . Addition of different combinations of these amino acids makes it possible to obtain the 20.2-56.3% higher biomass amounts with the simultaneous 26.8-89.6% increase of exopolysaccharide yield.

DNA Res, 1995, 2(2), 95 - 100
srb: a Bacillus subtilis gene encoding a homologue of the alpha-subunit of the mammalian signal recognition particle receptor; Oguro A et al.; We cloned a Bacillus subtilis gene (srb) encoding a homologue of the mammalian signal recognition particle receptor alpha-subunit (SR alpha) . The gene is 987 bp in length and encodes a 329-amino acid protein . The deduced amino acid sequence of the protein shared 26.6, 36.2 and 49.7% identity with those of mammalian SR alpha, archaebacterial DP alpha and Escherichia coli FtsY, respectively . The protein contains three conserved GTP-binding elements like the other three SRP receptor proteins, though the N-terminal portion of the putative B . subtilis protein was shorter than the others . Secondary structure prediction showed than an amphipathic alpha-helix is positioned in the N-terminal region . A defect in srb inhibited cell growth and protein translocation.

DNA Seq, 1995, 5(5), 283 - 90
The gtcRS operon coding for two-component system regulatory proteins is located adjacent to the grs operon of Bacillus brevis; Turgay K et al.; We identified, cloned and sequenced an operon comprising of two genes gtcR and gtcS located adjacent to the grs operon of Bacillus brevis, which encodes the multienzymes involved in the biosynthesis of the peptide antibiotic gramicidin S . The transcription initiation site of the gtcRS operon was determined in Bacillus brevis and Bacillus subtilis . The encoded proteins GtcR and GtcS were identified as members of the two-component system family of signal transducing proteins.

Gene, 1994 Dec 30, 151(1-2), 37 - 43
The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli; Zock J et al.; p-Nitrobenzyl esters serve as protecting groups on intermediates in the manufacture of clinically important oral beta-lactam antibiotics; de-esterification of the intermediates is required for synthesis of the final product . A Bacillus subtilis PNB carboxy-esterase (PNBCE) catalyzes hydrolysis of several beta-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol . This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleotide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli . The amino acid (aa) sequence, deduced from the nucleotide sequence of the pnbA ORF, matched an experimentally determined N-terminal aa sequence of B . subtilis PNBCE and also matched an active site sequence previously identified by biochemical analyses . Specific activity of PNBCE in crude extracts was more than 90-fold greater in recombinant E . coli, as compared to B . subtilis . This increase in expression led to more than a 500-fold improvement in the efficiency of purification of PNBCE.

Gene, 1994 Dec 30, 151(1-2), 161 - 6
Characterization of the macromolecular synthesis (MMS) operon from Listeria monocytogenes; Metzger R et al.; The macromolecular synthesis (MMS) operon consists of three genes: rpsU, which encodes the S21 ribosomal protein in Bacillus subtilis (Bs), rpsU is replaced by orfP23 which encodes a protein of unknown function), dnaG, encoding the DNA primase involved in the initiation of chromosome replication, and rpoD, which encodes the principal sigma subunit of RNA polymerase . The operon was cloned in three segments from Listeria monocytogenes (Lm), initially using a probe designed from a highly conserved region of RpoD . Analysis of the nucleotide sequence revealed three genes: orfP17 (whose product, P17, is homologous to Bs P23), dnaG and rpoD . The Lm DnaG resembles the primase from Escherichia coli through the first two-thirds of the sequence . C-terminal similarity was observed between DnaG from Lm and Bs . Lm RpoD is similar to Bs SigA, shares identical DNA-binding domains with SigA, and is a member of the sigma 43 subgroup of the sigma 70 family.

Biochemistry, 1994 Dec 27, 33(51), 15271 - 82
Structural consequences of histidine phosphorylation: NMR characterization of the phosphohistidine form of histidine-containing protein from Bacillus subtilis and Escherichia coli; Rajagopal P et al.; The bacterial phosphoenolpyruvate:sugar phosphotransferase system involves a series of reactions in which phosphoprotein intermediates are formed . Histidine-containing protein (HPr) is phosphorylated on the N delta 1 position of the imidazole ring of His15 by enzyme I and acts as a phosphoryl donor to the sugar-specific enzymes IIA . The structure of phosphorylated HPr from Bacillus subtilis, primarily, and from Escherichia coli has been studied by nuclear magnetic resonance (NMR) spectroscopy . Phosphorylation of His15 results in large downfield shifts in amide proton and nitrogen resonances for residues 16 and 17 but results in only modest or no shifts in other backbone resonances . The exchange rates of these two amide groups are decreased more than 10-fold upon phosphorylation . Analysis of the coupling constants 3JNH alpha revealed no significant changes throughout the protein, indicating that backbone phi dihedral angles do not change detectably . 3J alpha beta and 3JN beta patterns determined from P.E.COSY and HNHB spectra, respectively, revealed a change in one side chain, that of conserved Arg17 . Analysis of NOESY spectra revealed a limited number of changes in NOEs involving protons in Ser12, His15, Arg71, and Pro18 in B . subtilis HPr . The NMR results indicate that the Arg17 side chain becomes limited in its conformational range in the phosphorylated protein, taking on a conformation that points its guanidinium group toward the phosphoryl group on His15 . In addition, the tautomeric and ionization states of His15 have been determined using 15N and 31P NMR . At neutral pH, the imidazole is predominantly in the protonated form and the phosphoryl group is in the dianionic form in P-His15 . Altogether, the results indicate that phosphorylation of His15 yields only a local effect on the protein's structure . The data are consistent with a small change in the disposition of the histidine side chain, allowing phosphoryl group oxygens to serve as hydrogen bond acceptors for the amide protons of residues Ala16 and Arg17, which constitute the first two residues of an alpha-helix . Thus, similar to many proteins that bind phosphoryl moieties noncovalently, the phosphoryl group in P-His15-HPr is situated to allow for a favorable electrostatic interaction at the N-terminal end of an alpha-helix.

Nucleic Acids Res, 1994 Dec 25, 22(25), 5525 - 9
NRSub: a non-redundant data base for the Bacillus subtilis genome; Perriere G et al.; We have organized the DNA sequences of Bacillus subtillis from the EMBL collection to build the NRSub data base . This data base is free from duplications and all detected overlapping sequences are merged into contigs . Data on gene mapping and codon usage are also included . NRSub is publically available through anonymous FTP in flat file format or structured on the form of an ACNUC data base . Under this format, it is possible to use NRSub with the retrieval program Query--win . This program integrates a graphical interface and may be installed on any kind of UNX computer under X Window and on which the Vibrant and Motif libraries are available.

J Biol Chem, 1994 Dec 16, 269(50), 31450 - 6
Substrate specificity of an RNase III-like activity from Bacillus subtilis; Mitra S et al.; Bacillus subtilis bacteriophage SP82 codes for several early RNAs that were shown previously to be cleaved by an RNase III-like enzyme called "Bs-RNase III." Cloning of DNA fragments that encode these RNA sequences downstream of a T7 RNA polymerase promoter allowed the synthesis of substrates that were used to test the cleavage specificity of Bs-RNase III, which was purified from a protease-deficient strain of B . subtilis . Single nucleotide changes at or near the cleavage site and deletions upstream and downstream of the cleavage site were constructed . The effects of these changes on the rate of Bs-RNase III cleavage were measured . The activity of Bs-RNase III and Escherichia coli RNase III on heterologous substrates was also tested . Although the local environment of the site of Bs-RNase III cleavage appears very similar to that of E . coli RNase III, there are important differences in their substrate specificity.

FEMS Microbiol Lett, 1994 Dec 15, 124(3), 393 - 7
Isolation and characterization of a Bacillus subtilis secA mutant allele conferring resistance to sodium azide; Klein M et al.; A mutation has been isolated in the Bacillus subtilis secA gene (secA10) which allows cell growth and residual protein translocation in the presence of 1.5 mM sodium azide . Besides conferring resistance to sodium azide, the corresponding SecA10 mutant protein, in which glutamic acid at position 338 has been changed to glycine, seems to possess a secretion defect even in the absence of azide . In addition, the secA10 mutant protein was found to be recessive to wild-type secA with regard to azide resistance . Our results strongly suggest that, like the situation in Escherichia coli, the B . subtilis SecA protein is a main target for the lethal action of sodium azide.

FEMS Microbiol Lett, 1994 Dec 15, 124(3), 255 - 63
The oxidative stress response in Bacillus subtilis; Dowds BC; Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide . Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein . Induced protection against higher concentrations (10-30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant . Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide . A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide . This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type . The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.

Structure, 1994 Dec 15, 2(12), 1203 - 16
Refined structures of the active Ser83-->Cys and impaired Ser46-->Asp histidine-containing phosphocarrier proteins; Liao DI et al.; BACKGROUND: The histidine-containing phosphocarrier protein (HPr) functions in the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) . His15 on HPr accepts a phosphoryl group from Enzyme I and transfers it to the Enzyme IIA domain of a sugar-specific PTS permease . In addition, HPrs from Gram-positive bacteria undergo phosphorylation on a serine residue, Ser46, which inhibits phosphorylation at His15 and sugar transport . The questions to be addressed at the molecular level are: what is the mechanism of each of the phosphoryl transfers and what conformational transitions are associated with each event? RESULTS: Thus, the crystal structures of the mutants Ser83-->Cys HPr (fully active protein) and Ser46-->Asp HPr (impaired protein which mimics Ser46 approximately P HPr) from Bacillus subtilis have been determined at 2 A resolution . They have been crystallized from high-salt and low-salt solutions respectively, and in two different space groups . Analysis of the two crystal forms reveals some significant differences but these do not alter the overall fold of the protein . In each structure, the side chain of His15 caps the following helix . Two alternative side-chain conformations of Arg17 are observed; it either forms an ion pair with a sulfate ion, presumably resembling the phosphorylated state of the protein (high-salt crystal) or with Glu84 (low-salt crystal) . The main-chain conformation in the region of residue 46 is the same in the two crystal forms, with both Ser46 and Asp46 capping the following helix . CONCLUSIONS: The analysis suggests that phosphorylation of either His15 or Ser46 is not associated with main-chain conformational transitions . Rather, the protein is poised to accept the respective phosphoryl group with minor adjustments to side chains . The inhibitory effect of phosphorylation on Ser46 is attributed to the altered surface electrostatics, which impairs protein-protein interaction.

Gene, 1994 Dec 2, 150(1), 129 - 34
Construction of Tn917ac1, a transposon useful for mutagenesis and cloning of Bacillus subtilis genes; Chang LK et al.; A Tn917 derivative was constructed for the purposes of mutagenesis and cloning of Bacillus subtilis genes . This transposon, Tn917ac1 (4.6 kb), consisted of terminal inverted repeats of Tn917, the res sequence, a ColE1 origin of replication (ori) and two drug-resistance genes . The plasmid carrying this transposon, named pD917, contained the erm-tnpR-tnpA gene cluster of Tn917 and a temperature-sensitive ori of pE194 . For the purpose of mutagenesis, transposition of Tn917ac1 was induced by culturing strains harboring pD917 in a medium containing a low concentration of erythromycin . Cells with a Tn917ac1 insertion in the chromosome were selected on agar containing chloramphenicol after heat treatment to eliminate the plasmidic form of pD917 . DNA fragments adjacent to Tn917ac1 could be cloned by restriction digestion of the chromosomal DNA and by transforming the self-ligated restriction fragments into Escherichia coli . Sequence analysis revealed that Tn917ac1 was integrated into the chromosome of B . subtilis by transposition in a recE strain and by transposition or integration of pD917 in a wild-type strain . Tn917ac1 has been demonstrated to be useful for mutating and cloning of the genes involved in the biosynthesis of fengycin in B . subtilis F29-3.

J Bacteriol, 1994 Dec, 176(24), 7763 - 6
Isolation of a Bacillus subtilis spoIIGA allele that suppresses processing-negative mutations in the Pro-sigma E gene (sigE); Peters HK 3rd et al.; sigma E, a sporulation-essential sigma factor of Bacillus subtilis, is formed by a developmentally regulated proteolysis which removes 27 to 29 amino acids from the amino terminus of an inactive precursor protein (Pro-sigma E) . A mutation which facilitates the conversion of inefficiently processed Pro-sigma E variants into mature sigma E was identified and mapped to spoIIGA . The isolation of such a mutation argues that SpoIIGA is directly involved in the Pro-sigma E processing reaction.

J Bacteriol, 1994 Dec, 176(24), 7456 - 61
Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase; Okada M et al.; The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant . Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613) . This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product . The B . subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E . coli and S . cerevisiae, whereas no homology was found with that of the E . coli counterpart . A hydropathy analysis revealed that the B . subtilis synthase is very hydrophobic, in contrast to the hydrophilic E . coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane . A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B . subtilis enzyme, was found exclusively in the membrane fraction of E . coli (pssA1) cells harboring a B . subtilis pss plasmid . Overproduction of the B . subtilis synthase in E . coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E . coli counterpart . This finding suggested that the unusual cytoplasmic localization of the E . coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.

J Bacteriol, 1994 Dec, 176(23), 7252 - 9
Changes in wall teichoic acid during the rod-sphere transition of Bacillus subtilis 168; Pollack JH et al.; Wall teichoic acid (WTA) is essential for the growth of Bacillus subtilis 168 . To clarify the function of this polymer, the WTAs of strains 168, 104 rodB1, and 113 tagF1 (rodC1) grown at 32 and 42 degrees C were characterized . At the restrictive temperature, the rodB1 and tagF1 (rodC1) mutants undergo a rod-to-sphere transition that is correlated with changes in the WTA content of the cell wall . The amount of WTA decreased 33% in strain 104 rodB1 and 84% in strain 113 tagF1 (rodC1) when they were grown at the restrictive temperature . The extent of alpha-D-glucosylation (0.84) was not affected by growth at the higher temperature in these strains . The degree of D-alanylation decreased from 0.22 to 0.10 in the rodB1 mutant but remained constant (0.12) in the tagF1 (rodC1) mutant at both temperatures . Under these conditions, the degree of D-alanylation in the parent strain decreased from 0.27 to 0.21 . The chain lengths of WTA in strains 168 and 104 rodB1 grown at both temperatures were approximately 53 residues, with a range of 45 to 60 . In contrast, although the chain length of WTA from the tagF1 (rodC1) mutant at 32 degrees C was similar to that of strains 168 and 104 rodB1, it was approximately eight residues at the restrictive temperature . The results suggested that the rodB1 mutant is partially deficient in completed poly(glycerophosphate) chains . The precise biochemical defect in this mutant remains to be determined . The results for strain 113 tagF1(rodC1) are consistent with the temperature-sensitive defect in the CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (H . M . Pooley, F.-X . Abellan, and D . Karamata, J . Bacteriol . 174:646-649, 1992).

J Bacteriol, 1994 Dec, 176(23), 7197 - 205
Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4; Popham DL et al.; The gene encoding penicillin-binding protein 4 (PBP 4) of Bacillus subtilis, pbpD, was cloned by two independent methods . PBP 4 was purified, and the amino acid sequence of a cyanogen bromide digestion product was used to design an oligonucleotide probe for identification of the gene . An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight PBPs also identified this gene . DNA sequence analysis of the cloned DNA revealed that (i) the amino acid sequence of PBP 4 was similar to those of other class A high-molecular-weight PBPs and (ii) pbpD appeared to be cotranscribed with a downstream gene (termed orf2) of unknown function . The orf2 gene is followed by an apparent non-protein-coding region which exhibits nucleotide sequence similarity with at least two other regions of the chromosome and which has a high potential for secondary structure formation . Mutations in pbpD resulted in the disappearance of PBP 4 but had no obvious effect on growth, cell division, sporulation, spore heat resistance, or spore germination . Expression of a transcriptional fusion of pbpD to lacZ increased throughout growth, decreased during sporulation, and was induced approximately 45 min into spore germination . A single transcription start site was detected just upstream of pbpD . The pbpD locus was mapped to the 275 to 280 degrees region of the chromosomal genetic map.

J Bacteriol, 1994 Dec, 176(23), 7161 - 8
Biochemical characterization of the essential GTP-binding protein Obg of Bacillus subtilis; Welsh KM et al.; An essential guanine nucleotide-binding protein, Obg, of Bacillus subtilis has been characterized with respect to its enzymatic activity for GTP . The protein was seen to hydrolyze GTP with a Km of 5.4 microM and a kcat of 0.0061 min-1 at 37 degrees C . GDP was a competitive inhibitor of this hydrolysis, with an inhibition constant of 1.7 microM at 37 degrees C . The dissociation constant for GDP from the Obg protein was 0.5 microM at 4 degrees C and was estimated to be 1.3 microM at 37 degrees C . Approximately 80% of the purified protein was capable of binding GDP . In addition to hydrolysis of GTP, Obg was seen to autophosphorylate with this substrate . Subsequent release of the covalent phosphate proceeds at too slow a rate to account for the overall rate of GTP hydrolysis, indicating that in vitro hydrolysis does not proceed via the observed phosphoamidate intermediate . It was speculated that the phosphorylated form of the enzyme may represent either a switched-on or a switched-off configuration, either of which may be normally induced by an effector molecule . This enzyme from a temperature-sensitive mutant of Obg did not show significantly altered GTPase activity at the nonpermissive temperature.

J Bacteriol, 1994 Dec, 176(23), 7155 - 60
Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg; Kok J et al.; The obg gene is part of the spo0B sporulation operon and codes for a GTP-binding protein which is essential for growth . A temperature-sensitive mutant in the obg gene was isolated and found to be the result of two closely linked missense mutations in the amino domain of Obg . Temperature shift experiments revealed that the mutant was able to continue cell division for 2 to 3 generations at the nonpermissive temperature . Such experiments carried out during sporulation showed that Obg was necessary for the transition from vegetative growth to stage 0 or stage II of sporulation, but sporulation subsequent to these stages was unaffected at the nonpermissive temperature . Spores of the temperature-sensitive mutant germinated normally at the nonpermissive temperature but failed to outgrow . The primary consequence of the obg mutation may be an alteration in initiation of chromosome replication.

Anal Biochem, 1994 Dec, 223(2), 208 - 11
Specific labeling of diaminopimelate: a radioassay for the determination of the peptidoglycan cross-linking index; Roten CA et al.; A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised . It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting {14C}aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components . Purified {14C}DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC . The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts . The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems . A CLI of 50.8 +/- 1.3% and 55.5 +/- 0.9% was obtained for B . subtilis 168 cells growing exponentially in rich and minimal medium, respectively . Comparison of these to results previously obtained on B . subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.

Mol Gen Genet, 1994 Dec 1, 245(5), 529 - 36
A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage phi 29; Bravo A et al.; Protein p6 of the Bacillus subtilis phage phi 29 is essential for in vivo viral DNA replication . This protein activates the initiation of phi 29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins . The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations . We report on the development of an in vivo functional assay for protein p6 . This assay is based on the ability of protein p6-producing B . subtilis non-suppressor (su) cells to support growth of a phi 29 sus6 mutant phage . We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region . The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein . These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6 . Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.

Proteins, 1994 Dec, 20(4), 347 - 55
A P-loop-like motif in a widespread ATP pyrophosphatase domain: implications for the evolution of sequence motifs and enzyme activity; Bork P et al.; A conserved amino acid sequence motif was identified in four distinct groups of enzymes that catalyze the hydrolysis of the alpha-beta phosphate bond of ATP, namely GMP synthetases, argininosuccinate synthetases, asparagine synthetases, and ATP sulfurylases . The motif is also present in Rhodobacter capsulata AdgA, Escherichia coli NtrL, and Bacillus subtilis OutB, for which no enzymatic activities are currently known . The observed pattern of amino acid residue conservation and predicted secondary structures suggest that this motif may be a modified version of the P-loop of nucleotide binding domains, and that it is likely to be involved in phosphate binding . We call it PP-motif, since it appears to be a part of a previously uncharacterized ATP pyrophophatase domain . ATP sulfurylases, NtrL, and OutB consist of this domain alone . In other proteins, the pyrophosphatase domain is associated with amidotransferase domains (type I or type II), a putative citrulline-aspartate ligase domain or a nitrilase/amidase domain . Unexpectedly, statistically significant overall sequence similarity was found between ATP sulfurylase and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase, another protein of the sulfate activation pathway . The PP-motif is strongly modified in PAPS reductases, but they share with ATP sulfurylases another conserved motif which might be involved in sulfate binding . We propose that PAPS reductases may have evolved from ATP sulfurylases; the evolution of the new enzymatic function appears to be accompanied by a switch of the strongest functional constraint from the PP-motif to the putative sulfate-binding motif.

J Bioenerg Biomembr, 1994 Dec, 26(6), 639 - 46
Bacterial resistance to uncouplers; Lewis K et al.; Uncoupler resistance presents a potential challenge to the conventional chemiosmotic coupling mechanism . In E . coli, an adaptive response to uncouplers was found in cell growing under conditions requiring oxidative phosphorylation . It is suggested that uncoupler-resistant mutants described in the earlier literature might represent a constitutive state of expression of this "low energy shock" adaptive response . In the environment, bacteria are confronted by nonclassical uncoupling factors such as organic solvents, heat, and extremes of pH . It is suggested that the low energy shock response will aid the cell in coping with the effects of natural uncoupling factors . The genetic analysis of uncoupler resistance has only recently began, and is yielding interesting and largely unexpected results . In Bacillus subtilis, a mutation in fatty acid desaturase causes an increased content of saturated fatty acids in the membrane and increased uncoupler resistance . The protonophoric efficiency of uncouplers remains unchanged in the mutants, inviting nonorthodox interpretations of the mechanism of resistance . In E . coli, two loci conferring resistance to CCCP and TSA were cloned and were found to encode multidrug resistance pumps . Resistance to one of the uncouplers, TTFB, remained unchanged in strains mutated for the MDRs, suggesting a resistance mechanism different from uncoupler extrusion.

J Biochem (Tokyo), 1994 Dec, 116(6), 1287 - 94
A truncated Bacillus subtilis SecA protein consisting of the N-terminal 234 amino acid residues forms a complex with Escherichia coli SecA51(ts) protein and complements the protein translocation defect of the secA51 mutant; Takamatsu H et al.; Although wild-type Bacillus subtilis SecA barely complements the growth and protein translocation defect of Escherichia coli secA51(ts) at the non-permissive temperature, an N-terminal peptide of B . subtilis SecA complements the defects . To elucidate the mechanism of this complementation, a series of plasmids encoding truncated SecA proteins was constructed and their products were analyzed in E . coli cells . The truncated B . subtilis SecA protein consisting of the N-terminal 234 amino acid residues (BN234) complemented the growth and protein translocation defects of E . coli secA51 but not those of another secA amber mutant, E . coli secA13(ts) . BN234 existed in both a soluble form, possibly as a homodimer, and a higher-molecular-weight complex in E . coli strain MM52 (secA51) . The purified complex, consisting of at least BN234, SecA51, and ATP-dependent protease La, was held together by a cross-linking reagent, EDAC . The other truncated proteins consisting of the N-terminal 584 or 396 amino acid residues and the C-terminal 607 residues of B . subtilis SecA did not complement the two E . coli mutants or form a complex with SecA51 . These results suggest that BN234 and SecA51 proteins form a functional complex in vivo and complement the defects of E . coli MM52.

Bioorg Khim, 1994 Dec, 20(12), 1310 - 26
{Primary structure of the intracellular serine proteinase from Bacillus amyloliquefaciens . III . Amino acid sequence of peptides obtained by hydrolysis with a Glu,Asp-specific proteinase . Reconstruction of the entire amino acid sequence of the proteinase}; Surova IA et al.; Glu,Asp-specified protease hydrolysate of intracellular serine proteinase (ISP) was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield 30 individual peptides . Their sequences, spanning to 243 amino acid residues, were determined by the manual Edman procedure . Four overlapping fragments were reconstructed by comparing their sequences with those of tryptic and chymotryptic peptides . To arrange these fragments in the proteinase polypeptide chain and to reconstruct the enzyme's total sequence, additional peptides were isolated from the tryptic hydrolysate and analysed . Primary structure of ISP, corresponding to 297 amino acid residues, was reconstructed . Its comparison with related serine proteinases revealed the following levels of homology: with Bacillus subtilis intracellular serine proteinase, 88%; with secretory subtilisin BPN' produced by B . amyloliquefaciens, 46%.

J Bacteriol, 1994 Dec, 176(24), 7767 - 9
Transcriptional control of dacB, which encodes a major sporulation-specific penicillin-binding protein; Simpson EB et al.; Sporulation-specific sigma factor E (sigma E) of Bacillus subtilis is both necessary and sufficient for transcription of the dacB gene, which encodes penicillin-binding protein 5* . Evidence in support of this conclusion was obtained by primer extension analysis of dacB transcripts and the induction of active sigma E with subsequent synthesis of PBP 5* in vegetative cells.

Biochemistry, 1994 Nov 29, 33(47), 14207 - 12
Selection of circularly permuted ribozymes from Bacillus subtilis RNAse P by substrate binding; Pan T et al.; The effect of a single break in the phosphodiester backbone of Bacillus subtilis RNAse P RNA (P RNA) was examined using circular permutation analysis (CPA) . This method reveals that many of the phosphodiester bonds in this catalytic RNA can be broken with little or no effect on substrate binding . Phosphate positions that show strong effects are located mostly in regions conserved among all RNAse P RNAs, or they are in regions known to interact directly with the pre-tRNA substrate . Two circularly permuted isomers of P RNA were constructed and analyzed in detail . The KM for both circularly permuted isomers is nearly identical to that of the wild-type P RNA . Since the KM of the P RNA is essentially the same as the binding constant to the substrate, this finding confirms the CPA results . The implications of backbone breakage are discussed with respect to folding and catalysis of the RNAse P RNA.

Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11669 - 73
An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame; Bechhofer DH et al.; The thymidylate synthase gene (thy) (EC 2.1.1.45) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence . The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4 . In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent . The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron . The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures . These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event.

J Biol Chem, 1994 Nov 18, 269(46), 28752 - 6
A stable isotope-aided NMR study of the active site of an endoglucanase from a strain of Bacillus; Kawaminami S et al.; Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp . KSM-330, in which Trp are important for expression of the activity . Endoglucanase K, which was specifically labeled with {indole-2-13C}Trp, was prepared from recombinant Bacillus subtilis that carried the gene for this enzyme on an expression vector, pHSP-KC331 . Twelve cross-peaks originating from the C-2 position of Trp residues of endoglucanase K were separately observed in 1H-13C heteronuclear single-quantum coherence spectrum, and six of the cross-peaks have been assigned site-specifically by using site-directed mutagenesis . The chemical shifts of the cross-peaks originating from Trp-174 and Trp-243 were affected by the addition of cellotriose that was used as a competitive inhibitor of the enzyme . On the basis of the NMR data obtained after chemical modification of the enzyme by N-bromosuccinimide, it appears that Trp-174 was oxidized first with retention of 56% of the original activity and Trp-243 was then oxidized with complete loss of activity . Substitution of Trp-174 or Trp-243 by Tyr residue caused a decrease in the specific activity of the enzyme to 49 or 8% of that of the wild-type enzyme, respectively . Km values of these mutant enzymes for p-nitrophenyl beta-D-cellotrioside increased to 5 and 8 times those of the wild-type enzyme, respectively, while kcat values of both of the mutant enzymes decreased to one-fifth of those of the wild-type enzymes . These results suggest that Trp-174 and Trp-243 play an important role in binding of the substrate and/or in the catalytic activity.

J Mol Biol, 1994 Nov 18, 244(1), 1 - 5
11-fold symmetry of the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis determined by X-ray analysis; Antson AA et al.; The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis has been crystallized and examined by crystallography using X-ray synchrotron radiation diffraction data . Crystals of TRAP complexed with L-tryptophan belong to space group C2 with a = 156.8 A, b = 114.05 A, c = 105.9 A, beta = 118.2 degrees . Crystals of a potential heavy-atom derivative of TRAP complexed with 5-bromo-L-tryptophan grow in the same space group with similar cell dimensions . X-ray data for the native crystals and for the derivative have been collected to 2.9 A and 2.2 A resolution, respectively . Peaks in the self-rotation function and in the Patterson synthesis could only be explained by two 11-subunit oligomers (each formed by an 11-fold axis of symmetry) in the asymmetric unit lying with the 11-fold rotation axes parallel to each other . The consequence is that the TRAP molecule has 11-fold symmetry and contains 11 subunits.

J Biol Chem, 1994 Nov 11, 269(45), 28506 - 13
A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates; Ahmed M et al.; Multidrug transporters are membrane proteins which, by an unknown mechanism, recognize diverse toxic compounds and efflux them from cells . We found that two substrates of the Bacillus subtilis multidrug transporter Bmr, rhodamine 6G and tetraphenylphosphonium (TPP), enhance Bmr expression at the level of transcription . Gene knock-out experiments demonstrated that an open reading frame located immediately downstream of the bmr gene is required for this enhancement . The protein product of this open reading frame, BmrR, shows distinct sequence homology to several known bacterial transcription activator proteins, such as MerR and TipAL . Gel-mobility shift and DNase protection assays indicated that BmrR binds specifically, as a dimer, to the bmr gene promoter . Furthermore, the affinity of this binding was enhanced by rhodamine and TPP, thus suggesting that these structurally dissimilar molecules interact directly with BmrR . Indeed, we found that BmrR bound rhodamine 6G stoichiometrically, one rhodamine molecule/BmrR dimer, and that TPP competed with rhodamine for this binding . Our results indicate that the enhancement of Bmr expression by some of its substrates is due to the ability of the regulatory protein, BmrR, to bind structurally dissimilar compounds resulting in enhanced transcription of the transporter gene.

Proc Natl Acad Sci U S A, 1994 Nov 8, 91(23), 11143 - 7
The replication terminator protein of the gram-positive bacterium Bacillus subtilis functions as a polar contrahelicase in gram-negative Escherichia coli; Kaul S et al.; The replication terminator protein (RTP) of Bacillus subtilis is a dimer with a monomeric molecular mass of 14.5 kDa . The protein terminates DNA replication at a specific binding site . Although the protein has been crystallized and its crystal structure has been solved, the lack of an in vitro replication system in B . subtilis has been a serious impediment to the analysis of the mechanism of action of this protein . We have discovered that the protein is functional in the Gram-negative bacterium Escherichia coli in vivo and in vitro . RTP blocked replication forks initiated from a ColE1 replication origin at the cognate DNA-binding site (BS3) in a polar mode . The protein did not block rolling circle replication initiated from the pT181 origin in cell extracts of Staphylococcus aureus . RTP antagonized the helicase activity of DnaB but not that of helicase II of E . coli . Thus, RTP functioned as a polar contrahelicase blocking a helicase that participates in symmetric DNA replication but it did not impede rolling circle replication nor the action of a helicase involved in DNA repair.

Proc Natl Acad Sci U S A, 1994 Nov 8, 91(23), 10814 - 8
The crystal structure of allosteric chorismate mutase at 2.2-A resolution; Xue Y et al.; The crystal structure of an allosteric chorismate mutase, the Thr-226-->Ile mutant, from yeast Saccharomyces cerevisiae has been determined to 2.2-A resolution by using the multiple isomorphous replacement method . Solvent-flattening and electron-density modification were applied for phase improvement . The current crystallographic R factor is 0.196 . The final model includes 504 of the 512 residues and 97 water molecules . In addition, two tryptophan molecules were identified in the interface between monomers . The overall structure is completely different from the reported structure of chorismate mutase from Bacillus subtilis . This structure showed 71% helices with essentially no beta-sheet structures.

J Biol Chem, 1994 Nov 4, 269(44), 27365 - 71
The chromosomal tetracycline resistance locus of Bacillus subtilis encodes a Na+/H+ antiporter that is physiologically important at elevated pH; Cheng J et al.; The chromosomal tetB(L) gene of Bacillus subtilis encodes a transporter that catalyzes Na+/H+ antiport even more actively than tetracycline/H+ antiport, as shown by assays of membrane antiporter activity upon transformation of Na+/H+ antiporter-deficient Escherichia coli with the cloned gene; the transformation results in a substantial increase in Na+ resistance as well as detectable resistance to low tetracycline concentrations . Transpositional disruption of the chromosomal tetB(L) locus of B . subtilis led to reduced rates of electrogenic Na+ efflux and revealed a physiological role for this locus in Na+ resistance and Na(+)-dependent pH homeostasis at pH 8.5 . The mutant phenotype was reversed by transformation with a plasmid expressing the cloned tetB(L) gene . Energy-dependent tetracycline efflux rates in the wild type were greater than in the transposition mutant but were not sufficient to confer resistance to the antibiotic . TetB(L) is also inferred to have a modest capacity for K+ efflux, since the transposition mutant is slightly impaired in K(+)-dependent pH homeostasis at pH 8.5 and grew better than the wild type at pH 7 on limiting K+ concentrations.

J Appl Bacteriol, 1994 Nov, 77(5), 497 - 503
An investigation of enumeration and DNA partitioning in Bacillus subtilis L-form bacteria; Waterhouse RN et al.; Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-walled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e . deduced genome numbers) and viable cell counts (i.e . number of colony-forming units (cfu)) . The DNA hybridization method was shown to be a reliable and reproducible method for estimating genome numbers . Comparison of different L-form populations showed that the two methods of enumeration gave different values, with the deduced genome numbers much higher (by several orders of magnitude) than cell numbers deduced from viable cell counts . In contrast, when a culture of the cell-walled form was enumerated, the discrepancy between the two methods was low (by a factor of about 6) The combination of a high number of L-form genomes detected by DNA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replication and cell division processes in L-form bacteria . This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B . subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamycin resistance . The results are discussed with particular reference to cell division in cell wall-deficient, stable L-form bacteria.

J Antibiot (Tokyo), 1994 Nov, 47(11), 1258 - 65
Pyrroindomycins, novel antibiotics produced by Streptomyces rugosporus LL-42D005 . II . Biological activities; Singh MP et al.; The pyrroindomycins, a complex of novel antibiotics identified in fermentation broths of "Streptomyces rugosporus" LL-42D005, demonstrated excellent in vitro activity against Gram-positive bacteria . The semisynthetic diacetyl derivative of pyrroindomycin B (pyrroindomycin B-Ac2) was bactericidal for exponential-phase cells, but not for stationary-phase cells . This compound also exhibited marginal protection against a lethal Staphylococcus aureus challenge in mice . The poor in vivo activity of this antibiotic complex may be related to binding to blood components, as suggested by elevated MICs observed in blood-containing media . Incorporation of radiolabeled precursors into DNA, RNA, and protein was inhibited in an exponential-phase culture of Bacillus subtilis within ten minutes of exposure to pyrroindomycin B-Ac2 . Microscopic examinations of drug-treated cells revealed lysis within the same ten minute period . These data are consistent with an effect of pyrroindomycin B-Ac2 on the integrity of the bacterial membrane.

J Bacteriol, 1994 Nov, 176(22), 6992 - 8
23S rRNA domain V, a fragment that can be specifically methylated in vitro by the ErmSF (TlrA) methyltransferase; Kovalic D et al.; The DNA sequence that encodes 23S rRNA domain V of Bacillus subtilis, nucleotides 2036 to 2672 (C . J . Green, G . C . Stewart, M . A . Hollis, B . S . Vold, and K . F . Bott, Gene 37:261-266, 1985), was cloned and used as a template from which to transcribe defined domain V RNA in vitro . The RNA transcripts served as a substrate in vitro for specific methylation of B . subtilis adenine 2085 (adenine 2058 in Escherichia coli 23S rRNA) by the ErmSF methyltransferase, an enzyme that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics on Streptomyces fradiae NRRL 2702, the host from which it was cloned . Thus, neither RNA sequences belonging to domains other than V nor the association of 23S rRNA with ribosomal proteins is needed for the specific methylation of adenine that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics.

J Bacteriol, 1994 Nov, 176(22), 6802 - 11
Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants; Santana M et al.; We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase . The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species . The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms . We constructed two B . subtilis strains from which different parts of the atp operon were deleted . These B . subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source . ATP was synthesized in these strains only by substrate-level phosphorylation . The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio . In the absence of oxidative phosphorylation, B . subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate) . The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes . It therefore appears that B . subtilis and E . coli respond in similar ways to the absence of oxidative phosphorylation.

J Bacteriol, 1994 Nov, 176(21), 6744 - 8
Analysis of the dual regulatory mechanisms controlling expression of the vegetative catalase gene of Bacillus subtilis; Bol DK et al.; The expression of a vegetative catalase gene, katA (formerly the kat-19 gene), is necessary to protect Bacillus subtilis from H2O2, presumably by removing the oxidant from the environment . Genetic analysis of katA revealed that this gene is under two distinct forms of regulation, temporal and H2O2 inducible . The results reported here demonstrate that (i) the H2O2-inducible regulation of katA gene is not a component of the SOS regulon, (ii) the regulatory genes spo0A and abrB are involved in the temporal regulation but not the H2O2-specific induction of katA gene expression, and (iii) transcription initiation for the katA gene occurs at the same site under both forms of regulation.

J Bacteriol, 1994 Nov, 176(21), 6663 - 71
Bacillus subtilis CtaA is a heme-containing membrane protein involved in heme A biosynthesis; Svensson B et al.; Heme A is a prosthetic group of many respiratory oxidases . It is synthesized from protoheme IX (heme B) seemingly with heme O as a stable intermediate . The Bacillus subtilis ctaA and ctaB genes are required for heme A and heme O synthesis, respectively (B . Svensson, M . Lubben, and L . Hederstedt, Mol . Microbiol . 10:193-201, 1993) . Tentatively, CtaA is involved in the monooxygenation and oxidation of the methyl side group on porphyrin ring D in heme A synthesis from heme B . B . subtilis ctaA and ctaB on plasmids in both B . subtilis and Escherichia coli were found to result in a novel membrane-bound heme-containing protein with the characteristics of a low-spin b-type cytochrome . It can be reduced via the respiratory chain, and in the reduced state it shows light absorption maxima at 428, 528, and 558 nm and the alpha-band is split . Purified cytochrome isolated from both B . subtilis and E . coli membranes contained one polypeptide identified as CtaA by amino acid sequence analysis, about 0.2 mol of heme B per mol of polypeptide, and small amounts of heme A.

J Bacteriol, 1994 Nov, 176(21), 6528 - 37
Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G; Schmidt R et al.; The Bacillus subtilis RNA polymerase sigma factor sigma G is a cell-type-specific regulatory protein that governs the transcription of genes that are expressed at an intermediate to late stage of sporulation in the forespore compartment of the sporangium . Here we report the identification of a mutation (lon-1) that causes inappropriate transcription of genes under the control of sigma G under nutritional and genetic conditions in which sporulation is prevented . The mutation is located at 245 degrees on the genetic map and lies within a newly identified open reading frame that is predicted to encode a homolog to Lon protease . Inappropriate transcription of sigma G-controlled genes in the lon-1 mutant is not prevented by mutations in genes that are normally required for the appearance of sigma G during sporulation but is prevented by a mutation in the structural gene (spoIIIG) for sigma G itself . In light of previous work showing that spoIIIG is subject to positive autoregulation, we propose that Lon protease is responsible (possibly by causing degradation of sigma G) for preventing sigma G-directed transcription of spoIIIG and hence the accumulation of sigma G in cells that are not undergoing sporulation . An integrated physical and genetic map is presented that encompasses 36 kb of uninterrupted DNA sequence from the lon pheA region of the chromosome, corresponding to 245 degrees to 239 degrees on the genetic map.

J Bacteriol, 1994 Nov, 176(21), 6518 - 27
Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene; Riethdorf S et al.; The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes . In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis . The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined . The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues . A comparison of the deduced amino acid sequence with previously described lon gene products from E . coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins . Like the E . coli lon gene, the B . subtilis lon gene is induced by heat shock . Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin . The potential promoter region does not show similarities to promoters recognized by sigma 32 of E . coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B . subtilis . A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.

Genes Dev, 1994 Nov 1, 8(21), 2653 - 63
Role of interactions between SpoIIAA and SpoIIAB in regulating cell-specific transcription factor sigma F of Bacillus subtilis; Diederich B et al.; Genetic experiments have suggested that sigma F, the first compartment-specific transcription factor in sporulating B . subtilis, is regulated by an anti-sigma factor SpoIIAB and an anti-anti-sigma factor SpoIIAA . Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of sigma F-directed transcription . We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions . When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA-phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB . ADP is an inhibitor of the phosphorylation by ATP . Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine . SpoIIAAS58D, which apparently resembles SpoIIAA-phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-sigma F activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-sigma F activity both in vivo and in vitro . It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-sigma capacity of SpoIIAA and hence the activity of sigma F.

Ophthalmic Surg, 1994 Nov-Dec, 25(10), 690 - 4
Microbiologic evaluation of a hydrogen peroxide sterilization system; Wilkins DL et al.; The reliability of chemical sterilizers (acetone and/or 30-percent hydrogen peroxide at 25 degrees C and at 60 degrees C) was tested against Bacillus subtilis inoculated onto glass slides, commercial biological indicator discs (Bacillus stearothermophilus and B . subtilis), and B . subtilis spore survival . Acetone alone was not sporicidal . Hydrogen-peroxide-sterilized glass slides were sterile after 5 minutes . The indicator discs required 25 minutes at 25 degrees C, and less than 3 minutes at 60 degrees C (P < .0001) . The D value of B . subtilis in 27-percent hydrogen peroxide at 25 degrees C is 2 minutes, with z values of 22 degrees C and 26 degrees C at 25 degrees C and 40 degrees C, respectively . For delicate instruments, a 30-percent peroxide solution followed by an acetone rinse provides an effective alternative to classic heat sterilization.

Mol Microbiol, 1994 Nov, 14(3), 391 - 8
Biosynthesis and functional role of haem O and haem A; Mogi T et al.; Haem O and/or haem A are specifically synthesized for the haem-copper respiratory oxidases . A 17-carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen-reduction site . The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis . Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase) . It has recently been suggested that the ctaA gene adjacent to the ctaB-ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase) . In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.

Antimicrob Agents Chemother, 1994 Nov, 38(11), 2633 - 42
Inhibition of antibacterial activity of himastatin, a new antitumor antibiotic from Streptomyces hygroscopicus, by fatty acid sodium salts; Mamber SW et al.; Himastatin, a cyclohexadepsipeptide antibiotic, had in vivo antitumor activity against localized P388 leukemia and B16 melanoma but had no distal site antitumor activity . An in vitro Bacillus subtilis well-agar diffusion assay was employed to test the hypothesis that himastatin was enzymatically inactivated . The activity of himastatin against B . subtilis was inhibited when himastatin was mixed with mouse liver S9 fraction and microsomes . However, subsequent investigations demonstrated that the markedly decreased antibacterial activity was not enzymatic in nature but was related to the presence of certain fatty acid salts . Saturated fatty acid sodium salts with a carbon chain number of 8 or more reduced the antimicrobial activity of himastatin 50 to 100 times . If antibiotics such as ampicillin, bacitracin, chloramphenicol, and tunicamycin were used in place of himastatin, no meaningful reduction in antibacterial activity occurred . However, the antibacterial activity of the membrane-active peptide antibiotic polymyxin B, but not that of polymyxin E (colistin), was reduced in a manner similar to that of himastatin . Importantly, the activity of himastatin against HCT-116 colon adenocarcinoma cells in soft agar was markedly reduced in the presence of sodium palmitate as the reference fatty acid salt . The data indicate that himastatin may be trapped in micelles in vitro . It may be speculated that the lack of distal site antitumor activity resulted from similar complex formation between himastatin and lipids in vivo . The results also suggest that the cancer cytotoxic and antimicrobial effects of himastatin may result from interactions with the cell membrane.

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 925 - 31
Decrease in spermidine content during logarithmic phase of cell growth delays spore formation of Bacillus subtilis; Ishii I et al.; Bacillus subtilis 168M contained a large amount of spermidine during the logarithmic phase of growth, but the amount decreased drastically during the stationary phase . The extracts, prepared from B . subtilis cells harvested in the logarithmic phase, contained activity of arginine decarboxylase (ADC) rather than the activity of ornithine decarboxylase . In the presence of alpha-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of ADC, the amount of spermidine in B . subtilis during the logarithmic phase decreased to about 25% of the control cells . Under these conditions, spore formation of B . subtilis 168M delayed greatly without significant inhibition of cell growth . The decrease in spermidine content in the logarithmic phase rather than in the stationary phase was involved in the delay of sporulation . Electron microscopy of cells at 24 hrs . of culture confirmed the delay of spore formation by the decrease of spermidine content . Furthermore, the delay of sporulation was negated by the addition of spermidine . These data suggest that a large amount of spermidine existing during the logarithmic phase plays an important role in the sporulation of B . subtilis.

J Exp Biol, 1994 Nov, 196, 457 - 70
The role of monovalent cation/proton antiporters in Na(+)-resistance and pH homeostasis in Bacillus: an alkaliphile versus a neutralophile; Krulwich TA et al.; Both neutralophilic Bacillus subtilis and alkaliphilic Bacillus firmus OF4 depend upon electrogenic Na+/H+ antiporters, which are energized by the gradients established by respiration-coupled proton extrusion, to achieve Na(+)-resistance and pH homeostasis when the external pH is very alkaline . The interplay of proton and sodium cycles is discussed . In B . subtilis, pH homeostasis, up to pH9, can be achieved using K+ when Na+ is unavailable or when the gene encoding the Na+/H+ antiporter that is involved in Na(+)-dependent pH homeostasis is disrupted . That gene is a member of the tetracycline efflux family of genes . A second gene, encoding a Na+/H+ antiporter that functions in Na(+)-resistance, has been identified, and candidates for the K+/H+ antiporter genes are under investigation . Aggregate Na+/H+ antiport activity in B . subtilis is as much as 10 times lower than in the alkaliphile, and the neutralophile cannot regulate its internal pH upon a shift to pH 10.5 . Upon such a shift, there is a pronounced reduction in the generation of a primary electrochemical proton gradient . The alkaliphile, by contrast, maintains substantial driving forces and regulates its internal pH in an exclusively Na(+)-coupled manner upon shifts to either pH 8.7 or 10.5 . One gene locus has been identified and a second locus has been inferred as encoding relevant antiporter activities.

Microbiology, 1994 Nov, 140 ( Pt 11), 3079 - 83
Analysis of the expression and regulation of the gerB spore germination operon of Bacillus subtilis 168; Corfe BM et al.; The gerB spore germination operon of Bacillus subtilis 168 is a homologue of the gerA spore germination operon . The expression and regulation of the gerB operon has been examined using a lacZ transcriptional fusion and the transcriptional start defined . The gerB operon is expressed during sporulation under the control of RNA polymerase containing the forespore-specific sigma factor, delta G . This is a further homology to the gerA operon, which is similarly regulated . It is predicted from the localization of expression and the encoded primary sequences that the GerB proteins are located at the inner spore membrane.

Protein Sci, 1994 Nov, 3(11), 2144 - 7
Effect of pH and phosphate ions on self-association properties of the major cold-shock protein from Bacillus subtilis; Makhatadze GI et al.; The intermolecular interactions of the major cold-shock protein from Bacillus subtilis (CspB) in solution in the presence of different salts, including phosphate, have been studied by means of scanning calorimetry and size-exclusion chromatography . Calorimetric results indicate that, in all cases, protein unfolding can be approximated by a 2-state model, but the modes of unfolding can differ depending on the conditions . In the presence of phosphate, the cooperative folding unit is a monomer, whereas in the absence of phosphate, the cooperative unit is a dimer . The difference in the self-association of CspB in the presence and absence of phosphate was supported by size-exclusion chromatography . These results are compared with recent structural studies of CspB in crystal and in solution.

Protein Sci, 1994 Nov, 3(11), 2115 - 28
Unique dicistronic operon (ptsI-crr) in Mycoplasma capricolum encoding enzyme I and the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system: cloning, sequencing, promoter analysis, and protein characterization; Zhu PP et al.; The region of the genome of Mycoplasma capricolum encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced . Examination of the sequence revealed a unique arrangement of the pts operon . In all other bacterial species characterized thus far, the gene encoding Enzyme I (ptsI) in the pts operon is located immediately downstream of the gene (ptsH) encoding HPr, a general energy coupling protein of the PTS . In M . capricolum, ptsH and ptsI reside on 2 distinct operons at separate loci on the chromosome (Zhu PP, Reizer J, Reizer A, Peterkofsky A, 1993, J Biol Chem 268:26531-26540) . In the present work, it is shown that the Mycoplasma Enzyme I gene is preceded by an open reading frame homologous to the product of the Escherichia coli kdtB gene and is followed by the gene (crr) encoding Enzyme IIAglc . Northern blot analysis indicated that ptsI and crr constitute a dicistronic operon that includes an independent promoter for the crr gene . Primer extension studies established the transcription start sites for the ptsI and crr genes . The products of the ptsI and crr genes are homologous to previously sequenced Enzymes I and IIAglc proteins but are more similar to the counterpart proteins from gram-positive than to those from gram-negative organisms . The deduced amino acid sequence of the Mycoplasma Enzyme I shows that it differs from other Enzymes I by having fewer acidic amino acids and more basic, amidated, and aromatic amino acids . The deduced amino acid sequence of the Mycoplasma Enzyme IIAglc indicates that it is the shortest (154 residues) of the proteins in this class and it is the only Enzyme IIAglc with a tryptophan and a cysteine residue . In vitro sugar phosphorylation studies with extracts from E . coli and Bacillus subtilis and purified proteins indicated that the Mycoplasma HPr is not a phosphoacceptor from the E . coli Enzyme I, whereas the Mycoplasma Enzyme IIAglc accepts and transfers phosphate from both E . coli and B . subtilis PTS components.

Mol Microbiol, 1994 Nov, 14(3), 473 - 83
The copR gene product of plasmid pIP501 acts as a transcriptional repressor at the essential repR promoter; Brantl S; The amount of the rate-limiting replication initiator protein RepR of plasmid pIP501 is negatively controlled by an antisense RNA (RNAIII) and a dispensable protein (CopR) . Deletions or mutations in either component cause a 10-20-fold copy number increase . RNAIII induces transcription attenuation of the repR mRNA; the mode of CopR action remained unclear . To test the function of CopR, transcriptional fusions of promoters pI, pII and pIII with lacZ were integrated into the Bacillus subtilis chromosome . CopR and/or RepR were supplied in trans, and LacZ synthesis measured . The results show that CopR represses the repR promoter pII . Neither CopR nor RepR autoregulate their promoters . Gel mobility shift assays indicate that CopR binds to a 44 bp DNA fragment comprising the inverted repeat upstream of pII.

J Mol Biol, 1994 Oct 28, 243(3), 425 - 36
Sporulation regulatory protein SpoIIID from Bacillus subtilis activates and represses transcription by both mother-cell-specific forms of RNA polymerase; Halberg R et al.; Mother-cell-specific gene expression during sporulation of Bacillus subtilis is controlled by sigma E and sigma K RNA polymerases . sigma E is required for the expression of genes during stage III (engulfment of the forespore), while sigma K is required for the expression of genes during stage IV (formation of the spore cortex) and stage V (formation of the spore coat) . Previous studies indicated that SpoIIID could influence transcription by sigma K RNA polymerase in vitro . We demonstrate here that SpoIIID is a DNA-binding protein that recognizes specific sequences in the promoter regions and open reading frames of both sigma E- and sigma K-dependent genes . We also show that SpoIIID can activate or repress transcription by both forms of RNA polymerase . These results support the idea that the appearance and subsequent disappearance of SpoIIID plays a major role in controlling the mother-cell pattern fo gene expression during stages III to V of sporulation.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9808 - 12
Cryptic DNA-binding domain in the C terminus of RNA polymerase II general transcription factor RAP30; Tan S et al.; The C terminus of mammalian transcription factor RAP30 has been found to be a cryptic DNA-binding domain strikingly similar to the C-terminal DNA-binding domain present in conserved region 4 of members of the sigma 70 family of bacterial sigma factors . This RAP30 domain shares strongest sequence similarity with the DNA-binding domain present in region 4 of Bacillus subtilis sporulation-specific sigma K . Like the region 4 DNA-binding activity of Escherichia coli sigma 70, the RAP30 C-terminal DNA binding activity is masked in intact RAP30 but is readily detectable when the RAP30 C terminus is expressed as a fusion protein . Consistent with a role for RAP30 DNA-binding activity in transcription, mutations that abolish DNA binding also abolish transcription . Therefore, RAP30 may function at least in part through the action of an evolutionarily ancient DNA-binding domain that first appeared prior to the divergence of bacteria and eukaryotes.

Gene, 1994 Oct 11, 148(1), 107 - 12
In vivo functional relationships among terminal proteins of Bacillus subtilis phi 29-related phages; Bravo A et al.; Gene 3 of the Bacillus subtilis phage phi 29 encodes the terminal protein (TP), which acts as a primer in the initiation of viral DNA replication . We have developed an in vivo functional assay for the phi 29 TP based on the ability of TP-producing B . subtilis non-suppressor (su-) cells to support DNA replication of a phi 29 sus3 mutant phage . This trans-complementation assay has been used to study in vivo functional relationships between the TP of phi 29 and related phages . Our results demonstrate that phi 29 TP functionally substitutes the TP of phage PZA, whereas replication of phage Nf DNA cannot take place in vivo using the phi 29 TP as a primer.

Nucleic Acids Res, 1994 Oct 11, 22(20), 4087 - 94
Interaction of the 3'-end of tRNA with ribonuclease P RNA; Oh BK et al.; Ribonuclease P, which contains a catalytic RNA subunit, cleaves 5' precursor-specific sequences from pre-tRNAs . It was previously shown that the RNase P RNA optimally cleaves substrates which contain the mature, 3'-terminal CCA of tRNA . In order to determine the contributions of those individual 3'-terminal nucleotides to the interaction, pre-tRNAs that have CCA, only CC or C or are without CCA at the 3'-end were synthesized by run-off transcription, tested as substrates for cleavage by RNase P RNA and used in photoaffinity crosslinking experiments to examine contact sites in the ribozyme . In order to generalize the results, analyses were carried out using three different bacterial RNase P RNAs, from Escherichia coli, Bacillus subtilis and Thermotoga maritima . At optimal (Kcat/Km) ionic strength (1 M NH4+/25 mM Mg2+), Km increases incrementally 3- to 10-fold upon stepwise removal of each nucleotide from the 3'-end . At high ionic strength (2 M NH4+/50 mM Mg2+), which suppresses conformational effects, removal of the 3'-terminal A had little effect on Km, indicating that it is not a specific contact . Analysis of the deletion and substitution mutants indicated that the C residues act specially; their contribution to binding energy at high ionic strength (approximately 1 kcal/mol) is consistent with a non-Watson-Crick interaction, possibly irregular triple-strand formation with some component of the RNase P RNA . In agreement with previous studies, we find that the RNase P holoenzyme in vitro does not discriminate between tRNAs containing or lacking CCA . The structural elements of the three RNase P RNAs in proximity to the 3'-end of tRNA were examined by photoaffinity crosslinking . Photoagent-labeled tRNAs with 3'-terminal CCA, only CC or C, or lacking all these nucleotides were covalently conjugated to the three RNase P RNAs by irradiation and the sites of crosslinks were mapped by primer extension . The main crosslink sites are located in a highly conserved loop (probably an irregular helix) that is part of the core of the RNase P RNA secondary structure . The crosslinking results orient the CCA of tRNA with respect to that region of the RNase P RNA.

Microbiology, 1994 Oct, 140 ( Pt 10), 2567 - 75
Possible function and some properties of the CcpA protein of Bacillus subtilis; Miwa Y et al.; The ccpA mutations alsA1 (alsA1 is allelic to ccpA) and ccpA::Tn917 completely abolished catabolite repression of gluconate kinase and sorbitol dehydrogenase synthesis in Bacillus subtilis, whereas they only partially affected the catabolite repression of inositol dehydrogenase, histidase and xylose isomerase synthesis . The alsA1 mutation also partially affected catabolite repression of sporulation . Analysis of revertants from the alsA1 mutant by direct sequencing indicated that this mutation comprises a base substitution of guanine at nucleotide -14 to adenine within the Shine-Dalgarno sequence of the ccpA gene (ccpA translation starts at nucleotide +1) . A 1.37 kb EcoRI fragment carrying the ccpA gene was cloned into Escherichia coli plasmid pUC19 and B . subtilis plasmid pUB110, resulting in plasmids pCCPA19 and pCCPA110, respectively . The ccpA gene carried in pCCPA110 complemented the alsA1 mutation . Western blotting revealed that the level of the CcpA protein in B . subtilis cells, which seemed to be constitutively synthesized, was approximately 10 times lower for the alsA1 mutant than for the wild-type . The CcpA protein synthesized by either E . coli cells bearing pCCPA19 or B . subtilis cells bearing pCCPA110 was purified to over 90% homogeneity; the latter cells were grown in the presence of glucose . The molecular mass of the protein purified from E . coli was 74 kDa, suggesting that this protein exists as a dimer because its subunit molecular mass was 38 kDa as determined by SDS-PAGE . Gel retardation analysis indicated that the purified CcpA protein in both cases did not bind to the cis sequence for catabolite repression of the gnt operon, but it bound non-specifically to DNA.

Appl Environ Microbiol, 1994 Oct, 60(10), 3732 - 8
Evaluation of counting error due to colony masking in bioaerosol sampling; Chang CW et al.; Colony counting error due to indistinguishable colony overlap (i.e., masking) was evaluated theoretically and experimentally . A theoretical model to predict colony masking was used to determine colony counting efficiency by Monte Carlo computer simulation of microorganism collection and development into CFU . The computer simulation was verified experimentally by collecting aerosolized Bacillus subtilis spores and examining micro- and macroscopic colonies . Colony counting efficiency decreased (i) with increasing density of collected culturable microorganisms, (ii) with increasing colony size, and (iii) with decreasing ability of an observation system to distinguish adjacent colonies as separate units . Counting efficiency for 2-mm colonies, at optimal resolution, decreased from 98 to 85% when colony density increased from 1 to 10 microorganisms cm-2, in contrast to an efficiency decrease from 90 to 45% for 5-mm colonies . No statistically significant difference (alpha = 0.05) between experimental and theoretical results was found when colony shape was used to estimate the number of individual colonies in a CFU . Experimental colony counts were 1.2 times simulation estimates when colony shape was not considered, because of nonuniformity of actual colony size and the better discrimination ability of the human eye relative to the model . Colony surface densities associated with high counting accuracy were compared with recommended upper plate count limits and found to depend on colony size and an observation system's ability to identify overlapped colonies . Correction factors were developed to estimate the actual number of collected microorganisms from observed colony counts.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1994 Oct, 176(20), 6397 - 401
Analysis of expression of the argC and argD genes in the cyanobacterium Anabaena sp . strain PCC 7120; Floriano B et al.; A cloned DNA fragment from Anabaena sp . strain PCC 7120 that complements an arginine auxotrophic mutant from the same organism was found to include an open reading frame encoding a 427-residue polypeptide that is homologous to N-acetylornithine aminotransferase from Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae . The gene encoding N-acetylornithine aminotransferase in bacteria has been named argD . The expression of Anabaena sp . strain PCC 7120 argD, as well as of argC, was analyzed at the mRNA level . Both genes were transcribed as monocistronic mRNAs, and their expression was not affected by exogenously added arginine . Primer extension analysis identified transcription start points for both genes which were preceded by sequences similar to that of the E . coli RNA polymerase sigma 70 consensus promoter . A second transcription start point for the argD gene that is not preceded by a sigma 70 consensus promoter was detected in dinitrogen-grown cultures.

J Bacteriol, 1994 Oct, 176(19), 5962 - 70
Bacillus subtilis HemY is a peripheral membrane protein essential for protoheme IX synthesis which can oxidize coproporphyrinogen III and protoporphyrinogen IX; Hansson M et al.; The hemY gene of the Bacillus subtilis hemEHY operon is essential for protoheme IX biosynthesis . Two previously isolated hemY mutations were sequenced . Both mutations are deletions affecting the hemY reading frame, and they cause the accumulation of coproporphyrinogen III or coproporphyrin III in the growth medium and the accumulation of trace amounts of other porphyrinogens or porphyrins intracellularly . HemY was found to be a 53-kDa peripheral membrane-bound protein . In agreement with recent findings by Dailey et al . (J . Biol . Chem . 269:813-815, 1994) B . subtilis HemY protein synthesized in Escherichia coli oxidized coproporphyrinogen III and protoporphyrinogen IX to coproporphyrin and protoporphyrin, respectively . The protein is not a general porphyrinogen oxidase since it did not oxidize uroporphyrinogen III . The apparent specificity constant, kcat/Km, for HemY was found to be about 12-fold higher with coproporphyrinogen III as a substrate compared with protoporphyrinogen IX as a substrate . The protoporphyrinogen IX oxidase activity is consistent with the function of HemY in a late step of protoheme IX biosynthesis, i.e., HemY catalyzes the penultimate step of the pathway . However, the efficient coproporphyrinogen III to coproporphyrin oxidase activity is unexplained in the current view of protoheme IX biosynthesis.

Wei Sheng Wu Xue Bao, 1994 Oct, 34(5), 339 - 44
{The effect of a promoter P28-1 of Bacillus subtilis on Streptomyces differentiation}; Tan H et al.; A 1.1kb promoter P28-1 was inserted into pUC19 . After then, the P28-1 was subcloned into the HindIII-EcoRI sites of the high copy number Streptomyces promoter probe plasmid pIJ4083 containing xy1E reporter gene . This recombinant plasmid was designated as pIJ4498 . When pIJ4498 was introduced into Streptomyces coelicolor J1501 protoplasts, transformants conferred a white phenotype, whereas the vector pIJ4083 gave rise to colonies of normal, dark grey appearance which is the same as that of J1501 itself . After confirming pIJ4498 structure with some restriction enzymes, it was also introduced into whiG mutant (C71) . Crude enzyme extracts were isolated from J1501/pIJ4498, J1501/pIJ4083 and C71/pIJ4498 respectively, the crude enzyme extract from J1501/pIJ4498 could oxidize catechol (colourless) to 2-hydroxy muconic semialdehyde (yellow colour), but crude enzyme extracts from J1501/pIJ4083 and C71/pIJ4498 could not oxidize catechol to 2-hydroxymuconic semialdehyde . The results indicated that P28-1 might be recognised by sigma whiG RNA polymerase, and activated the xylE reporter gene expression and reduced J1501 sporulation.

Res Microbiol, 1994 Oct, 145(8), 579 - 83
New shuttle vector for cloning in Bacillus stearothermophilus; De Rossi E et al.; Cloning vector plasmid pRP9 was constructed on the basis of the broad host-range plasmid pLM6 . pRP9 was a small plasmid (2.9 kb), possessed a convenient polyrestriction site sequence and efficiently transformed Bacillus subtilis, Bacillus stearothermophilus and Escherichia coli . Furthermore, pRP9 presented a very high segregational stability in Bacillus hosts . Also, the structural stability in Bacillus strains, grown under selective pressure, of pRP9 carrying a 3-kb fragment, was high . No single-stranded and high-molecular weight pRP9 DNA was found in B . stearothermophilus . The host/vector systems described possessed all the properties required for efficient gene cloning.

Curr Opin Genet Dev, 1994 Oct, 4(5), 630 - 6
Establishment of cell type specific gene transcription during sporulation in Bacillus subtilis; Duncan L et al.; Asymmetric cell division during the process of sporulation in the bacterium Bacillus subtilis generates dissimilar progeny that exhibit distinct programs of gene transcription . Recent work reveals a partner switching mechanism that governs the activity of the sporulation regulatory protein sigma F and that may be responsible for the establishment of cell type specific gene transcription.

Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1845 - 50
The rapid degradation of mutant SecA protein in the Bacillus subtilis secA341 (ts) mutant causes a protein translocation defect in the cell; Takamatsu H et al.; To study the function of SecA protein and the protein translocation system of Bacillus subtilis, wild-type and mutant SecA proteins were characterized in vivo and in vitro . SecA protein was abundant in a wild-type strain (168) and existed in a stable homodimer . In contrast to this, SecA341 (ts) protein having an amino acid replacement from proline to leucine at residue 431 was undetectable by immunoblotting in the cell lysate of a secA341 mutant (TB301) at the nonpermissive temperature, 42 degrees C . Pulse-chase studies using 35S-methionine showed that newly synthesized SecA protein was rapidly degraded in the mutant at 42 degrees C . Purified SecA341 protein was more sensitive to trypsin and subtilisin than purified wild-type SecA protein in the presence of ATP . These results indicate that the secA341 mutation causes the rapid degradation of mutant SecA protein and a concomitant protein translocation defect in the cell.

Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1836 - 41
First evidence for homologous recombination-mediated large DNA inversion on the Bacillus subtilis 168 chromosome; Itaya M; A Bacillus subtilis 168 strain carrying an inversion of about 1600 kb-long chromosomal DNA was isolated . Physical and genetic analyses demonstrated that the inversion was generated as a result of homologous recombination between two homologous sequences integrated at the met and leuB loci . This is the first clear evidence of a large stable chromosomal inversion induced by homologous recombination in B . subtilis.

Nat Struct Biol, 1994 Oct, 1(10), 717 - 23
The structure of Bacillus subtilis pectate lyase in complex with calcium; Pickersgill R et al.; We have solved the structure of the Bacillus subtilis pectate lyase (BsPel) in complex with calcium . The structure consists of a parallel beta-helix domain and a loop region . The alpha L-bounded beta-strand seen in BsPel is a new element of protein structure and its frequent occurrence suggests it is an important characteristic of the parallel beta-helix . A pronounced cleft is formed between the loops and the parallel beta-helix domain and we propose that this is the active site cleft . Calcium, essential for the activity of the enzyme, binds at the bottom of this cleft and an arginine residue close to the calcium, which is conserved across all pectin and pectate lyases, may be involved in catalysis.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9397 - 401
Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis; D'Souza C et al.; Genetic competence (the ability to internalize exogenous DNA) in Bacillus subtilis is dependent on a regulatory pathway that activates the expression of a battery of competence-specific genes . The srfA operon, encoding the subunits of surfactin synthetase, which catalyzes the nonribosomal synthesis of the peptide antibiotic surfactin, also functions in the competence regulatory pathway . The DNA encoding only one of the seven amino acid-activating domains of surfactin synthetase, the valine-activating domain (srfAB1), is necessary for competence . Deletion analysis revealed that a 569-bp fragment of srfAB1, fused to the srfA promoter, complements a srfA deletion mutation (delta srfA) with respect to competence . This fragment contains an open reading frame consisting of 46 amino acids (orf46), which is out of frame with srfAB1 . A frameshift mutation in srfAB upstream of orf46 has no effect on competence but a frameshift and nonsense mutation in orf46 resulted in failure to complement the delt srfA mutation . These results indicate that orf46 encodes the srfA-associated competence regulatory factor . Computer-aided analysis of the putative orf46 product (ComS) shows similarity to the homeodomain of the POU domain class of eukaryotic transcriptional regulators.

Biochemistry, 1994 Sep 27, 33(38), 11501 - 6
Mutations in sigma factor that affect the temperature dependence of transcription from a promoter, but not from a mismatch bubble in double-stranded DNA; Aiyar SE et al.; Specificity of promoter utilization in bacterial RNA polymerases is imparted by a class of proteins referred to as sigma factors . Conserved region 2.3 of these proteins is thought to play a role in the strand separation process that occurs during the formation of an initiation-competent RNA polymerase-promoter complex . We have used a heterologous system consisting of Escherichia coli core RNA polymerase and Bacillus subtilis sigma A to probe the effects of amino acid substitutions in region 2.3 . In agreement with previous work {Juang & Helmann (1994) J . Mol . Biol . 235, 1470-1488} we observe that several amino acid substitutions exacerbate the deleterious effect of low temperature on promoter-dependent initiation . On the other hand, no such enhanced cold sensitivity is found with double-stranded templates that contain short "bubbles" of single-stranded DNA, indicating that the DNA-melting defect imposed by these mutant sigma factors can be suppressed by the use of such bubble templates . These results support the involvement of region 2.3 in the strand separation process that accompanies open complex formation at promoters.

Gene, 1994 Sep 15, 147(1), 95 - 100
A pho regulon promoter induced under sporulation conditions; Birkey SM et al.; Sporulation-induced alkaline phosphatases (APases) of Bacillus subtilis require the products of the sporulation stage-0 genes and certain stage-II genes, including the spoIIA operon, for induction . Mutations in either sapA or sapB bypass this requirement {Piggot and Taylor, J . Gen . Microbiol . 10 (1977) 69-80}, resulting in APase production in a spoIIA sapA or spoIIA sapB strain, under sporulation conditions . B . subtilis has multiple structural genes encoding APases, which are induced either by phosphate starvation or during sporulation, or under both conditions . We report studies designed to determine which APase(s) were being expressed in the sap mutants, and from which promoters . phoB (formerly phoAIII), one of the structural genes encoding an APase in B . subtilis, is expressed under both sporulation and phosphate starvation conditions, but from separate promoters {Chesnut et al., Mol . Microbiol . 5 (1991) 2181-2190} . The spoIIA sapA and spoIIA sapB strains express phoB under sporulation conditions . Interestingly, the expression of phoB during sporulation was from Pv, the phosphate starvation-inducible promoter of phoB, rather than from Ps, the sporulation-specific promoter . Since the induction of phosphate starvation-inducible promoters during phosphate limitation requires the phoPR operon {Miki et al., Genetics 52 (1965) 1093-1100}, we asked if the phoPR products were involved in regulating Pv expression under sporulation conditions . The phoPR genes are transcribed under sporulation conditions, regulated by sapA and sapB under sporulation conditions, and required for expression from Pv under sporulation conditions.

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