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 t