Bacillus subtilis

Biochemistry, 1999 Jan 5, 38(1), 22 - 32
His68 and His141 are critical contributors to the intersubunit catalytic site of adenylosuccinate lyase of Bacillus subtilis; Lee TT et al.; Mutant adenylosuccinate lyases of Bacillus subtilis were prepared by site-directed mutagenesis with replacements for His141, previously identified by affinity labeling as being in the active site {Lee, T . T., Worby, C., Dixon, J . E., and Colman, R . F . (1997) J . Biol . Chem . 272, 458-465} . Four substitutions (A, L, E, Q) yield mutant enzyme with no detectable catalytic activity, while the H141R mutant is about 10(-)5 as active as the wild-type enzyme . Kinetic studies show, for the H141R enzyme, a Km that is only 3 times that of the wild-type enzyme . Minimal activity was also observed for mutant enzymes with replacements for His68 {Lee, T . T., Worby, C., Bao, Z . -Q., Dixon, J . E., and Colman, R . F . (1998) Biochemistry 37, 8481-8489} . Measurement of the reversible binding of radioactive adenylosuccinate by inactive mutant enzymes with substitutions at either position 68 or 141 shows that their affinities for substrate are decreased by only 10-40-fold . These results suggest that His141, like His68, plays an important role in catalysis, but not in substrate binding . Evidence is consistent with the hypothesis that His141 and His68 function, respectively, as the catalytic base and acid . Circular dichroism spectroscopy and gel filtration chromatography conducted on wild-type and all His141 and His68 mutants reveal that none of the mutant enzymes exhibits major structural changes and that all the enzymes are tetramers . Mixing inactive His141 with inactive His68 mutant enzymes leads to striking increases in catalytic activity . This complementation of mutant enzymes indicates that His141 and His68 come from different subunits to form the active site . A tetrameric structure of adenylosuccinate lyase was constructed by homology modeling based on the known structures in the fumarase superfamily, including argininosuccinate lyase, delta-crystallin, fumarase, and aspartase . The model suggests that each active site is constituted by residues from three subunits, and that His141 and His68 come from two different subunits.

EMBO J, 1998 Nov 16, 17(22), 6730 - 8
Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor; Turgay K et al.; Competence is a physiological state, distinct from sporulation and vegetative growth, that enables cells to bind and internalize transforming DNA . The transcriptional regulator ComK drives the development of competence in Bacillus subtilis . ComK is directly required for its own transcription as well as for the transcription of the genes that encode DNA transport proteins . When ComK is sequestered by binding to a complex of the proteins MecA and ClpC, the positive feedback loop leading to ComK synthesis is interrupted . The small protein ComS, produced as a result of signaling by a quorum-sensing two-component regulatory pathway, triggers the release of ComK from the complex, enabling comK transcription to occur . We show here, based on in vivo and in vitro experiments, that ComK accumulation is also regulated by proteolysis and that binding to MecA targets ComK for degradation by the ClpP protease in association with ClpC . The release of ComK from binding by MecA and ClpC, which occurs when ComS is synthesized, protects ComK from proteolysis . Following this release, the rates of MecA and ComS degradation by ClpCP are increased in our in vitro system . In this novel system, MecA serves to recruit ComK to the ClpCP protease and connects ComK degradation to the quorum-sensing signal-transduction pathway, thereby regulating a key developmental process . This is the first regulated degradation system in which a specific targeting molecule serves such a function.

Chem Biol, 1999 Jan, 6(1), 31 - 41
Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3; Steller S et al.; BACKGROUND: Bacillus subtilis strains produce a broad spectrum of lipopeptides that are potent biosurfactants and have specific antimicrobial and antiviral activities . The cyclic lipodecapeptide fengycin is one such compound . Although the fengycin biosynthetic genes in B . subtilis 168 (pps genes) and F29-3 (fen genes) have been well characterized, only limited information is available about the biochemical features of the fengycin synthetase multienzyme system . RESULTS: Five multifunctional peptide synthetases (Fen1-5) that catalyze biosynthesis of the peptide portion of fengycin have been purified from crude extracts of the B . subtilis b213 and A1/3 strains . These enzymes activate all fengycin amino-acid components as aminoacyl adenylates or aminoacyl thioesters . Fen1, Fen2 and Fen3 are each approximately 286 kDa, Fen4 is approximately 400 kDa and Fen 5 is approximately 140kDa; each enzyme activates a different set of L-amino acids . A five-gene cluster (fen1-5) was detected in the B . subtilis A1/3 genome that shows high homology to the pps and fen genes in B . subtilis strains 168 and F29-3 . Disruption of fen4 resulted in a loss of fengycin production . The fengycin synthetase enzymes isolated from B . subtilis b213 were assigned to the corresponding A1/3 fen genes by their amino-terminal sequences . CONCLUSIONS: The structural and functional organization of the fengycin synthetase system from B . subtilis b213 has been characterized in detail and correlated with the corresponding pps and fen genes in B . subtilis strains 168, A1/3 and F29-3 . Biosynthesis of the peptide part of fengycin involves five multifunctional modular proteins that assemble the lipopeptide chain using a nonribosomal, multiple carrier thiotemplate mechanism.

Biochemistry, 1999 Jan 12, 38(2), 629 - 35
Cloning and characterization of the 23S RNA pseudouridine 2633 synthase from Bacillus subtilis; Niu L et al.; A Bacillus subtilis ORF, ypul, 41% homologous to rsuA, the gene for the synthase which forms pseudouridine 516 in Escherichia coli 16S rRNA, was cloned and the protein expressed and affinity-purified by the His tag procedure . Reactions with E . coli 16S and 23S rRNA transcripts were performed in vitro . The protein did not form pseudouridine 516 as expected but did produce pseudouridine 552 in 16S rRNA and pseudouridines 1199, 2605, and 2833 in 23S rRNA . Of these, only pseudouridine 2605 is found naturally in either E . coli or B . subtilis rRNA . Kinetic experiments confirmed that pseudouridine 2605 was the primary target . Comparison of the four pseudouridine sites yielded a consensus recognition sequence for the synthase . This consensus sequence was not present at any other site in either E . coli or B . subtilis 16S or 23S RNA . We propose that YpuL is the B . subtilis pseudouridine 2633 (2605 in E . coli) synthase . Since the closest gene sequence homologue in E . coli is yciL, we suggest that its gene product is the corresponding E . coli pseudouridine 2605 synthase.

Biochemistry, 1999 Jan 12, 38(2), 605 - 18
Identification of the genes encoding Mn2+-dependent RNase HII and Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families; Ohtani N et al.; Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues . The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively . RNases HI and HII show no significant sequence similarity . These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E . coli RNases HI and HII . We found that the ypdQ gene product showed no RNase H activity . The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E . coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well . In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E . coli RNase HII, whereas the ysgB (rnhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity . Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B . subtilis and E . coli RNases HII . Likewise, B . subtilis RNase HIII and E . coli RNase HI have generated similar products . These results suggest that B . subtilis RNases HII and HIII may be functionally similar to E . coli RNases HII and HI, respectively . We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.

Plasmid, 1999 Jan, 41(1), 17 - 29
Regulation of initiation of Bacillus subtilis chromosome replication; Moriya S et al.; Bacterial chromosome replication is tightly regulated at the initiation stage to coordinate with mass increase . Together with chromosome partition at cell division, this regulation mechanism ensures the proper number of chromosomes in daughter cells at any growth rate . Therefore, elucidation of this regulation mechanism is important for understanding the bacterial cell cycle . Despite much effort in Escherichia coli and Bacillus subtilis for many years, the mechanism remains to be completely elucidated . In E . coli, it is proposed that a critical amount of DnaA protein determines the time of initiation of replication in the cell cycle . Our study strongly suggested that this might not be the case in B . subtilis . Recently, remarkable progress has been made in bacterial cytology . The new techniques enable us to examine the subcellular location of proteins of interest and DNA regions of the chromosome (for example, the replication origin) and, therefore, to determine directly when in the cell division cycle and where within the cell initiation of chromosome replication takes place . Using the techniques, we detected the initiation complex by examining subcellular location of several Dna-initiation proteins in B . subtilis . Based on our new findings, we propose a novel model for regulation of the time of initiation of chromosome replication in the cell cycle .

J Mol Biol, 1999 Jan 22, 285(3), 917 - 29
Interaction of a repressor and its binding sites for regulation of the Bacillus subtilis iol divergon; Yoshida KI et al.; Transcription of the Bacillus subtilis iol divergon is negatively regulated by a repressor encoded by iolR, which belongs to the DeoR family of bacterial regulators . Gel retardation analysis involving the IolR protein synthesized in Escherichia coli revealed that IolR bound specifically and independently to each of the iol and iolRS promoter regions, with higher affinity to iol . DNase I footprinting revealed that IolR affected DNase I sensitivity either in the iol promoter region between nucleotides -46 and +51 or in iolRS between -79 and -2 (+1 is the transcription initiation nucleotide of both iol and iolRS), indicating its interaction with the extended regions of the iol and iolRS promoters . Deletion analysis indicated that the iol region between -23 and +21 is involved mainly in IolR binding and negative regulation, while the iolRS region between -70 and -44 comprises at least part of the cis-acting sequences for IolR binding and negative regulation . Sequence examination of the extended regions revealed that a tandem direct repeat consisting of two relatively conserved 11-mer sequences, WRAYCAADARD (where D is A, G or T; R is A or G; W is A or T; and Y is C or T), found in each of the iol and iolRS regions might be a determinant sequence for the IolR-DNA interaction . Actual involvement of the direct repeats in the IolR-DNA interaction was shown by the deficiency of IolR-binding and negative regulation that was caused by substitution of the conserved bases within the conserved sequences . These results imply a unique mode of interaction of IolR with the target DNA .

J Bacteriol, 1999 Jan, 181(2), 685 - 8
Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein; Bengtsson J et al.; The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein . CtaC is the subunit II of cytochrome caa3, which is a cytochrome c oxidase . Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at the N-terminal part of the protein . Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochrome caa3 enzyme activity . Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.

J Bacteriol, 1999 Jan, 181(2), 600 - 9
Organized cell swimming motions in Bacillus subtilis colonies: patterns of short-lived whirls and jets; Mendelson NH et al.; The swimming motions of cells within Bacillus subtilis colonies, as well as the associated fluid flows, were analyzed from video films produced during colony growth and expansion on wet agar surfaces . Individual cells in very wet dense populations moved at rates between 76 and 116 microm/s . Swimming cells were organized into patterns of whirls, each approximately 1,000 microm2, and jets of about 95 by 12 microm . Whirls and jets were short-lived, lasting only about 0.25 s . Patterns within given areas constantly repeated with a periodicity of approximately 1 s . Whirls of a given direction became disorganized and then re-formed, usually into whirls moving in the opposite direction . Pattern elements were also organized with respect to one another in the colony . Neighboring whirls usually turned in opposite directions . This correlation decreased as a function of distance between whirls . Fluid flows associated with whirls and jets were measured by observing the movement of marker latex spheres added to colonies . The average velocity of markers traveling in whirls was 19 microm/s, whereas those traveling in jets moved at 27 microm/s . The paths followed by markers were aligned with the direction of cell motion, suggesting that cells create flows moving with them into whirls and along jets . When colonies became dry, swimming motions ceased except in regions close to the periphery and in isolated islands where cells traveled in slow whirls at about 4 microm/s . The addition of water resulted in immediate though transient rapid swimming (> 80 microm/s) in characteristic whirl and jet patterns . The rate of swimming decreased to 13 microm/s within 2 min, however, as the water diffused into the agar . Organized swimming patterns were nevertheless preserved throughout this period . These findings show that cell swimming in colonies is highly organized.

J Bacteriol, 1999 Jan, 181(2), 501 - 7
CtaA of Staphylococcus aureus is required for starvation survival, recovery, and cytochrome biosynthesis; Clements MO et al.; A Staphylococcus aureus mutant (SPW3) apparently unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to ctaA of Bacillus subtilis which encodes a heme A synthase . Analysis of the cytochrome profiles of SPW3 revealed the absence of heme A-containing cytochromes compared to the parental 8325-4 strain . SPW3 demonstrated a 100-fold reduction in the ability to survive starvation induced by glucose limitation, under aerated conditions, compared to 8325-4 . Analysis of starved cultures revealed that greater than 90% of the cells which demonstrated metabolism (as shown by rhodamine 123 accumulation) were unable to recover and form colonies on agar . Analysis of the lag phase and initial growth kinetics of those cells which could recover also showed a defect . This recovery defect could be partially alleviated by the inclusion of catalase in the recovery medium, indicating the probable involvement of oxidative stress . SPW3 also exhibited reduced colony size similar to that of a small-colony variant, increased resistance to aminoglycoside antibiotics, and reduced hemolysin and toxic shock syndrome toxin 1 production, but no alteration in the ability to form lesions in a subcutaneous mouse infection model.

J Bacteriol, 1999 Jan, 181(2), 493 - 500
Temporal expression of the Bacillus subtilis secA gene, encoding a central component of the preprotein translocase; Herbort M et al.; In Bacillus subtilis, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase . It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity . In this study, we analyzed the transcriptional organization and temporal expression of the secA gene, encoding a central component of the B . subtilis preprotein translocase . We found that secA and the downstream gene (prfB) constitute an operon that is transcribed from a vegetative (sigmaA-dependent) promoter located upstream of secA . Furthermore, using different independent methods, we found that secA expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase . Following to this maximum, the de novo transcription of secA sharply decreased to a low basal level . Since at the time of maximal secA transcription the secretion activity of B . subtilis strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the B . subtilis protein export apparatus is adapted to the increased demand for protein secretion . Possible mechanistic consequences are discussed.

J Bacteriol, 1999 Jan, 181(2), 418 - 25
Cell wall teichoic acid glycosylation in Listeria monocytogenes serotype 4b requires gtcA, a novel, serogroup-specific gene; Promadej N et al.; We have identified a novel gene, gtcA, involved in the decoration of cell wall teichoic acid of Listeria monocytogenes serotype 4b with galactose and glucose . Insertional inactivation of gtcA brought about loss of reactivity with the serotype 4b-specific monoclonal antibody c74.22 and was accompanied by a complete lack of galactose and a marked reduction in the amounts of glucose on teichoic acid . Interestingly, the composition of membrane-associated lipoteichoic acid was not affected . Complementation of the mutants with the cloned gtcA in trans restored galactose and glucose on teichoic acid to wild-type levels . The complemented strains also recovered reactivity with c74.22 . Within L . monocytogenes, sequences homologous to gtcA were found in all serogroup 4 isolates but not in strains of any other serotypes . In serotype 4b, gtcA appears to be the first member of a bicistronic operon which includes a gene with homology to Bacillus subtilis rpmE, encoding ribosomal protein L31 . In contrast to gtcA, the latter gene appears conserved among all screened serotypes of L . monocytogenes.

J Bacteriol, 1999 Jan, 181(2), 411 - 7
Differential stabilities of phosphorylated response regulator domains reflect functional roles of the yeast osmoregulatory SLN1 and SSK1 proteins; Janiak-Spens F et al.; Osmoregulation in Saccharomyces cerevisiae involves a multistep phosphorelay system requiring three proteins, SLN1, YPD1, and SSK1, that are related to bacterial two-component signaling proteins, in particular, those involved in regulating sporulation in Bacillus subtilis and anaerobic respiration in Escherichia coli . The SLN1-YPD1-SSK1 phosphorelay regulates a downstream mitogen-activated protein kinase cascade which ultimately controls the concentration of glycerol within the cell under hyperosmotic stress conditions . The C-terminal response regulator domains of SLN1 and SSK1 and full-length YPD1 have been overexpressed and purified from E . coli . A heterologous system consisting of acetyl phosphate, the bacterial chemotaxis response regulator CheY, and YPD1 has been developed as an efficient means of phosphorylating SLN1 and SSK1 in vitro . The homologous regulatory domains of SLN1 and SSK1 exhibit remarkably different phosphorylated half-lives, a finding that provides insight into the distinct roles that these phosphorylation-dependent regulatory domains play in the yeast osmosensory signal transduction pathway.

Arch Biochem Biophys, 1999 Jan 15, 361(2), 231 - 40
Activity and cellular location in Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus subtilis/yeast ferrochelatases; Gora M et al.; We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilis ferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane . These genes were expressed in a Saccharomyces cerevisiae mutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized . Exchanging the approximately 40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in Escherichia coli . The water-soluble ferrochelatase of B . subtilis did not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol . Addition of the N-terminal leader sequence of yeast ferrochelatase to the B . subtilis enzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactive in vivo and had residual activity when measured in vitro . An internal approximately 45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of the B . subtilis enzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein . The fusion protein was inactive in vivo and had residual activity in vitro . We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane .

Anal Biochem, 1998 Dec 15, 265(2), 351 - 5
Predictable deuteration of recombinant proteins expressed in Escherichia coli; Leiting B et al.; Random deuteration of recombinant proteins in Escherichia coli is widely used for protein structure determination by nuclear magnetic resonance (NMR) . It is desirable to predict accurately the degree of deuteration because each NMR experiment benefits from a different level of deuteration . The method described here which uses {2H}2O and glucose as the sole carbon and deuterium sources is an alternative for a previously published procedure using acetate and {2H}2O (Venter et al., J . Biomol . NMR 5, 339-344, 1995) and it is of advantage for proteins that do not express well using acetate . While the deuteration degree with acetate is approximately linear with the {2H}2O content in the medium, the use of glucose leads to deviations up to 19%, which is analyzed systematically here . With {2H}2O as the sole deuterium source 0-86% of the chemically nonexchangeable hydrogen atoms can be deuterated . Higher levels of deuteration require perdeuterated glucose in combination with {2H}2O . As an example, recombinant peptide deformylase from Bacillus subtilis was overexpressed, deuterated to various degrees, purified, and analyzed by mass spectrometry and NMR .

J Biochem (Tokyo), 1999 Jan, 125(1), 151 - 9
Enhancing effect of Bacillus subtilis Ffh, a homologue of the SRP54 subunit of the mammalian signal recognition particle, on the binding of SecA to precursors of secretory proteins in vitro; Bunai K et al.; The precursors of beta-lactamase fusion proteins having the signal peptide of Bacillus subtilis alkaline protease (pAprE-BlaH6) or penicillin binding protein 5(*) (pPBP5(*)-BlaH6) accumulated in B . subtilis cells in the absence of SecA or Ffh . Using the five purified precursors of secretory proteins including the two fusion proteins, B . subtilis Ffh and SecA, we analyzed the protein targeting mechanism of B . subtilis in vitro . B . subtilis SecA recognized the completely translated precursors of secretory proteins to which Ffh also bound . Moreover, B . subtilis SecA-precursor complex formation was enhanced 15-to 30-fold when the precursor and Ffh were incubated first and then SecA was added, but not vice versa . We also found that B . subtilis SecA directly interacted with Ffh in vitro . These results indicate that B . subtilis SecA and Ffh interact to function cooperatively in a protein translocation pathway including other protein factors, and that Ffh, as well as SecB in Escherichia coli, enhances the binding of SecA to presecretory proteins in B . subtilis cells.

Biochim Biophys Acta, 1999 Jan 5, 1409(3), 171 - 5
Analyses of a Bacillus subtilis homologue of the Na+/H+ antiporter gene which is important for pH homeostasis of alkaliphilic Bacillus sp . C-125; Kosono S et al.; Bacillus subtilis was revealed to have a homologous region to the DNA fragment responsible for alkaliphily of alkaliphilic Bacillus sp . C-125 on the genome, as reported previously {1} . The yufT gene on the B . subtilis genome showed a significant similarity with ORF1 of Bacillus sp . C-125, which is related to membrane potential (DeltaPsi)-driven Na+/H+ antiport activity and is important for pH homeostasis in an alkaline condition . Disruption of the yufT gene resulted in the decrease of Na+/H+ antiport activity, and the growth of the yufT disrupted strain was impaired with an increase in the external Na+ concentration . We conclude that the yufT gene encodes a Na+/H+ antiporter, which has a dominant role in the extrusion of cytotoxic Na+.

Curr Microbiol, 1999 Feb, 38(2), 107 - 12
Sequence analysis of small cryptic plasmids isolated from Selenomonas ruminantium S20; Nakamura M et al.; Two small cryptic plasmids designated pONE429 and pONE430 were isolated from a rumen bacterium, Selenomonas ruminantium S20 . The complete sequence of pONE429 was 2100 bp and contained one open reading frame (ORF) of 201 amino acids . The sequence of pONE430 had 1527 bp and one ORF of 171 amino acids with the similarity of replication protein (Rep protein) of pOM1, pSN2, and pIM13 isolated from Butyrivibrio fibrisolvens, Staphylococcus aureus, and Bacillus subtilis, respectively . In these plasmids, the upstream nucleotide sequence of Rep protein had the conserved nucleotides which could be double-strand origin (DSO) of rolling circle replication (RCR) mechanism . The plasmids of pONE429, pONE430, pJJMI, pJDB21, and pS23 were isolated from S . ruminantium strains and had similar regions that were located within a <450-bp nucleotide . These similar regions may be the location that was recognized by the host strain, S . ruminantium.

FEMS Microbiol Lett, 1998 Dec 15, 169(2), 341 - 7
Characterization of a large motility gene cluster containing the cheR, motAB genes of Listeria monocytogenes and evidence that PrfA downregulates motility genes; Michel E et al.; Through the analysis of a non-motile mutant of Listeria monocytogenes, we identified and characterized a locus containing the cheR, motA and motB genes . These three genes are homologous to the cheR, and motA/B genes of Bacillus subtilis which in this organism are 954 kb apart . The gene organization in Listeria is also not similar either to that of Escherichia coli in which cheR and motAB are 5.9 kb apart . CheR and motA/B, as previously reported for flaA, the flagellin gene, are thermoregulated with a higher expression at 25 degrees C and low expression at 37 degrees C . In a delta prfA strain, motA expression was derepressed at 37 degrees C, suggesting that PrfA, the transcriptional activator of virulence genes, downregulates motility genes in Listeria at 37 degrees C.

J Biotechnol, 1998 Dec 11, 66(2-3), 157 - 63
Quantitative assay system for specific enzyme activity using antibody: the case of protease, subtilisin BPN'; Miyota Y et al.; Assay on a high-quality microtiter plate was found to allow for quantitative analysis of bacterial serine protease, subtilisin BPN', and its mutant enzymes which had been genetically engineered to be adapted to low-temperatures (Taguchi et al., 1998 . Appl . Environ . Microbiol . 64, 492-495), by using polyclonal antibody against subtilisin BPN' . The use of polyclonal antibody was crucial in normalizing the number of various different enzyme molecules in culture supernatant samples of recombinant strains of Bacillus subtilis, giving rise to the performance of specific activity assay of the enzymes . Relative activity of each mutant subtilisin BPN' to wild-type enzyme was estimated by monitoring the increased value of absorbance caused by enzymatic hydrolysis of a chromogenic substrate . The relative activity in each enzyme estimated by this method showed good coincidence with that estimated by kinetic parameters, kcat/K(m) of the purified enzymes . We termed the system as 'ABEA' (antibody-bound enzyme assay) . The efficient ABEA system developed here would be useful for the determination of specific activity of other enzymes of interest and provide us versatile applications in the field of evolutionary engineering.

J Bacteriol, 1999 Jan, 181(1), 353 - 6
A novel Bacillus subtilis gene, antE, temporally regulated and convergent to and overlapping dnaE; Wang LF et al.; A Bacillus subtilis promoter, Px, that functions in a convergent manner with the sigA operon promoter P3 has been found in the sigA operon . Promoter Px is turned on at the same time as promoter P3 during early sporulation . The transcript from promoter Px codes for a small protein with partial homology to the OmpR protein from Escherichia coli and also carries an untranslated sequence at its 3' end that is complementary to the 5' end of the P3 transcript, which codes for the ribosome binding site of dnaE . The gene controlled by Px has been called antE . The expression of antE does not require sigmaB, sigmaE, or sigmaH . Px was transcribed in vitro by the sigmaA holoenzyme and is the seventh promoter to be recognized in the sigmaA operon . A possible role for the antE gene during early sporulation is proposed.

J Bacteriol, 1999 Jan, 181(1), 204 - 11
Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2); Paget MS et al.; The sigE gene of Streptomyces coelicolor A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily . Constructed sigE deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from Bacillus subtilis . This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan . The sigE mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence . Together, these data suggest that sigE is required for normal cell wall structure . The role of sigmaE was further investigated by analyzing the expression of hrdD, which is partially sigE dependent . The hrdD gene, which encodes the sigmaHrdD subunit of RNA polymerase, is transcribed from two promoters, hrdDp1 and hrdDp2, both similar to promoters recognized by other ECF sigma factors . The activities of hrdDp1 and hrdDp2 were reduced 20- and 3-fold, respectively, in sigE mutants, although only hrdDp1 was recognized by EsigmaE in vitro . Growth on media deficient in magnesium caused the induction of both hrdD promoters in a sigE-dependent manner.

J Bacteriol, 1999 Jan, 181(1), 126 - 32
Roles of low-molecular-weight penicillin-binding proteins in Bacillus subtilis spore peptidoglycan synthesis and spore properties; Popham DL et al.; The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration . A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by D,D-carboxypeptidases . We report here the construction of mutant B . subtilis strains lacking all combinations of two and three of the four apparent D, D-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains . The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan . The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance . A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.

Endod Dent Traumatol, 1998 Jun, 14(3), 124 - 6
Effectiveness of four chemical solutions in eliminating Bacillus subtilis spores on gutta-percha cones; Siqueira JF Jr et al.; Gutta-percha cones should be free of pathogenic micro-organisms before being used for root canal filling . This study was carried out to evaluate the effectiveness of four chemical agents in eliminating Bacillus subtilis spores from gutta-percha cones . The solutions tested were 5.25% sodium hypochlorite, 2% glutaraldehyde, 2% chlorhexidine digluconate, and 70% ethyl alcohol . The gutta-percha cones coated with spores were placed into contact with the chemical agents for 1, 3, 5 and 10 min . The results showed that 5.25% sodium hypochlorite was effective in destroying the spores after 1 min of contact . Glutaraldehyde, chlorhexidine and ethyl alcohol did not decontaminate the gutta-percha cones even after 10 min of contact.

FEMS Microbiol Rev, 1998 Oct, 22(4), 305 - 22
Hydropathy profile alignment: a tool to search for structural homologues of membrane proteins; Lolkema JS et al.; Hydropathy profile alignment is introduced as a tool in functional genomics . The architecture of membrane proteins is reflected in the hydropathy profile of the amino acid sequence . Both secondary and tertiary structural elements determine the profile which provides enough sensitivity to detect evolutionary links between membrane proteins that are based on structural rather than sequence similarities . Since structure is better conserved than amino acid sequence, the hydropathy profile can detect more distant evolutionary relationships than can be detected by the primary structure . The technique is demonstrated by two approaches in the analysis of a subset of membrane proteins coded on the Escherichia coli and Bacillus subtilis genomes . The subset includes secondary transporters of the 12 helix type . In the first approach, the hydropathy profiles of proteins for which no function is known are aligned with the profiles of all other proteins in the subset to search for structural paralogues with known function . In the second approach, family hydropathy profiles of 8 defined families of secondary transporters that fall into 4 different structural classes (SC-ST1-4) are used to screen the membrane protein set for members of the structural classes . The analysis reveals that over 100 membrane proteins on each genome fall in only two structural classes . The largest structural class, SC-ST1, correlates largely with the Major Facilitator Superfamily defined before, but the number of families within the class has increased up to 57 . The second large structural class, SC-ST2 contains secondary transporters for amino acids and amines and consists of 12 families.

FEMS Microbiol Rev, 1998 Oct, 22(4), 207 - 27
Indigo: a World-Wide-Web review of genomes and gene functions; Nitschke P et al.; The present article describes a genome database reviewing gene-related knowledge of two model bacteria, Bacillus subtilis and Escherichia coli . The database, Indigo, is open through the World-Wide Web . The concept used for organising the data, the concept of neighbourhood, allows one to explore the database content in an efficient although somewhat unusual way . Here, genes are related to each other by a variety of neighbourhoods, including proximity in the chromosome, phylogenetic kinship, participation in a common metabolic pathway, common presence in an article of the literature, or similar use of the genetic code . Several examples illustrate how this concept of neighbourhood permits one to review the available knowledge about a given gene or gene family, and elaborate unexpected, but revealing, analyses about gene functions.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15212 - 7
Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA; Niranjanakumari S et al.; The protein component of ribonuclease P (RNase P) binds to the RNA subunit, forming a functional ribonucleoprotein complex in vivo and enhancing the affinity of the precursor tRNA (pre-tRNA) substrate . Photocrosslinking experiments with pre-tRNA bound to RNase P reconstituted with the protein component of Bacillus subtilis ribonuclease P (P protein) site specifically modified with a crosslinking reagent indicate that: (i) the central cleft of P protein directly interacts with the single-stranded 5' leader sequence of pre-tRNA, and (ii) the orientation and register of the pre-tRNA leader sequence in the central cleft places the protein component in close proximity to the active site . This unique mode of interaction suggests that the catalytic active site in RNase P occurs near the interface of RNA and protein . In contrast to other ribonucleoprotein complexes where the protein mainly stabilizes the active tertiary fold of the RNA, a critical function of the protein component of RNase P is to alter substrate specificity and enhance catalytic efficiency.

Biochemistry, 1998 Dec 15, 37(50), 17618 - 28
Identification of individual nucleotides in the bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-linking; Christian EL et al.; The bacterial RNase P ribozyme is a site-specific endonuclease that catalyzes the removal of pre-tRNA leader sequences to form the 5' end of mature tRNA . While several specific interactions between enzyme and substrate that direct this process have been determined, nucleotides on the ribozyme that interact directly with functional groups at the cleavage site are not well-defined . To identify individual nucleotides in the ribozyme that are in close proximity to the pre-tRNA cleavage site, we introduced the short-range photoaffinity cross-linking reagent 6-thioguanosine (s6G) at position +1 of tRNA and position -1 in a tRNA bearing a one-nucleotide leader sequence {tRNA(G-1)} and examined cross-linking in representatives of the two structural classes of bacterial RNase P RNA (from Escherichia coli and Bacillus subtilis) . These photoagent-modified tRNAs bind with similar high affinity to both ribozymes, and the substrate bearing a single s6G upstream of the cleavage (-1) site is cleaved accurately . Interestingly, s6G at position +1 of tRNA cross-links with high efficiency to homologous positions in J5/15 in both E . coli and B . subtilis RNase P RNAs, while s6G at position -1 of tRNA(G-1) cross-links to homologous nucleotides in J18/2 . Both cross-links are detected over a range of ribozyme and substrate concentrations, and importantly, ribozymes cross-linked to position -1 of tRNA(G-1) accurately cleave the covalently attached substrate . These data indicate that the conserved guanosine at the 5' end of tRNA is adjacent to A248 (E . coli) of J5/15, while the base upstream of the substrate phosphate is adjacent to G332 (E . coli) of J18/2 and, along with available biochemical data, suggest that these nucleotides play a direct role in binding the substrate at the cleavage site.

Mikrobiol Z, 1998 Jul-Aug, 60(4), 25 - 32
{The effect of the acidity of the medium and of the temperature on the growth and polysaccharide excretion of Bacillus subtilis under submerged cultivation}; Osadchaia AI et al.; Growth of Bacillus subtilis strains and excretion of polysaccharides have been studied under different environmental condition . These strains were studied as active antagonists of pathogenic microflora and were used for development of the biopreparation (probiotic), being efficient in treatment and prophylaxis of a number of bacterial infections of farm animals; the drug soon will be put into series production . The indices of accumulation of biomass of viable cells and exopolysaccharides (EPS) were used to study productivity of the strains . A possibility to increase yield in the production process was shown using the temperature factor and optimal pH of the culture medium under batch cultivation . The processes of the biomass and EPS accumulation were noncompetitive, almost coincided in time and were independent of the initial level of the pH of medium.

Gene, 1998 Nov 26, 223(1-2), 135 - 42
Bacteriophage phi29 early protein p17 is conditionally required for the first rounds of viral DNA replication; Crucitti P et al.; The gene 17 of the Bacillus subtilis phage phi29 is known to be involved in the viral DNA replication in vivo . In this paper, we show that the presence of protein p17 is required when phage infection occurs at a low multiplicity of infection (moi), which is probably the natural condition for infection, but is dispensable at a high moi . Gene 17 has been cloned in an Escherichia coli expression vector and protein p17 purified . A stimulatory effect of protein p17 was demonstrated under in vitro conditions required to amplify phi29 DNA, starting with a low amount of input DNA . We propose that p17, which is synthesized early after infection, is required at the very beginning of the phage amplification, conditions in which a low number of viral DNA molecules enter the host cell, possibly to recruit the limiting amount of initiation factors at the replication origins . Once the infection process is established and the other replication proteins reach optimal concentration, p17 becomes dispensable.

J Bacteriol, 1998 Dec, 180(24), 6649 - 54
Expression of the Bacillus subtilis acsA gene: position and sequence context affect cre-mediated carbon catabolite repression; Zalieckas JM et al.; In Bacillus subtilis, carbon catabolite repression (CCR) of many genes is mediated at cis-acting carbon repression elements (cre) by the catabolite repressor protein CcpA . Mutations in transcription-repair coupling factor (mfd) partially relieve CCR at cre sites located downstream of transcriptional start sites by abolishing the Mfd-mediated displacement of RNA polymerase stalled at cre sites which act as transcriptional roadblocks . Although the acsA cre is centered 44.5 bp downstream of the acsA transcriptional start site, CCR of acsA expression is not affected by an mfd mutation . When the acsA cre is centered 161.5 bp downstream of the transcriptional start site for the unregulated tms promoter, CCR is partially relieved by the mfd mutation . Since CCR mediated at an acsA cre centered 44.5 bp downstream of the tms start site is not affected by the mfd mutation, the inability of Mfd to modulate CCR of acsA expression most likely results from the location of the acsA cre . Higher levels of CCR were found to occur at cre sites flanked by A+T-rich sequences than at cre sites bordered by G and C nucleotides . This suggests that nucleotides adjacent to the proposed 14-bp cre consensus sequence participate in the formation of the CcpA catabolite repression complex at cre sites . Examination of CCR of acsA expression revealed that this regulation required the Crh and seryl-phosphorylated form of the HPr proteins but not glucose kinase.

J Pept Sci, 1998 Nov, 4(7), 449 - 58
Tuning micelles of a bioactive heptapeptide biosurfactant via extrinsically induced conformational transition of surfactin assembly; Osman M et al.; We have studied the effects of extrinsic environmental conditions on the conformation of surfactin, a heptapeptide biosurfactant from Bacillus subtilis, in aqueous solutions . It has been made clear that temperature, pH, Ca2+ ions and the synthetic nonionic surfactant hepta-ethylene glycol (C12E7) affect the conformation of surfactin in aqueous solutions . The beta-sheet formation reached a maximum at 40 degrees C both in presence and absence of (C12E7) and the nonionic surfactant enhances the beta-sheet formation even at 25 degrees C . Ca2 + induced the formation of alpha-helices and caused this transition at 0.3 mM with surfactin monomers or at 0.5 mM with surfactin micelles, but above these transition concentrations of Ca2+ beta-sheets were observed . In micellar solution the beta-sheet structure was stabilized at pH values below 7 or upon addition of Ca2+ in concentrations above 0.5 mM . Our results indicated that the bioactive conformation of surfactin is most likely the beta-sheets when the molecules are assembled in micelles . The beta-sheet structure in micelles could be retained by tuning the micelles . Surfactin micelles could be tuned in the bioactive conformation by manipulating pH, temperature, Ca2+ or (C12E7) concentrations in surfactin solutions . Our results strongly indicated that Ca2+ and other molecules (such as C12E7) may function as directing templates in the assembly and conformation of surfactin in micelles . Thus, we suggest environmental manipulation and template-aided micellation (TAM) as a new approach for preparing predesigned micelles, microemulsions or micro-spheres for specific application purposes.

J Bacteriol, 1998 Dec, 180(24), 6729 - 35
Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants; Clements MO et al.; Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to L-alanine, but the most rapid germination response is elicited by a combination of these germinants . Mutants defective in their germination response to either inosine or to L-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to L-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to L-alanine . These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination . Stimulation of germination in L-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of L-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci . Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome . This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis . The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC . The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.

J Bacteriol, 1998 Dec, 180(24), 6704 - 12
New small, acid-soluble proteins unique to spores of Bacillus subtilis: identification of the coding genes and regulation and function of two of these genes; Bagyan I et al.; Eleven small, acid-soluble proteins (SASP) which are present in spores but not in growing cells of Bacillus subtilis were identified by sequence analysis of proteins separated by acrylamide gel electrophoresis of acid extracts from spores which lack the three major SASP (alpha, beta, and gamma) . Six of these proteins are encoded by open reading frames identified previously or by analysis of the complete sequence of the B . subtilis genome, including two minor alpha/beta-type SASP (SspC and SspD) and a putative spore coat protein (CotK) . Five proteins are encoded by short open reading frames that were not identified as coding regions in the analysis of the complete B . subtilis genomic sequence . Studies of the regulation of two of the latter genes, termed sspG and sspJ, showed that both are expressed only in sporulation . The sspG gene is transcribed in the mother cell compartment by RNA polymerase with the mother cell-specific sigma factor for RNA polymerase, sigmaK, and is cotranscribed with a downstream gene, yurS; sspG transcription also requires the DNA binding protein GerE . In contrast, sspJ is transcribed in the forespore compartment by RNA polymerase with the forespore-specific sigmaG and appears to give a monocistronic transcript . A mutation eliminating SspG had no effect on sporulation or spore properties, while loss of SspJ caused a slight decrease in the rate of spore outgrowth in an otherwise wild-type background.

J Bacteriol, 1998 Dec, 180(24), 6681 - 8
The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes; Kruger E et al.; The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter . When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction . This result strongly indicated negative regulation of the clpC operon by one of its gene products . Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif . Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions . Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter . Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion . Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro . Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.

J Bacteriol, 1998 Dec, 180(24), 6674 - 80
The yvyD gene of Bacillus subtilis is under dual control of sigmaB and sigmaH; Drzewiecki K et al.; During a search by computer-aided inspection of two-dimensional (2D) protein gels for sigmaB-dependent general stress proteins exhibiting atypical induction profiles, a protein initially called Hst23 was identified as a product of the yvyD gene of Bacillus subtilis . In addition to the typical sigmaB-dependent, stress- and starvation-inducible pattern, yvyD is also induced in response to amino acid depletion . By primer extension of RNA isolated from the wild-type strain and appropriate mutants carrying mutations in the sigB and/or spo0H gene, two promoters were mapped upstream of the yvyD gene . The sigmaB-dependent promoter drives expression of yvyD under stress conditions and after glucose starvation, whereas a sigmaH-dependent promoter is responsible for yvyD transcription following amino acid limitation . Analysis of Northern blots revealed that yvyD is transcribed monocistronically and confirmed the conclusions drawn from the primer extension experiments . The analysis of the protein synthesis pattern in amino acid-starved wild-type and relA mutant cells showed that the YvyD protein is not synthesized in the relA mutant background . It was concluded that the stringent response plays a role in the activation of sigmaH . The yvyD gene product is homologous to a protein which might modify the activity of sigma54 in gram-negative bacteria . The expression of a sigmaL-dependent (sigmaL is the equivalent of sigma54 in B . subtilis) levD-lacZ fusion is upregulated twofold in a yvyD mutant . This indicates that the yvyD gene product, being a member of both the sigmaB and sigmaH regulons, might negatively regulate the activity of the sigmaL regulon . We conclude that (i) systematic, computer-aided analysis of 2D protein gels is appropriate for the identification of genes regulated by multiple transcription factors and that (ii) YvyD might form a junction between the sigmaB and sigmaH regulons on one side and the sigmaL regulon on the other.

J Bacteriol, 1998 Dec, 180(24), 6571 - 80
Cytochrome bd biosynthesis in Bacillus subtilis: characterization of the cydABCD operon; Winstedt L et al.; Under aerobic conditions Bacillus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases . At present there is evidence for three types of terminal oxidases in B . subtilis: a caa3-, an aa3-, and a bd-type oxidase . We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex . Downstream of the structural genes, cydC and cydD are located . These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters . Analysis of isolated cell membranes showed that inactivation of cydA or deletion of cydABCD resulted in the loss of spectral features associated with cytochrome bd . Gene disruption experiments and complementation analysis showed that the cydC and cydD gene products are required for the expression of a functional cytochrome bd complex . Disruption of the cyd genes had no apparent effect on the growth of cells in broth or defined media . The expression of the cydABCD operon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions . Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message . The operon was found to be expressed maximally under conditions of low oxygen tension.

J Bacteriol, 1998 Dec, 180(24), 6493 - 502
Analysis of outgrowth of Bacillus subtilis spores lacking penicillin-binding protein 2a; Murray T et al.; The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore . Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.

FEMS Microbiol Lett, 1998 Dec 1, 169(1), 165 - 70
The leader sequence of the Escherichia coli lysC gene is involved in the regulation of LysC synthesis; Patte JC et al.; In Escherichia coli and Bacillus subtilis, long leader sequences are found upstream of the lysC coding sequences which encode lysine-sensitive aspartokinase . Highly conserved regions exist between these sequences . Mutations leading to constitutive expression of the E . coli lysC gene have been localised within these conserved regions, indicating that they participate in the lysine-mediated repression mechanism of lysC expression.

FEBS Lett, 1998 Nov 13, 439(1-2), 59 - 62
Bacillus subtilis DnaG primase stabilises the bacteriophage SPP1 G40P helicase-ssDNA complex; Ayora S et al.; Purified Bacillus subtilis DnaG primase (predicted molecular mass 68.8 kDa) behaves as a monomer in solution . We demonstrate that DnaG physically interacts with bacteriophage SPP1 hexameric helicase G40P (G40P6) in the absence of ATP . G40P6-ATP forms an unstable complex with ssDNA, and by itself carries out ATP-driven translocation along a ssDNA template with low processivity . The presence of DnaG in the reaction mixture increased the helicase activity of G40P6 about 3-fold, but not the ATPase activity . The results presented here suggest that the DnaG protein stabilises the G40P6-ssDNA complexes.

Indian J Ophthalmol, 1998 Jun, 46(2), 97 - 101
Microbiological assay of ampicillin in serum and aqueous humor of patients given ampicillin-sulbactam injection; Madhavan HN et al.; The aim of this study was to determine the bacterial growth inhibitory activities of ampicillin in aqueous humor and serum of patients administered ampicillin-sulbactam combination intramuscularly prior to cataract surgery . 43 patients received a combination of both antibiotics intramuscularly at varying periods (60-140 minutes) prior to surgery . Aqueous humor and venous blood were collected at the beginning of the surgery . For microbiological assay, spores of Bacillus subtilis were incorporated in the agar . The test sample and the standard solutions (calibrators) of ampicillin and ampicillin-sulbactam combination were placed in 3 mm wells in the agar . The diameter zones of growth inhibitory activities of ampicillin of the calibrators and the test samples measured in mm were extrapolated to the standard curve and were recorded as ampicillin activity in micrograms/ml . The results of the assay were placed in 5 groups according to the time intervals between injection and collection of serum and aqueous humor (< or = 70, 75, 80, 90, > 90 minutes) . Ampicillin activities in sera and aqueous humor of group 5 (> 90 minutes) were significantly higher than the others (p < 0.001) . The ratio of ampicillin activities of sera and aqueous humor in group 5 patients was significantly lower indicating higher concentration of ampicillin activity in aqueous humor during this period . Bacterial growth inhibitory activities of ampicillin-sulbactam combination were adequate in aqueous humor of all patients with highest activity being 90 minutes after intramuscular administration indicating the potential usefulness of this antibiotic combination as chemoprophylaxis prior to cataract surgery.

J Colloid Interface Sci, 1998 Aug 1, 204(1), 1 - 8
Separation and Characterization of Surfactin Isoforms Produced by Bacillus subtilis OKB 105
Kowall M, Vater J, Kluge B, Stein T, Franke P, Ziessow D.
Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a beta-hydroxy fatty acid with chain lengths of 13-15 carbon atoms . The lipopeptide biosurfactant was produced by Bacillus subtilis OKB 105 and part of the material subjected to esterification of its Glu and Asp residues . High-resolution preparative reversed phase HPLC on EnCaPharm 100 of surfactin and its monomethyl and dimethyl esters yielded 44 fractions which were characterized by NMR and MS methods . Among the separated isoforms are the known surfactin variants with l-Leu, l-Val, or l-Ile in position 7 of the peptide ring and three hitherto unknown variants showing replacements of the leucine residues in position 2 and/or 7 by l-Val and l-Ile . Our work makes available lipoheptapeptide compounds with modified structures and different hydrophobicities which promise to have potential for biotechnological and pharmaceutical applications .

Microbiology, 1998 Nov, 144 ( Pt 11), 3111 - 8
The kdgRKAT operon of Bacillus subtilis: detection of the transcript and regulation by the kdgR and ccpA genes; Pujic P et al.; Transcription of a new catabolic operon in Bacillus subtilis, involved in the late stages of galacturonic acid utilization, has been studied . The operon consists of four genes: kdgR, encoding the putative regulator protein; kdgK, encoding 2-keto-3-deoxygluconate kinase; kdgA, encoding 2-keto-3-deoxygluconate-6-phosphate aldolase; and kdgT, encoding a transporter . These four genes are organized in one transcriptional unit and map at 198 degrees of the B . subtilis chromosome . Primer extension experiments and Northern blot analysis show that an active sigmaA-dependent promoter precedes kdgR and transcription is terminated at the putative p-independent terminator downstream of kdgT . The operon is negatively regulated by the kdgR and ccpA gene products, which belong to the LacI family of transcription regulators . The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium . Knockout mutations in genes kdgR and ccpA remove, respectively, the effects of galacturonate and glucose on the transcription of this operon.

Microbiology, 1998 Nov, 144 ( Pt 11), 3105 - 9
The product of the yvoC (gerF) gene of Bacillus subtilis is required for spore germination; Robinson C et al.; All known gerF mutations affecting Bacillus subtilis spore germination have been mapped, by a combination of recombination and complementation analysis, to yvoC (Igt), a gene belonging to the yvoB (ptsK) yvoC (Igt) yvoDEF operon . Examination of the properties of null mutants confirmed that the only gene in the operon that affects germination is yvoC, which encodes a homologue of known prelipoprotein diacylglyceryl transferases . As several germination proteins (GerAC, GerBC, GerKC, GerD) are predicted lipoproteins, it is not unreasonable to assume that a defect in prelipoprotein processing will affect spore germination . Two other null mutants in this chromosomal region showed a clear phenotype: the nagA gene is required for growth on N-acetylglucosamine, whereas a null mutation in yvoB (ptsK) affects colony formation from single cells.

Microbiology, 1998 Nov, 144 ( Pt 11), 3097 - 104
A vector for systematic gene inactivation in Bacillus subtilis; Vagner V et al.; To study the functions of the uncharacterized open reading frames identified in the Bacillus subtilis genome, several vectors were constructed to perform insertional mutagenesis in the chromosome . All the pMUTIN plasmids carry a lacZ reporter gene and an inducible Pspac promoter, which is tightly regulated and can be induced about 1000-fold . The integration of a pMUTIN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lacZ becomes transcriptionally fused to the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion . This last feature is important because B . subtilis genes are often organized in operons . The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG . Also, conditional mutants of essential genes can be obtained by integrating pMUTIN vectors upstream of the target gene . The vectors are currently being used for systematic inactivation of genes without known function within the B . subtilis European consortium . pMUTIN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.

EMBO J, 1998 Dec 1, 17(23), 7139 - 48
ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer; Hirano M et al.; SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya . Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair . In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex . Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits . We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis . Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits . It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity . In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner . Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA . The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.

Biochemistry, 1998 Nov 24, 37(47), 16538 - 45
Characterization of interactions between a two-component response regulator, Spo0F, and its phosphatase, RapB; Tzeng YL et al.; The phosphorelay signal transduction pathway controls sporulation initiation in Bacillus subtilis . Transfer of a phosphoryl group from multiple kinases (KinA and KinB) through a single domain response regulator homologue (Spo0F), a phosphotransferase (Spo0B), and ultimately to a transcriptional regulator, (Spo0A) activates sporulation . Counteracting this response are phosphatases (RapA and RapB), which can short-circuit this phosphorelay via dephosphorylation of Spo0F . In vitro assays of RapB activity on phosphorylated Spo0F alanine-scanning mutants have been used to identify Spo0F residues critical for interactions between these proteins . The Spo0F surface comprised of the beta1-alpha1 loop and N-terminal half of helix alpha1 has the largest number of residues in which an alanine substitution leads to resistance or decreased sensitivity to RapB phosphatase activity . Other mutations desensitizing Spo0F to RapB are also located near the site of phosphorylation on the beta3-alpha3 and beta4-alpha4 loops . This surface is similar to but not the same as the surface identified for KinA and Spo0B interactions with Spo0F . Divalent metal ions were shown to be required for RapB activity, and this activity was insensitive to vanadate, suggesting that Rap phosphatases catalyze acyl phosphate hydrolysis by inducing conformational changes in phosphorylated Spo0F, which results in increased autodephosphorylation . Arginine 16 of Spo0F is proposed to play a role in catalysis, and similarities between the mechanisms for RapB catalyzed Spo0F approximately P hydrolysis and GAP (GTPase activating protein)-assisted GTP hydrolysis of Ras are discussed.

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 323 - 8
pH-dependent activation of the alternative transcriptional factor sigmaB in Bacillus subtilis; Kovacs T et al.; The expression of the sigB gene of Bacillus subtilis was analysed in response to a mild acid shock . This gene is subject to sigmaB-dependent regulation . It has been found that the expression of sigB is induced as part of the acid-tolerant response . In that respect sigB is similar to the previously described gene gsiB which is also a member of the sigmaB regulon . Through this induction, the sigmaB regulon provides protection against acid shock . Besides its protective role against acid shock, no other general function could be directly associated with the sigmaB regulon . An acidification of the cytoplasmic environment induces synthesis of general stress proteins in B . subtilis.

Biosens Bioelectron, 1998 Oct 15, 13(9), 931 - 8
Luminescent bacterial sensor for cadmium and lead; Tauriainen S et al.; A sensor plasmid was constructed by inserting the regulation unit from the cadA determinant of plasmid pI258 to control the expression of firefly luciferase . The resulting sensor plasmid pTOO24 is capable of replicating in Gram-positive and Gram-negative bacteria . The expression of the reporter gene as a function of added extracellular heavy metals was studied in Staphylococcus aureus strain RN4220 and Bacillus subtilis strain BR151 . Strain RN4220(pTOO24) mainly responded to cadmium, lead and antimony, the lowest detectable concentrations being 10 nM, 33 nM and 1 nM respectively . Strain BR151(pTOO24) responded to cadmium, antimony, zinc and tin at concentrations starting from 3.3 nM, 33 nM, 1 microM, and 100 microM, respectively . The luminescence ratios between induced and uninduced cells, the induction coefficients, of strains RN4220(pTOO24) and BR151(pTOO24) were 23-50 and about 5, respectively . These results were obtained with only 2-3 h incubation times . Freeze-drying of the sensor strains had only moderate effects on the performance with respect to sensitivity or induction coefficients.

Lett Appl Microbiol, 1998 Nov, 27(5), 292 - 6
Evaluation of the efficacy of glutaraldehyde and peroxygen for disinfection of dental instruments; Angelillo IF et al.; The antimicrobial and sporicidal activities in vitro and in vivo of 1% peroxygen (Virkon) and 2% alkaline glutaraldehyde (Asporin) were evaluated on dental instruments before and after cleaning . The in vitro antimicrobial activity against vegetative bacteria, bacterial spores and fungi indicated that glutaraldehyde is more active against these organisms than peroxygen . Asporin killed all vegetative bacteria within 1 min after cleaning, whereas Virkon was active, in the majority of cases, within 15 min and obtained a greater than 10(5)-fold reduction in count before killing for the vast majority of instruments, and for all micro-organisms . The spores of Bacillus subtilis were killed by Asporin within 4-5 h after cleaning, whereas Virkon required almost 20 h . A meticulous instrument cleaning process followed by an appropriate disinfection treatment assures a shorter disinfection time . Asporin should be recommended for chemical sterilization or high-level disinfection of dental instruments, and Virkon, if only disinfection is required, would seem to be a possible alternative, even if used with a higher exposure time.

J Appl Microbiol, 1998 Nov, 85(5), 849 - 54
Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment; Raso J et al.; The inactivation of Bacillus subtilis spores by ultrasonic treatments under static pressure (Mano-Sonication, MS) and a combined MS/heat treatment (Mano-Thermo-Sonication) was investigated . The sporicidal effect of MS treatments depended on static pressure, amplitude of ultrasonic waves and treatment temperature . At 70 degrees C, pressure increments up to 500 kPa caused progressively more inactivation . An MS treatment at 500 kPa and 117 microns of amplitude for 12 min inactivated approximately 99% of the B . subtilis spore population . Over 500 kPa, further increments in pressure did not increase the percentage of inactivation . In the range 90-150 microns, an exponential relationship was observed between the amplitude of ultrasonic waves under pressure and the number of survivors . While an MS treatment (20 kHz, 300 kPa, 70 degrees C, 12 min) at 90 microns inactivated 75% of the B . subtilis spore population, the same treatment at 150 microns inactivated 99.9% of this population . The MS treatments at temperatures higher than 70 degrees C (MTS) led to more spore inactivation . In the range 70-90 degrees C, the combination of heat with an MS treatment (20 kHz, 300 kPa, 117 microns, 6 min) had a synergistic effect on spore inactivation . The inactivating effect of ultrasound was due neither to titanium particles eroded from the sonication tip, nor to free radicals released during ultrasonic treatment . The MS treatments sensitized spores of B . subtilis to lysozyme.

J Bacteriol, 1998 Dec, 180(23), 6316 - 24
A homolog of CcpA mediates catabolite control in Listeria monocytogenes but not carbon source regulation of virulence genes; Behari J et al.; Readily utilizable sugars down-regulate virulence gene expression in Listeria monocytogenes, which has led to the proposal that this regulation may be an aspect of global catabolite regulation (CR) . We recently demonstrated that the metabolic enzyme alpha-glucosidase is under CR in L . monocytogenes . Here, we report the cloning and characterization from L . monocytogenes of an apparent ortholog of ccpA, which encodes an important mediator of CR in several low-G+C-content gram-positive bacteria . L . monocytogenes ccpA (ccpALm) is predicted to encode a 335-amino-acid protein with nearly 65% identity to the gene product of Bacillus subtilis ccpA (ccpABs) . Southern blot analysis with a probe derived from ccpALm revealed a single strongly hybridizing band and also a second band of much lower intensity, suggesting that there may be other closely related sequences in the L . monocytogenes chromosome, as is the case in B . subtilis . Disruption of ccpALm resulted in the inability of the mutant to grow on glucose-containing minimal medium or increase its growth rate in the presence of preferred sugars, and it completely eliminated CR of alpha-glucosidase activity in liquid medium . However, alpha-glucosidase activity was only partially relieved from CR on solid medium . These results suggest that ccpA is an important element of carbon source regulation in L . monocytogenes . Nevertheless, utilizable sugars still down-regulate the expression of hly, which encodes the virulence factor hemolysin, in a ccpALm mutant, indicating that CcpA is not involved in carbon source regulation of virulence genes.

J Bacteriol, 1998 Dec, 180(23), 6298 - 305
Role and regulation of Bacillus subtilis glutamate dehydrogenase genes; Belitsky BR et al.; The complete Bacillus subtilis genome contains two genes with the potential to encode glutamate dehydrogenase (GlutDH) enzymes . Mutations in these genes were constructed and characterized . The rocG gene proved to encode a major GlutDH whose synthesis was induced in media containing arginine or ornithine or, to a lesser degree, proline and was repressed by glucose . A rocG null mutant was impaired in utilization of arginine, ornithine, and proline as nitrogen or carbon sources . The gudB gene was expressed under all growth conditions tested but codes for a GlutDH that seemed to be intrinsically inactive . Spontaneous mutations in gudB that removed a 9-bp direct repeat within the wild-type gudB sequence activated the GudB protein and allowed more-efficient utilization of amino acids of the glutamate family.

J Bacteriol, 1998 Dec, 180(23), 6154 - 63
Catabolite regulation of the Bacillus subtilis ctaBCDEF gene cluster; Liu X et al.; Bacillus subtilis cytochrome c oxidase caa3 is encoded by the ctaCDEF genes at the ctaABCDEF locus, with the ctaBCDEF genes organized as an operon-like unit . A dyad symmetry sequence and a catabolite response element homolog can be recognized in the 240-bp intercistronic region between ctaB and ctaC . ctaB'-lacZ and ctaBCD'-lacZ transcriptional fusions integrated at the native locus were used to study catabolite effects on transcription of the ctaB and ctaCDEF genes . In Schaeffer's medium lacking glucose, ctaBCD'-lacZ was expressed at a very low level during the exponential phase, and expression increased about 30-fold 2 h after entry into the stationary phase . In the presence of 0.5% glucose, ctaBCD'-lacZ expression was totally repressed . In contrast to ctaBCD'-lacZ, ctaB'-lacZ was constitutively expressed regardless of carbon source . The ctaCDEF genes were separated from ctaB by insertion of plasmids carrying selectable markers in such a way that the ctaCDEF and ctaB transcription units remained intact . Enzymatic assays of caa3 with these constructs, showed that ctaCDEF was not expressed independently of ctaB . Also, when a 'ctaB-ctaC'-lacZ fusion (containing the ctaB-ctaC intercistronic region) was placed at a remote nonessential locus, beta-galactosidase activity could not be detected . The absence of a promoter in the ctaB-ctaC intercistronic space also was indicated by the inability to detect ctaC-specific transcripts with RNase protection assays, primer extension, and rapid amplification of 5' cDNA ends . Direct mRNA measurements showed that, in the presence of 0.5% glucose, ctaBCDEF transcripts terminated at the 3' end of the putative stem-loop structure and the distal portion was down-regulated . A possible mechanism for ctaCDEF gene regulation is suggested . Catabolite repression of ctaBCD'-lacZ was partly dependent on CcpA but was independent of HPr . The expression of ctaBCDEF also appears to require the strC, ctaA, and resD-resE gene products.

J Bacteriol, 1998 Dec, 180(23), 6077 - 81
Transcriptional analysis of the Staphylococcus aureus penicillin binding protein 2 gene; Pinho MG et al.; Sequencing of the vicinity of the staphylococcal pbp2 gene and transcriptional analysis by primer extension and promoter fusions were used to show that pbp2 is part of an operon that also includes a gene with high homology to prfA of Bacillus subtilis . Two distinct promoters were identified directing transcription of pbp2 either alone or together with prfA . It was recently reported that transposon inactivation of pbp2 causes a reduction in methicillin resistance, but complementation experiments were not fully successful . We now show that introduction of the intact pbp2 gene with its two newly identified promoters into the chromosome of the transposon mutant resulted in the full recovery of high-level methicillin resistance.

Mol Gen Genet, 1998 Oct, 260(1), 48 - 55
Molecular analysis of the interaction between the Bacillus subtilis trehalose repressor TreR and the tre operator; Burklen L et al.; The trehalose operon of Bacillus subtilis is subject to regulation by induction, mediated by the repressor TreR, and by carbon catabolite repression (CCR) . For in vitro investigations, TreR from B . subtilis was overproduced and purified . Its molecular mass, as estimated by SDS-PAGE, is 27 kDa . Size fractionation under native conditions yielded a size estimate of 56 kDa, indicating that TreR exists as a dimer in its native state . Analysis of its interaction with various DNA fragments shows that TreR is able to recognize two tre operators with different efficiencies, and indicates cooperative binding . Previous results have suggested that CCR of the tre operon occurs by a mechanism in which the specific regulator, TreR, may be involved independently of the central component, CcpA . The data presented here indicate that the TreR-tre operator interaction is influenced by several effectors . Thus, the presence of trehalose-6-phosphate, as well as glucose-1-phosphate and sodium chloride, inhibits tre operator binding . Glucose-6-phosphate can act as an anti-inducer, which might reflect its additional role in CCR exerted by glucose.

Food Addit Contam, 1998 Jul, 15(5), 528 - 34
Optimization of an antibiotic residue screening test, based on inhibition of Bacillus subtilis BGA, with experimental design; Koenen-Dierick K et al.; Experimental design was applied to an agar-diffusion inhibition test for the screening of residues of veterinary antimicrobial drugs in slaughter animals . The effects and interactions of four independent parameters were studied: the dextrose content of the culture medium, the trimethoprim concentration in the culture medium, the thickness of the agar-layer and the preincubation time . The effects on the inhibition zones of sulphadimidine, oxytetracycline, streptomycin and tylosin, substances of four different antibiotic groups were measured . An optimized multi-residue test was established with the parameter values that yielded the best sensitivity for the four substances.

FEBS Lett, 1998 Nov 6, 438(3), 263 - 6
Purification and characterization of a novel glycine oxidase from Bacillus subtilis; Nishiya Y et al.; The open reading frame yjbR which had been sequenced as a part of the Bacillus subtilis genome project encodes a putative 40.9-kDa protein . The yjbR-coding sequence was slightly similar to those of bacterial sarcosine oxidases and possibly compatible with the tertiary structure of the porcine kidney D-amino acid oxidase . The yjbR gene product was overproduced in Escherichia coli, purified to homogeneity from the recombinant strain, and characterized . This protein effectively catalyzed the oxidation of sarcosine (N-methylglycine), N-ethylglycine and glycine . Lower activities on D-alanine, D-valine, and D-proline were detected although no activities were shown on L-amino acids and other D-amino acids . Since glycine is a product and not a substrate for sarcosine oxidase, this protein is not a type of demethylating enzymes but a novel deaminating oxidase, named glycine oxidase as a common name . Several enzymatic properties of the B . subtilis glycine oxidase were also investigated.

Nucleic Acids Res, 1998 Dec 1, 26(23), 5379 - 87
In vitro and in vivo secondary structure probing of the thrS leader in Bacillus subtilis; Luo D et al.; The Bacillus subtilis thrS gene is a member of the T-box gene family in Gram-positive organisms whose expression is regulated by a tRNA-mediated transcriptional antitermination mechanism involving a direct tRNA:mRNA interaction . The complex leader sequences of these genes share only short stretches of primary sequence homology, but a common secondary structure has been proposed by comparing the leaders of many genes of this family . The proposed mechanism forthe tRNA:mRNA interaction depends heavily on the secondary structure model, but is so far only supported by genetic evidence . We have studied the structure of the B.subtilis thrS leader in solution, in protection experiments using both chemical and enzymatic probes . The thrS leader structure was also probed in vivo using dimethylsulphate and the in vitro and in vivo data are in good accordance . We have organized the thrS leader into three major domains comprising six separate stem-loops . All but one of the short sequences conserved in this gene family are present in loop structures . The ACC specifier codon proposed to interact with the tRNAThrGGUisoacceptor is present in a bulge and probably exists in a stacking conformation . The proposed antiterminator structure is not visible in transcripts containing the terminator, but was probed using a transcript with the 3'-half of the terminator deleted and its folding appears consistent with the regulatory model . The leader sequences, and in particular the specifier domains, of the other genes of this family can be folded similarly to the experimentally solved thrS structure.

J Mol Biol, 1998 Dec 4, 284(3), 569 - 78
The kinase activity of the antisigma factor SpoIIAB is required for activation as well as inhibition of transcription factor sigmaF during sporulation in Bacillus subtilis; Garsin DA et al.; The activity of the developmental transcription factor sigmaF in the spore-forming bacterium Bacillus subtilis is controlled by SpoIIAB, which sequesters sigmaF in an inactive complex . sigmaF is released from the SpoIIAB-sigmaF complex by the action of SpoIIAA, which triggers the dissociation of the complex . SpoIIAB is also a protein kinase that phosphorylates SpoIIAA on serine residue 58 (S58) . This phosphorylation inactivates SpoIIAA and thus indirectly prevents the activation of sigmaF . Here, we report the identification of a patch of amino acid residues located in the vicinity of the adenosine nucleotide binding pocket of SpoIIAB that is required for the phosphorylation of SpoIIAA . A lysine substitution (E104K) at one of these residues (Glu104) markedly impaired the capacity of SpoIIAB to phosphorylate SpoIIAA in vitro as well as during sporulation . Kinetic analysis and evidence from the construction of alanine substitution mutants indicates that the side-chains of these amino acids could be contact sites for the SpoIIAA substrate during the phosphorylation reaction . Importantly, E104K and other kinase mutants blocked the activation of sigmaF during sporulation . This is paradoxical, because a mutant of SpoIIAA (S58A) that cannot be phosphorylated is known to cause higher than normal levels of sigmaF activity during sporulation . In resolution of this paradox, we present biochemical evidence indicating that SpoIIAA directly attacks the SpoIIAB-sigmaF complex and that SpoIIAA is phosphorylated as a result of this reaction . Consistent with this idea, mutations impairing kinase function of SpoIIAB were found to be epistatic to a mutation causing the S58A substitution in SpoIIAA; that is, cells producing mutant forms of both proteins were blocked in the activation of sigmaF . We conclude that phosphorylation of SpoIIAA plays a dual role in the sigmaF pathway, and that the kinase function of SpoIIAB is required for the activation as well as the inhibition of sigmaF during sporulation .

J Mol Biol, 1998 Dec 4, 284(3), 557 - 68
Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor sigmaF in Bacillus subtilis; Garsin DA et al.; The activity of the developmental transcription factor sigmaF in Bacillus subtilis is governed by a switch involving the dual function protein SpoIIAB . SpoIIAB is an antisigma factor that forms complexes with sigmaF and with an alternative partner protein SpoIIAA . SpoIIAB is also a protein kinase that can inactivate SpoIIAA by phosphorylating it on a serine residue . We sought to identify amino acids in SpoIIAB that are involved in the formation of the SpoIIAB-SpoIIAA complex by screening for mutants that were defective in the activation of sigmaF . This genetic screen, in combination with biochemical analysis and the construction of loss-of-side-chain (alanine substitution) mutants, led to the identification of amino acid side-chains in the N-terminal region of SpoIIAB that could contact SpoIIAA . Unexpectedly, the same amino acid side-chains (R20 and N50) that appear to touch SpoIIAA are required for binding to, and may represent sites of contact with, sigmaF . We propose that the N-terminal region of SpoIIAB forms a binding surface that is responsible for the formation of both the SpoIIAB-SpoIIAA and the SpoIIAB-sigmaF complexes, and that in some cases the same amino acid side-chains contact both partner proteins . N50 is also the defining residue of a region of amino acid sequence homology known as the N-box that is shared by SpoIIAB and related serine protein kinases, as well as by members of a mechanistically dissimilar family of protein kinases that undergo autophosphorylation at a histidine residue . We discuss the implications of this finding for the mechanism of histidine autophosphorylation .

Eur J Biochem, 1998 Oct 15, 257(2), 472 - 8
Biochemical characterization of the SecA protein of Streptomyces lividans--interaction with nucleotides, binding to membrane vesicles and in vitro translocation of proAmy protein; Blanco J et al.; The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J . J . R . & Martin, J . F . (1996) Gene (Amst.) 176, 61-65) was expressed from the strong promoter of the Streptomyces griseus saf gene . The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration . It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins . Purified S . lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S . lividans lipid fraction . SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS) . {Alpha-32P}ATP could be crosslinked by ultraviolet radiation at the high-affinity site . The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 microM (the range of saturation at the NBS I) . The calculated Kd of the high-affinity binding site for ADP was 150 nM . Millimolar concentrations of ATP or ADP did not render the S . lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E . coli and B . subtilis SecA proteins . SecA was found to bind to urea-washed S . lividans membrane vesicles with high-affinity, i.e . 10 nM . SecA-dependent binding of E . coli SecB to membrane vesicles was observed when E . coli SecA was used, but not with the S . lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria . An in vitro translocation system has been developed using inverted membrane vesicles of S . lividans . SecA supported in vitro translocation of proAmy into S . lividans membrane vesicles in an ATP-dependent manner.

J Biotechnol, 1998 Sep 17, 64(1), 3 - 13
Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli; Bron S et al.; Bacillus subtilis is one of the best known Gram-positive bacteria at both the genetic and physiological level . The entire sequence of its chromosome is known and efficient tools for the genetic modification of this bacterium are available . Moreover, B . subtilis and related Bacillus species are widely used in biotechnology, in particular for the production of secreted enzymes . Although bacilli can secrete large amounts of several native enzymes, the use of these bacteria for the production of heterologous enzymes has frequently resulted in low yields . Here we describe the identification of several components of the Bacillus protein secretion machinery . These components can now be engineered for optimal protein secretion . Special emphasis is given on type I signal peptidases, which remove signal peptides from secretory precursor proteins . Five genes specifying such enzymes (sip, for signal peptidase) are present on the B . subtilis chromosome . Although none of the sip genes is essential by itself, a specific combination of mutations in these genes is lethal . The expression pattern of some of the sip genes coincides with that of many secretory proteins, which seems to reflect an adaptation to high demands on the secretion machinery . Although the various B . subtilis type I signal peptidases have at least partially overlapping substrate specificities, clear differences in substrate preferences are also evident . These observations have implications for the engineering of the processing apparatus for improved secretion of native and heterologous proteins by Bacillus.

Science, 1998 Nov 20, 282(5393), 1516 - 9
Localization of bacterial DNA polymerase: evidence for a factory model of replication; Lemon KP et al.; Two general models have been proposed for DNA replication . In one model, DNA polymerase moves along the DNA (like a train on a track); in the other model, the polymerase is stationary (like a factory), and DNA is pulled through . To distinguish between these models, we visualized DNA polymerase of the bacterium Bacillus subtilis in living cells by the creation of a fusion protein containing the catalytic subunit (PolC) and green fluorescent protein (GFP) . PolC-GFP was localized at discrete intracellular positions, predominantly at or near midcell, rather than being distributed randomly . These results suggest that the polymerase is anchored in place and thus support the model in which the DNA template moves through the polymerase.

Biodegradation, 1998, 9(2), 133 - 41
Aerobic chromate reduction by Bacillus subtilis; Garbisu C et al.; We have studied the reduction of hexavalent chromium (chromate) to the less toxic trivalent form by using cell suspensions and cell-free extracts from the common soil bacterium, Bacillus subtilis . B . subtilis was able to grow and reduce chromate at concentrations ranging from 0.1 to 1 mM K2CrO4 . Chromate reduction was not affected by a 20-fold excess of nitrate-compound that serves as alternate electron acceptor and antagonizes chromate reduction by anaerobic bacteria . Metabolic poisons including sodium azide and sodium cyanide inhibited chromate reduction . Reduction was effected by a constitutive system associated with the soluble protein fraction and not with the membrane fraction . The reducing activity was heat labile and showed a Km of 188 microns CrO4(2)- . The reductase can mediate the transfer of electrons from NAD(P)H to chromate . The results suggest that chromate is reduced via a detoxification system rather than dissimilatory electron transport.

Mol Gen Mikrobiol Virusol, 1998, (3), 30 - 2
{Cloning and expression of thermostable secretory metalloproteinase genes from two Bacillus brevis strains in Bacillus subtilis cells}; Akimkina TV et al.; Two closely related genes of thermostable Bac . brevis metalloproteases were cloned and expressed in Bac . subtilis cells . Their restriction maps and directions of transcription were determined . Thermostability and thermal optimum of proteolytic activity of cloned gene products are significantly lower than those of native enzymes . The authors believe that alteration of the enzymes' characteristics may be due to uncorrected folding of thermostable protein in case of its expression in mesophilic bacterial strains.

J Antimicrob Chemother, 1998 Oct, 42(4), 519 - 22
Antimicrobial activity of the semisynthetic compound, hexahydrocolupulone; Stephan TE et al.; In this study we demonstrate that hexahydrocolupulone (HHC) more effectively inhibits the growth in vitro of Gram-positive organisms than Mycobacterium tuberculosis or Escherichia coli . Vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and coagulase-negative staphylococci were inhibited by HHC at concentrations < or = 4.06 mg/L . Growth inhibition profiles varied according to the microorganism evaluated (static for S . aureus and bactericidal for Bacillus subtilis).

Arch Microbiol, 1998 Oct, 170(5), 319 - 30
Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments; Kempf B et al.; All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion . Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm . To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes . These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine . The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya . Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions . Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis . Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria.

Cell, 1998 Oct 30, 95(3), 431 - 7
Assembly of a tailed bacterial virus and its genome release studied in three dimensions; Tao Y et al.; We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy . The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29 . The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA . The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry . Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

J Bacteriol, 1998 Nov, 180(22), 6048 - 51
Effect of minCD on FtsZ ring position and polar septation in Bacillus subtilis; Levin PA et al.; We examined the pattern of FtsZ localization in a Bacillus subtilis minCD mutant . When grown in minimal medium, the majority (approximately 89%) of the minCD mutant cells with an FtsZ ring had a single, medially positioned FtsZ ring . These results indicate that genes in addition to minCD function to restrict the number and position of FtsZ rings . When grown in rich medium, greater than 50% of the minCD mutant cells had multiple FtsZ rings, indicating significant differences in regulation of FtsZ ring formation based on growth medium.

J Bacteriol, 1998 Nov, 180(22), 5968 - 77
Decay of ermC mRNA in a polynucleotide phosphorylase mutant of Bacillus subtilis; Bechhofer DH et al.; ermC mRNA decay was examined in a mutant of Bacillus subtilis that has a deleted pnpA gene (coding for polynucleotide phosphorylase) . 5'-proximal RNA fragments less than 400 nucleotides in length were abundant in the pnpA strain but barely detectable in the wild type . On the other hand, the patterns of 3'-proximal RNA fragments were similar in the wild-type and pnpA strains . Northern blot analysis with different probes showed that the 5' end of the decay intermediates was the native ermC 5' end . For one prominent ermC RNA fragment, in particular, it was shown that formation of its 3' end was directly related to the presence of a stalled ribosome . 5'-proximal decay intermediates were also detected for transcripts encoded by the yybF gene . These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments . However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.

J Bacteriol, 1998 Nov, 180(22), 5961 - 7
Transcriptional activation of the Bacillus subtilis ackA gene requires sequences upstream of the promoter; Turinsky AJ et al.; Transcriptional activation of the Bacillus subtilis ackA gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the ackA promoter . CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in B . subtilis and other gram-positive bacteria, recognizes a cis-acting consensus sequence, designated cre (catabolite response element), generally located within or downstream of the promoter of the repressed gene . Two sites resembling this sequence are centered at positions -116.5 and -56.5 of the ackA promoter and have been termed cre1 and cre2, respectively . Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose . In this study, cre2, the site closer to the promoter, and the region upstream of cre2 were shown to be indispensable for CcpA-dependent transcriptional activation of ackA, whereas cre1 was not required . In addition, insertion of 5 bp between cre2 and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of cre2 and/or upstream sequences . DNase footprinting experiments demonstrated binding of CcpA in vitro to cre2 but not cre1, consistent with the genetic data . Activation of ackA transcription was blocked in a ptsH1/crh double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.

J Bacteriol, 1998 Nov, 180(22), 5815 - 21
Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis; Gaballa A et al.; Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation . We have identified three of the key components acting to maintain zinc homeostasis in Bacillus subtilis . Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake, yciC and ycdHIyceA . A zur mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the yciC control region . A similar operator precedes the ycdH-containing operon, which encodes an ABC transporter . Two lines of evidence suggest that the ycdH operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway . First, a ycdH mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a yciC mutation . Second, mutation of ycdH, but not yciC, alters the regulation of both the yciC and ycdH operons such that much higher levels of exogenous zinc are required for repression . We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc . Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.

Planta Med, 1998 Oct, 64(7), 588 - 93
Study of three sesquiterpene lactones from Tithonia diversifolia on their anti-inflammatory activity using the transcription factor NF-kappa B and enzymes of the arachidonic acid pathway as targets; Rungeler P et al.; In Central America leaf extracts from the Asteraceae Tithonia diversifolia are used externally for the treatment of haematomas and wounds . Therefore, the main sesquiterpene lactones (Sls) of this species growing in Costa Rica, diversifolin (1), diversifolin methyl ether (2), and tirotundin (3), were studied for their anti-inflammatory activity . We determined whether these compounds inhibit cyclooxygenase-I, phospholipase A2, or the transcription factor NF-kappa B . Here we show that these Sls do not influence the enzymes of the arachidonic acid pathway, but inhibit the activation of NF-kappa B . Thereby, the synthesis of inflammatory mediators such as cytokines and chemokines is reduced . Our results indicate that the inhibitory activity of compounds 1-3 is due to alkylation of cysteine residues, which are probably located in the DNA binding domain of NF-kappa B . The Sls were also studied for their antibacterial activity, but only Sl 1 was moderately active against Bacillus subtilis in the agar plate diffusion test.

FEMS Microbiol Lett, 1998 Oct 15, 167(2), 315 - 20
Normal induction of the SOS response in Bacillus subtilis is prevented by the mutant repressor from phage phi 105cts23; Rubinstein CP et al.; The presence of the phi 105cts23 mutant prophage in Bacillus subtilis induces a series of pleiotropic effects that could be ascribed to an anti-SOS activity . In order to circumvent the phage function responsible for this phenomenon, the cts23 mutant repressor was cloned and sequenced . The isolated repressor reduced the survival capacity of the host cells after mitomycin C or nalidixic acid treatments and lowered the spontaneous reversion frequency . When SOS induction kinetics were studied, low or null induction of the damage-inducible din22::LacZ fusion was observed . In contrast, the presence of the wild-type prophage amplified the SOS response . Sequencing of the mutant repressor revealed that the cts23 mutation is a T-->C transition affecting the 5' closest codon to one of the two reported DNA binding domains.

Nat Struct Biol, 1998 Nov, 5(11), 959 - 64
A novel DNA-binding motif shares structural homology to DNA replication and repair nucleases and polymerases; Yuan YC et al.; A novel class of DNA-binding domains has been established from at least sixteen recently identified DNA-binding proteins . The three-dimensional structure of one of these domains, Mrf-2, has been solved using NMR methods . This structure is significantly different from known DNA-binding domain structures . The mechanism of DNA recognition by this motif has been suggested based on conserved residues, surface electrostatic potentials and chemical shift changes . This new DNA-binding motif shares structural homology with T4 RNase H, E . coli endonuclease III and Bacillus subtilis DNA polymerase I . The structural homology suggests a mechanism for substrate recognition by these enzymes.

Genes Dev, 1998 Nov 1, 12(21), 3419 - 30
Polar localization of the MinD protein of Bacillus subtilis and its role in selection of the mid-cell division site; Marston AL et al.; Cell division in rod-shaped bacteria is initiated by formation of a ring of the tubulin-like protein FtsZ at mid-cell . Division site selection is controlled by a conserved division inhibitor MinCD, which prevents aberrant division at the cell poles . The Bacillus subtilis DivIVA protein controls the topological specificity of MinCD action . Here we show that DivIVA is targeted to division sites late in their assembly, after some MinCD-sensitive step requiring FtsZ and other division proteins has been passed . DivIVA then recruits MinD to the division sites preventing another division from taking place near the newly formed cell poles . Sequestration of MinD to the poles also releases the next mid-cell sites for division . Remarkably, this mechanism of DivIVA action is completely different from that of the equivalent protein MinE of Escherichia coli, even though both systems operate via the same division inhibitor MinCD.

Curr Microbiol, 1998 Dec, 37(6), 368 - 72
In Bacillus subtilis DegU-P is a positive regulator of the osmotic response; Ruzal SM et al.; The osmosensitivity presented by spo0A and degU null mutant strains of Bacillus subtilis pointed to their protein products as essential regulators for the osmotic response . This was further investigated by analyzing their transcription activity . The results showed that both spo0A-lacZ and degSU-lacZ were induced by the hypertonic medium . The actual phosphorylation state of these proteins was also analyzed by the bias of two reporter gene promoters activity (abrB-lacZ and degQ-lacZ) . The absence of repression of abrB in hypertonic conditions suggested that Spo0A was not phosphorylated while the derepression of degQ promoter suggested that DegU-P was formed . These results were in accordance with the observed absence of sporulation in hyperosmotic media and semi-constitutive osmotolerance of degUh mutant strains known to retain phosphorylated DegU . The failure to secrete proteases and to sporulate in hypertonic media suggested that Spo0A acts through abrB regulation in the prevention of alternate responses . The role of DegU-P as positive regulator of the osmotic response seems to be settled and is discussed.

Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1720 - 5
Hyperexpression of the gene for a Bacillus alpha-amylase in Bacillus subtilis cells: enzymatic properties and crystallization of the recombinant enzyme; Ikawa K et al.; We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis {Sumitomo et al., Biosci . Biotech . Biochem., 59, 2172-2175 (1995)} . The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp . KSM-1378 was amplified by PCR . It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B . subtilis . The transformed B . subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture . The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield . No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp . KSM-1378 . The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions . The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).

Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1707 - 13
Restricted transcription from sigma H or phosphorylated spo0A dependent promoters in the temperature-sensitive secA341 mutant of Bacillus subtilis; Asai K et al.; The temperature-sensitive secA341 mutation of Bacillus subtilis affects sporulation and sporulation-associated events as well as protein secretion and cell septation . With lacZ or bgaB fusion genes, we examined the expression of the early sporulation genes in the mutant strain . Transcriptional expression of delta H dependent kinA, spo0A (Ps), phrC, spoVG, and citG (p2) genes was blocked by the secA341 mutation at 37 degrees C . On the other hand, neither repression of the abrB gene nor induction of the spoH (delta H) gene was affected . Active RNA polymerase containing delta H was, however, found to be produced in the mutant cells . Expression of the phosphorylated Spo0A dependent spoIIG operon was also blocked . Thus the secA341 mutation blocks some step(s) or factor(s) required for delta H-dependent transcription in vivo.

Microbiology, 1998 Oct, 144 ( Pt 10), 2809 - 17
The conjugative plasmid pSG5 from Streptomyces ghanaensis DSM 2932 differs in its transfer functions from other Streptomyces rolling-circle-type plasmids; Maas RM et al.; The Streptomyces ghanaensis plasmid pSG5 is self-transmissible but does not form the growth-retardation zones (pocks) normally characteristic of the Streptomyces plasmid-transfer process . The complete nucleotide sequence of pSG5 was determined on both strands . pSG5 is 12,208 bp in length and has a GC content of 68 mol% . Characterization of the open reading frames by insertion and deletion analysis revealed that only a single gene, traB, is involved in the transfer of pSG5 . The deduced amino acid sequence of TraB is similar to the SpoIIIE protein that is responsible for chromosome translocation during prespore formation of Bacillus subtilis . In contrast to the tra genes of the other Streptomyces plasmids, the pSG5 traB does not represent a kill function . Although pSG5 transfer is not associated with pock formation, pSG5 was shown to possess putative spd genes that are responsible for the pock phenotype of other Streptomyces plasmids . However, promoter-probe experiments revealed that the spd genes of pSG5 are not transcribed, thus explaining the deficiency in pock formation.

J Mol Biol, 1998 Nov 13, 283(5), 907 - 12
An evolutionary link between sporulation and prophage induction in the structure of a repressor:anti-repressor complex; Lewis RJ et al.; Spore formation is an extreme response of some bacteria to adversity . In Bacillus subtilis the proteins of the sin, sporulation inhibition, region form a component of an elaborate molecular circuitry that regulates the commitment to sporulation . SinR is a tetrameric repressor protein that binds to the promoters of genes essential for entry into sporulation and prevents their transcription . This repression is overcome through the activity of SinI, which disrupts the SinR tetramer through the formation of a SinI-SinR heterodimer . The interactions governing this curious quaternary transition are revealed in the crystal structure of the SinI-SinR complex . The most striking, and unexpected, finding is that the tertiary structure of the DNA-binding domain of SinR is identical with that of the corresponding domains of the repressor proteins, CI and Cro, of bacteriophage 434 that regulate lysis/lysogeny . This structural similarity greatly exceeds that between SinR and any bacterial protein or between the 434 repressor proteins and their homologues in the closely related bacteriophage lambda . The close evolutionary relationship implied by the structures of SinR and the 434 repressors provokes both comparison of their functions and a speculative consideration of the intriguing possibility of an evolutionary link between the two adaptive responses, sporulation and prophage induction .

Biochemistry, 1998 Nov 3, 37(44), 15466 - 73
Recognition of a pre-tRNA substrate by the Bacillus subtilis RNase P holoenzyme; Loria A et al.; The holoenzyme of the bacterial RNase P has broader selectivity for biological substrates compared to the RNA alone (denoted P RNA) reaction . The structural basis of the substrate selectivity is investigated using a pre-tRNA substrate containing single-atom modifications by single turnover kinetics . Hydroxyl radical protection of the holoenzyme in the absence of the substrate shows that the RNase P protein binds to several regions in P RNA . The holoenzyme interacts with a subset of functional groups in the T stem-loop region of a pre-tRNA substrate previously identified to directly contact P RNA . The subtle change in structural recognition allows the holoenzyme to recognize RNA structures with only a small perturbation in an A-form helix at the corresponding position of the T stem-loop . This altered profile may permit the holoenzyme to bind non-tRNA substrates with little change in catalytic efficiency . The holoenzyme recognizes the same set of functional groups as the P RNA reaction in the region around the cleavage site and shows similar cleavage site selection compared to the P RNA reaction . These results suggest that the holoenzyme does not alter the fundamental mechanism of this enzymatic reaction . Rather, the holoenzyme significantly affects the binding affinity of an RNA substrate through additional interactions with the 5' leader {Kurz, C . A., Niranjanakumari, S., and Fierke, C . A . (1998) Biochemistry 37, 2393} and through altered recognition of the substrate structure.

Eur J Biochem, 1998 Oct 1, 257(1), 210 - 5
Menaquinone-dependent succinate dehydrogenase of bacteria catalyzes reversed electron transport driven by the proton potential; Schirawski J et al.; Succinate dehydrogenases from bacteria and archaea using menaquinone (MK) as an electron acceptor (succinate/menaquinone oxidoreductases) contain, or are predicted to contain, two heme-B groups in the membrane-anchoring protein(s), located close to opposite sides of the membrane . All succinate/ubiquinone oxidoreductases, however, contain only one heme-B molecule . In Bacillus subtilis and other bacteria that use MK as the respiratory quinone, the succinate oxidase activity (succinate-->O2), and the succinate/menaquinone oxidoreductase activity were specifically inhibited by uncoupler (CCCP, carbonyl cyanide m-chlorophenylhydrazone) or by agents dissipating the membrane potential (valinomycin) . Other parts of the respiratory chains were not affected by the agents . Succinate oxidase or succinate/ubiquinone oxidoreductase from bacteria using ubiquinone as an acceptor were not inhibited . We propose that the endergonic electron transport from succinate (Eo' = +30 mV) to MK (Eo' approximately/= -80 mV) in succinate/menaquinone oxidoreductase includes a reversed electron transport across the cytoplasmic membrane from the inner (negative) to the outer (positive) side via the two heme-B groups . The reversed electron transport is driven by the proton or electrical potential, which provides the driving force for MK reduction.

Antimicrob Agents Chemother, 1998 Nov, 42(11), 2906 - 13
Mutations in Bartonella bacilliformis gyrB confer resistance to coumermycin A1; Battisti JM et al.; This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species . The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced . The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of approximately 77.5 kDa . Sequence alignment indicates that B . bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date . The cloned B . bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E . coli Cour gyrB mutant (strain N4177) . We isolated and characterized spontaneous mutants of B . bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB . Sequence analysis of gyrB from 12 Cour mutants of B . bacilliformis identified single nucleotide transitions at three separate loci in the ORF . The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124-->Ser), Arg184-->Gln, and Thr214-->Ala or Thr214-->Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E . coli, Staphylococcus aureus, and Haloferax sp . The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.

Biochim Biophys Acta, 1998 Oct 23, 1425(2), 387 - 97
Stability of water-soluble and lipid-soluble paramagnetic probes in Bacillus subtilis; Jung K et al.; Batch cultures of the Gram-positive bacterium Bacillus subtilis PB19 have been investigated for their metabolic action to electron spin resonance (ESR) probes . Five- and six-membered water-soluble and lipid-soluble nitroxides have been used, which were reduced most probably to the corresponding hydroxylamine derivatives . The reduction was followed by the ESR signal intensity and found to be dependent on chemical structure and stability, lipophilic/hydrophilic character, charge, concentration, and temperature . Water-soluble nitroxides did not show apparent toxicity towards B . subtilis, in contrast with n-DXSA (n=5, 12, 16) which were found to be strongly cytotoxic . The cytotoxicity depended on the position of the doxyl unit along the hydrocarbon chain . The hydrophilic nitroxides were reduced at a much slower rate relative to the lipophilic ones . Membrane diffusion was suggested to be a slower process relative to chemical reduction for water-soluble nitroxides . The lipophilic nitroxides were solubilized into the membrane where they were rapidly reduced with a reduction maximum at 303-310 K, which is the optimal growth temperature of B . subtilis, while an inactivation at higher temperatures was observed . Both toxicity and reduction rates of nitroxides strongly indicated that the reduction was an enzyme-mediated process taking place near the outer surface of the periplasmic membrane.

Gene, 1998 Oct 23, 221(2), 185 - 90
Rapid isolation of RNA polymerase from sporulating cells of Bacillus subtilis; Fujita M et al.; A highly ordered program of temporal and spatial gene activation during sporulation in Bacillus subtilis is governed by the principal RNA polymerase, and RNA polymerases containing at least five developmental sigma factors appearing successively during sporulation . This report describes a rapid procedure for extracting RNA polymerase from sporulating B . subtilis cells, which involves the construction of hexahistidine tagged beta' subunit of RNA polymerase and the isolation of RNA polymerase holoenzyme with Ni2+-NTA resin . In in vitro transcription of various promoters with the RNA polymerase thus purified, we observed the temporal change of each RNA polymerase activity during sporulation . This procedure enables isolation of RNA polymerase within 4h, starting with cell pellets . Our results indicated that a principal sigma factor, sigmaA, could be detected in a holoenzyme form during all the stages of growth and sporulation, while the other sigma factors sigmaH, sigmaE, sigmaF, sigmaG, and sigmaK involved in sporulation could be detected sequentially during sporulation . Moreover, Spo0A, the central transcription factor of commitment to sporulation, was also co-purified with RNA polymerase at early stages of sporulation.

Gene, 1998 Sep 14, 217(1-2), 31 - 40
A novel sporulation-control gene (spo0M) of Bacillus subtilis with a sigmaH-regulated promoter; Han WD et al.; A novel sporulation-control gene (spo0M) of Bacillus subtilis was cloned, sequenced and analyzed . The spo0M gene is located at the end of large tRNA gene clusters including rrnD and codes for a 257-amino-acid protein with a calculated size of 29.6kDa . The protein Spo0M has a strong negative charge (calculated pI=4.3) and shows no significant sequence homology to any known proteins . Gene disruption experiments revealed that spo0M is not essential for cell viability, but its disruption results in considerable impairments (decreasing by 20- to 100-fold) in sporulation . The morphological stage blocked in sporulation was stage 0 as observed by electron microscopy, and expression analysis using spo0Aps-bgaB fusion revealed an impaired gene expression of spo0A in the spo0M mutant . In contrast, spo0M disruption had no effect on antibiotic productivity . Propagation of the spo0M gene in wild-type cells using a high-copy-number plasmid also impaired sporulation, indicating that overproduction of Spo0M exerts certain negative effects on sporulation . spo0M gene expression is controlled by sigmaH, as demonstrated: (1) by monitoring expression of a bgaB transcriptional fusion integrated into the amyE locus on the chromosome of the wild-type or spo0H mutant cells, and (2) by in-vitro transcription of spo0M gene with EsigmaH.

J Bacteriol, 1998 Nov, 180(21), 5749 - 55
Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation; Graumann PL et al.; We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis . Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B . subtilis cell cycle . Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell . DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci . The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci . Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures . In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation . Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.

J Mol Biol, 1998 Nov 6, 283(4), 809 - 19
Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G40 P); Barcena M et al.; The Bacillus subtilis bacteriophage SPP1 gene 40 product (G40P), which belongs to the DnaB-like family of helicases, is essential for SPP1 genome replication . The active form of the enzyme is the hexamer, capable of DNA unwinding with a 5' to 3' polarity fueled by the hydrolysis of a nucleoside 5'-triphosphate . We have used electron microscopy of negatively stained G40P samples and image processing techniques to study the structural characteristics of the hexameric assemblies of this protein . Our results provide the first low resolution data on a hexameric helicase of a Gram-positive bacterial origin . A novel approach has been adopted to analyze possible symmetry heterogeneities, an unsupervised method based on a neural network self-organizing algorithm, which has led to the detection of different subclasses of G40P views . Two different quaternary states of G40P homohexamers sharing a C3 symmetry organization have been found, as well as a minor class that seems to reflect an alternative C6 symmetry architecture . These forms show general features known for other hexameric helicases, such as the ring-like arrangement of monomers around a central hole . A clear structural handedness has also been detected in some of these forms . An analysis of these quaternary states and a model for the structural organization of G40P are presented .

J Mol Biol, 1998 Nov 6, 283(4), 771 - 83
RNase P RNA structure and cleavage reflect the primary structure of tRNA genes; Brannvall M et al.; The function of RNase P RNA depends on its folding in space . A majority of RNase P RNAs from various bacteria show a similar secondary structure to that of Escherichia coli (M1 RNA) . However, there are exceptions as exemplified by the RNase P RNA derived from the low GC-content Gram-positive bacteria Bacillus subtilis and Mycoplasma hyopneumoniae (Hyo P RNA) . Previous studies using M1 RNA and Hyo P RNA suggest differences both with respect to the kinetics of cleavage as well as to cleavage site recognition . Here we have studied cleavage by these two structurally different RNase P RNAs as a function of changes in the 5' leader and the 3'-terminal CCA motif in the substrate . Our data suggest that the nucleotide at the -2 position in the 5' leader plays a role both for cleavage site recognition and for the rate of cleavage . However, depending on the identity of the -2 residue differences in the cleavage pattern comparing these two types of RNase P RNAs were observed . The results also suggest that the identity of the -1/+73 base-pair in the substrate influences the cleavage site recognition process . These findings will be related to differences in structure comparing these types of RNase P RNAs and the "RCCA-RNase P RNA" interaction . In addition, our findings will be discussed with respect to the primary structure of the tRNA genes in different bacteria .

Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 12838 - 43
Glutamyl-tRNA(Gln) amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis; Curnow AW et al.; Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis . They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS) . The alternative route involves transamidation of incorrectly charged tRNA . Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation . Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS) . Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present . Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB . However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D . radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate . The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D . radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D . radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 12809 - 13
Directed evolution of a thermostable esterase; Giver L et al.; We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase . Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility . Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14 degreesC increase in Tm) without compromising its catalytic activity at lower temperatures . Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated . Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important . Mutations that increase thermostability while maintaining low-temperature activity are very rare . Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

Mol Microbiol, 1998 Oct, 30(1), 189 - 96
Serine kinase activity of a Bacillus subtilis switch protein is required to transduce environmental stress signals but not to activate its target PP2C phosphatase; Kang CM et al.; The RsbT serine kinase has two known functions in the signal transduction pathway that activates the general stress factor sigmaB of Bacillus subtilis . First, RsbT can phosphorylate and inactivate its specific antagonist protein, RsbS . Second, upon phosphorylation of RsbS, RsbT is released to stimulate RsbU, a PP2C phosphatase, thereby initiating a signalling cascade that ultimately activates sigmaB . Here we describe a mutation that separates these two functions of RsbT . Although the mutant RsbT protein had essentially no kinase activity, it still retained the capacity to stimulate the RsbU phosphatase in vitro and to activate sigmaB when overexpressed in vivo . These results support the hypothesis that phosphatase activation is accomplished via a long-lived interaction between RsbT and RsbU . In contrast, RsbT kinase activity was found to be integral for the transmission of external stimuli to sigmaB . Thus, one route by which environmental stress signals could enter the sigmaB network is by modulation of the RsbT kinase activity, thereby controlling the magnitude of the partner switch between the RsbS-RsbT complex and the RsbT-RsbU complex.

J Mol Biol, 1998 Oct 30, 283(3), 595 - 603
Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer; Steinmetzer K et al.; The CopR protein is one of the two regulators of pIP501 copy number . It acts as transcriptional repressor at the essential repR promoter pII . Previously, we found that CopR contacts two consecutive major grooves (site I and site II) on the same face of the DNA . In spite of identical sequence motifs in these sites, neighboring bases were contacted differently . Furthermore, we showed that CopR can dimerize in solution . We demonstrate by two independent methods that CopR binds the DNA as a dimer . We present data that suggest that the sigmoidal CopR-DNA binding curve published previously is the result of two coupled equilibria: dimerization of CopR monomers and CopR dimer-DNA binding . A KD-value of 1.44(+/-0.49)x10(-6) M for CopR dimers was determined by analytical ultracentrifugation . Based on this value and the binding curve, the equilibrium dissociation constant K2 for the CopR-DNA complex was calculated to be 4(+/-1 . 3)x10(-10) M . Quantitative Western blot analysis was used to determine the intracellular concentration of CopR in Bacillus subtilis . This value, 20x10(-6) to 30x10(-6) M, is 10 to 20-fold higher than the equilibrium constant for dimer dissociation, suggesting that CopR binds in vivo as a preformed dimer .

J Mol Biol, 1998 Oct 30, 283(3), 559 - 69
Binding of phage phi29 protein p4 to the early A2c promoter: recruitment of a repressor by the RNA polymerase; Monsalve M et al.; Regulatory protein p4 from Bacillus subtilis phage Phi29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase alpha subunit . This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented . Here, the binding of protein p4 to the A2c promoter has been studied . In the absence of RNA polymerase, protein p4 was found to bind with low affinity to a site centered at position -39 relative to the transcription start site . When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position -71 . Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase alpha-subunit . Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high affinity . A mutational analysis revealed that the site at -71 is critical for a stable interaction between protein p4 and RNA polymerase, and for efficient repression, whereas mutation of the site at -39 had only a small effect on repression efficiency . Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance .

Arch Biochem Biophys, 1998 Oct 15, 358(2), 251 - 6
Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis; Corrigall AV et al.; The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His6 tag and characterized . The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor . The Michaelis constants (Km) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 microM, respectively . Polyclonal antibody to B . subtilis protoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase . B . subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 microM and is weakly inhibited by methylacifluorfen at the same concentration . Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme .

Extremophiles, 1998 Aug, 2(3), 217 - 22
pH homeostasis and ATP synthesis: studies of two processes that necessitate inward proton translocation in extremely alkaliphilic Bacillus species; Krulwich TA et al.; Alkaliphilic Bacillus species that are isolated from nonmarine, moderate salt, and moderate temperature environments offer the opportunity to explore strategies that have developed for solving the energetic challenges of aerobic growth at pH values between 10 and 11 . Such bacteria share many structural, metabolic, genomic, and regulatory features with nonextremophilic species such as Bacillus subtilis . Comparative studies can therefore illuminate the specific features of gene organization and special features of gene products that are homologs of those found in non-extremophiles, and potentially identify novel gene products of importance in alkaliphily . We have focused our studies on the facultative alkaliphile Bacillus firmus OF4, which is routinely grown on malate-containing medium at either pH 7.5 or 10.5 . Current work is directed toward clarification of the characteristics and energetics of membrane-associated proteins that must catalyze inward proton movements . One group of such proteins are the Na+/H+ antiporters that enable cells to adapt to a sudden upward shift in pH and to maintain a cytoplasmic pH that is 2-2.3 units below the external pH in the most alkaline range of pH for growth . Another is the proton-translocating ATP synthase that catalyzes robust production of ATP under conditions in which the external proton concentration and the bulk chemiosmotic driving force are low . Three gene loci that are candidates for Na+/H+ antiporter encoding genes with roles in Na(+)-dependent pH homeostasis have been identified . All of them have homologs in B . subtilis, in which pH homeostasis can be carried out with either K+ or Na+ . The physiological importance of one of the B . firmus OF4 loci, nhaC, has been studied by targeted gene disruption, and the same approach is being extended to the others . The atp genes that encode the alkaliphile's F1F0-ATP synthase are found to have interesting motifs in areas of putative importance for proton translocation . As an initial step in studies that will probe the importance and possible roles of these motifs, the entire atp operon from B . firmus OF4 has been cloned and functionally expressed in an Escherichia coli mutant that has a full deletion of its atp genes . The transformant does not exhibit growth on succinate, but shows reproducible, modest increases in the aerobic growth yields on glucose as well as membrane ATPase activity that exhibits characteristics of the alkaliphile enzyme.

Microbiology, 1998 Sep, 144 ( Pt 9), 2555 - 61
Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates; van der Ploeg JR et al.; A 5 kb region upstream of katA at 82 degrees on the Bacillus subtilis chromosome contains five ORFs organized in an operon-like structure . Based on sequence similarity, three of the ORFs are likely to encode an ABC transport system (ssuBAC) and another to encode a monooxygenase (ssuD) . The deduced amino acid sequence of the last ORF (ygaN) shows no similarity to any known protein . B . subtilis can utilize a range of aliphatic sulfonates such as alkanesulfonates, taurine, isethionate and sulfoacetate as a source of sulfur, but not when ssuA and ssuC are disrupted by insertion of a neomycin-resistance gene . Utilization of aliphatic sulfonates was not affected in a strain lacking 3'-phosphoadenosine 5'-phosphosulfate (PAPS) sulfotransferase, indicating that sulfate is not an intermediate in the assimilation of sulfonate-sulfur . Sulfate or cysteine prevented expression of beta-galactosidase from a transcriptional ssuD::lacZ fusion . It is proposed that ssuBACD encode a system for ATP-dependent transport of alkanesulfonates and an oxygenase required for their desulfonation.

Mol Microbiol, 1998 Sep, 29(6), 1369 - 77
A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis; Chedin F et al.; Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme . This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme . From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E . coli . Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation . We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme . Finally, inspection of the complete B . subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication . Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B . subtilis analogue of the E . coli chi sequence.

Syst Appl Microbiol, 1998 Aug, 21(3), 398 - 407
A DNA probe for the detection and identification of Bacillus sporothermodurans using the 16S-23S rDNA spacer region and phylogenetic analysis of some field isolates of Bacillus which form highly heat resistant spores; de Silva S et al.; The spacer regions between the 16S and 23S rRNA genes (spacer regions 1) of Bacillus sporothermodurans were PCR-amplified, cloned and sequenced . Six unique spacer sequences in four size classes were recovered from two strains, rrnA (about 190 bp), rrnB (about 303 bp), rrnC (355 bp) and rrnD (554 bp) . rrnD contained two tRNA genes which were deciphered as tRNA(ala) and tRNA(ile) separated from each other by 13 nucleotides . The primary structures of the tRNA molecules clearly resembled those found in Bacillus subtilis; the tRNA(ala) genes were identical and the tRNA(ile) genes were 95% similar . The mixed rrnA and rrnB spacers when PCR-amplified from chromosomal DNA were effective as a hybridization probe for identification of B . sporothermodurans strains . However, high background signals with DNA from some other bacilli were encountered . A more discriminating probe was prepared from the cloned rrnB spacer region . Of eight aerobic, endospore-forming bacteria isolated from silage following heat enrichment, one was identified as B . sporothermodurans using the probe and its identity was confirmed from partial 16S rDNA analysis (phylotyping) . This indicated that contamination in milk and dairies by B . sporothermodurans could originate from cattle feeds such as silage . Of the other seven silage strains, only two were identified conclusively by phylotyping and three represented probable new species . The latter three strains were subjected to phylogenetic analysis using almost complete 16S rDNA sequences . Branch lengths, bootstrap percentage values, and 16S rDNA similarity to other Bacillus species suggested that these isolates are likely to constitute new species within the genus Bacillus.

Vet Res, 1998 Sep-Oct, 29(5), 441 - 56
In vivo studies on lysosubtilin . 3 . Efficacy for treatment of mastitis and superficial lesions of the udder and teats in cows; Biziulevichius GA et al.; Lysosubtilin is a broad-spectrum preparation of lytic enzymes from Bacillus subtilis designed for veterinary medicine . This study demonstrates its efficacy for the treatment of reproductive system diseases (mastitis, superficial lesions of the udder and teats) in cows . Prior to determination of optimal therapeutic doses, samples taken from the milk and udder skin of sick animals were examined microbiologically . The examinations revealed a high incidence of polymicrobial infections (26.9 and 84.9% for mastitis and superficial udder lesions, respectively) caused by various mixtures of bacteria (both Gram-positive and Gram-negative) and fungi/yeasts . Dose determination studies involved 115 cows with clinical signs of mastitis . The optimal dose for mastitis treatment was found to be 3.5 x 10(6) U lysosubtilin dissolved in 100 mL of distilled water, which was then administered into the mammary gland via the teat canal once daily until recovery . Such a dose yielded statistically significant decreases (P < 0.05) both in the length of time before clinical recovery (2 d versus 4 and 4.5 d with either of the two antibiotic-based traditional drugs) and in the percentage of animals who suffered relapses within a 2-month period following treatment (5% versus 60%, with one of the two drugs) . A field experiment involving 106 cows was designed to compare the efficacy of 1% lysosubtilin water-glycerin solution (1:9 v/v) and other traditional medications for the topical treatment of superficial lesions of the udder and teats as well as its potential for mastitis prevention . All drugs used yielded a 100% cure rate, but lysosubtilin application made it possible to achieve a statistically significant decrease (P < 0.05) in the duration of the recovery period (2.5 d versus 4.5 to 5.5 d) when compared with any of the four other drugs tested . Its efficacy for mastitis prevention was at least 3.4 times higher than the efficacy of the other medications used (statistically significant, P < 0.05, with regards to two of the four drugs) as well . We therefore conclude that lytic enzyme preparations are prospective antimicrobial drugs and when used to combat animal diseases they may serve as a possible alternative to common antibiotics.

Trends Microbiol, 1998 Sep, 6(9), 366 - 70
Kinase-phosphatase competition regulates Bacillus subtilis development; Perego M; The major regulator of sporulation initiation in Bacillus subtilis is the phosphorelay, a multicomponent signal transduction system . A myriad of signals, both positive and negative, from the environment, cell cycle and metabolism is received and interpreted by the phosphorelay and integrated through the opposing activity of protein kinases and protein aspartate phosphatases to create an extremely sophisticated regulatory network.

Antibiot Khimioter, 1998, 43(8), 11 - 5
{Antimicrobial and membranolytic activity of sterically hindered phenols}; Petrykina ZM et al.; Antimicrobial activity of some complicated space phenols (screened) was studied . The compounds had different activities against grampositive bacteria and were inactive against gramnegative microbes . Di-tertiary butyl derivatives of pyrocatechol and resorcin showed the highest activities . The MICs of such derivatives for the collection strains of Bacillus megaterium, Bacillus subtilis and Staphylococcus aureus were 8 to 30 micrograms/ml and exceeded 6-25 times those of the nonsubstituted analogs . The derivatives of pyrocatechol and resorcin impaired the membrane permeability in susceptible intact cells of B.megaterium and S.aureus 209P and had no effect on the membrane permeability of the Escherichia coli resistant cells . In concentrations up to 200 micrograms/ml the nonsubstituted analogs of pyrocatechol and resorcin did not impair the membrane permeability in the intact cells of the above bacteria . Di-tertiary butyl derivatives of pyrocatechol and resorcin had lytic activity with respect to cytoplasmic membranes (protoplasts) of B.megaterium and had no lytic action on E.coli spheroplasts . The antimicrobial spectrum correlated with the membranotropic properties of the compounds . It was suggested that the target of the antimicrobial action of the screened phenols was the bacterial cell cytoplasmic membrane.

EMBO J, 1998 Oct 15, 17(20), 6096 - 105
Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets; Bravo A et al.; Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication . In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33 . By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays . These structures were constituted by a core of protein p1 surrounded by MalE subunits . After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles . The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution . Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets . These structures resemble eukaryotic tubulin and bacterial FtsZ polymers . In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner . We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane . This structure would provide an anchoring site for the viral DNA replication machinery.

FEMS Microbiol Lett, 1998 Sep 15, 166(2), 361 - 7
Cloning of a novel gene yrbB, encoding a protein located in the spore integument of Bacillus subtilis; Takamatsu H et al.; A DNA fragment (2.7 kbp) containing three deduced open reading frames, orf1, orf2 and orf3 (partial sequence), was isolated from the genomic library of Bacillus subtilis using an antiserum raised against spore integument, and was sequenced . orf2 was 519 nucleotides long and encoded a protein of 172 amino acids with a predicted molecular size of 19,552, corresponding to the protein which reacted with the antiserum . Immunoelectron microscopic observation indicated that YrbB, the product of orf2, was located within the spore integument, mainly in the cortex layer with a part in the inner region of the coat layer.

J Mol Biol, 1998 Oct 23, 283(2), 371 - 81
Structure and expression of elongation factor Tu from Bacillus stearothermophilus; Krasny L et al.; The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stearothermophilus was cloned and sequenced . This gene maps in the same context as the tufA gene of Escherichia coli str operon . Northern-blot analysis and primer extension experiments revealed that the transcription of the tuf gene is driven from two promoter regions . One of these is responsible for producing a 4.9-kb transcript containing all the genes of B . stearothermophilus str operon and the other, identified adjacent to the stop codon of the fus gene and designated tufp, for producing a 1.3-kb transcript of the tuf gene only . In contrast to the situation in E . coli, the ratio between the transcription products was found to be about 10:1 in favour of the tuf gene transcript . This high transcription activity from the tufp promoter might be accounted for by the presence of an extremely A+T-rich block consisting of 29 nucleotides which immediately precedes the consensus -35 region of the promoter . A very similar tuf gene transcription strategy and the same tufp promoter organization with the identical A/T block were found in Bacillus subtilis . The tuf gene specifies a protein of 395 amino acid residues with a molecular mass of 43,290 Da, including the N-terminal methionine . A computer-generated three-dimensional homology model shows that all the structural elements essential for binding guanine nucleotides and aminoacyl-tRNA are conserved . The presence of serine at position 376 and a low affinity for kirromycin determined by zone-interference gel electrophoresis (Kd approximately 8 microM) and by polyacrylamide gel electrophoresis under non-denaturing conditions are in agreement with the reported resistance of this EF-Tu to the antibiotic . The replacement of the highly conserved Leu211 by Met was identified as a possible cause of pulvomycin resistance .

Mol Microbiol, 1998 Sep, 29(5), 1215 - 24
De novo fatty acid synthesis is required for establishment of cell type-specific gene transcription during sporulation in Bacillus subtilis; Schujman GE et al.; A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric septum . The developmental programme of these two cells involves the compartmentalized activities of sigmaE in the larger mother cell and of sigmaF in the smaller prespore . A potential role of de novo lipid synthesis on development was investigated by treating B . subtilis cells with cerulenin, a specific inhibitor of fatty acid biosynthesis . These experiments demonstrated that spore formation requires de novo fatty acid synthesis at the onset of sporulation . The transcription of the sporulation genes that are induced before the formation of two cell types or that are under the exclusive control of sigmaF occurred in the absence of fatty acid synthesis, as monitored by spo-lacZ fusions . However, expression of lacZ fusions to genes that required activation of sigmaE for transcription was inhibited in the absence of fatty acid synthesis . The block in sigmaE-directed gene expression in cerulenin-treated cells was caused by an inability to process pro-sigmaE to its active form . Electron microscopy revealed that these fatty acid-starved cells initiate abnormal polar septation, suggesting that de novo fatty acid synthesis may be essential to couple the activation of the mother cell transcription factors with the formation of the differentiating cells.

Mol Microbiol, 1998 Sep, 29(5), 1129 - 36
Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the sigmaB regulon; Hecker M et al.; Bacillus subtilis cells respond almost immediately to different stress conditions by increasing the production of general stress proteins (GSPs) . The genes encoding the majority of the GSPs that are induced by heat, ethanol, salt stress or by starvation for glucose, oxygen or phosphate belong to the sigmaB-dependent general stress regulon . Despite a good understanding of the complex regulation of the activity of sigmaB and knowledge of a very large number of general stress genes controlled by sigmaB, first insights into the physiological role of this nonspecific stress response have been obtained only very recently . To explore the physiological role of this reguIon, we and others identified sigmaB-dependent general stress genes and compared the stress tolerance of wild-type cells with mutants lacking sigmaB or general stress proteins . The proteins encoded by sigmaB-dependent general stress genes can be divided into at least five functional groups that most probably provide growth-restricted B . subtilis cells with a multiple stress resistance in anticipation of future stress . In particular, sigB mutants are impaired in non-specific resistance to oxidative stress, which requires the sigmaB-dependent dps gene encoding a DNA-protecting protein . Protection against oxidative damage of membranes, proteins or DNA could be the most essential component of sigmaB mediated general stress resistance in growth-arrested aerobic gram-positive bacteria . Other general stress genes have both a sigmaB-dependent induction pathway and a second sigmaB-independent mechanism of stress induction, thereby partially compensating for a sigmaB deficiency in a sigB mutant . In contrast to sigB mutants, null mutations in genes encoding those proteins, such as cIpP or cIpC, cause extreme sensitivity to salt or heat.

Res Microbiol, 1997 Jun, 148(5), 397 - 403
The recA gene from Streptomyces rimosus R6: sequence and expression in Escherichia coli; Mikoc A et al.; The recA gene from Streptomyces rimusus encodes a 376-amino acids polypeptide (M(r) 39,702) that is one of the largest bacterial RecA proteins observed . Detailed analyses of the Streptomyces RecA proteins showed that all possess an additional and unique C-terminal, rich in lysines and alanines, which can form an additional terminal alpha helix . Expression of the S . rimosus RecA protein in Escherichia coli FR333 (delta recA306) was demonstrated using antibodies raised against E . coli RecA protein; expression was possible only from the S . rimosus promoter . A Streptomyces-E . coli-like promoter sequence (TTGACA-18bp-TCTTAT) was found in the A+ T-rich region 135-165 base pairs upstream from the initiation codon and was related to Bacillus subtilis DNA damage-inducible promoters.

Radiats Biol Radioecol, 1998 Jul-Aug, 38(4), 595 - 600
{Effect of substances, absorbing ultraviolet irradiation on Bacillus subtilis resistance to inactivation and mutagenic action of natural sunlight}; Lotareva OV et al.; Substances, absorbing UV-irradiation from sunlight (triptophan, cistein, catalase) decrease Bacillus subtilis resistance to sunlight-induced lethal and mutagenic damages . Possible, they act as exogenous photosensitizers . Tryptophan was found to be the most active photosensitizer . Cistein and catalase are not so active . Casaminoacids being present in the bacterial suspensions during illumination protect bacteria from mutagenic action sunlight . In this studies we can demonstrate, that sunlight damage B . subtilis membranes and allows substances to penetration into cells . Following, that polishing of the environmental by chemical agents can strength genetic risk for organisms permanently exposure to sunlight.

Radiats Biol Radioecol, 1998 Jul-Aug, 38(4), 495 - 501
{Quantitative analysis of inducible systems of Escherichia coli and Bacillus subtilis using radioimmunological methods}; Suslov AV et al.; By RIA dot-blot method two main cell system, system SOS-repair and heat shock system, was showed possibility to receive as well qualitative as quantitative results influence of different induced agents . Was showed quantitative differences in initial levels enzymes of RecA and CroEL in different strains of E . coli, as well of principle possibility to using received antibody, which has high specific to this enzymes, for investigation reactions this systems in the other bacterial species, such as Bac . subtilis.

J Bacteriol, 1998 Oct, 180(20), 5344 - 50
Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE; Nakano MM et al.; The nitrate and nitrite reductases of Bacillus subtilis have two different physiological functions . Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules . They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors . Two distinct nitrate reductases, encoded by narGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively . However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes . The nasDE genes, together with nasBC (encoding assimilatory nitrate reductase) and nasF (required for nitrite reductase siroheme cofactor formation), constitute the nas operon . Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC and nasD genes . Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA . However, under conditions of oxygen limitation, nasDEF expression and nitrite reductase activity were significantly induced . Anaerobic induction of nasDEF required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration.

J Bacteriol, 1998 Oct, 180(20), 5327 - 33
MinCD proteins control the septation process during sporulation of Bacillus subtilis; Barak I et al.; Mutation of the divIVB locus in Bacillus subtilis causes misplacement of the septum during cell division and allows the formation of anucleate minicells . The divIVB locus contains five open reading frames (ORFs) . The last two ORFs (minCD) are homologous to minC and minD of Escherichia coli but a minE homolog is lacking in B . subtilis . There is some similarity between minicell formation and the asymmetric septation that normally occurs during sporulation in terms of polar septum localization . However, it has been proposed that MinCD has no essential role in sporulation septum formation . We have used electron microscopic studies to show septation events during sporulation in some minD strains . We have observed an unusually thin septum at the midcell position in minD and also in minD spoIIE71 mutant cells . Fluorescence microscopy also localized a SpoIIE-green fluorescent protein fusion protein at the midcell site in minD cells . We propose that the MinCD complex plays an important role in asymmetric septum formation during sporulation of B . subtilis cells.

J Bacteriol, 1998 Oct, 180(20), 5319 - 26
Regulation of the Bacillus subtilis GlcT antiterminator protein by components of the phosphotransferase system; Bachem S et al.; Bacillus subtilis utilizes glucose as the preferred source of carbon and energy . The sugar is transported into the cell by a specific permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI operon . Expression of this operon is induced by glucose and requires the action of a positive transcription factor, the GlcT antiterminator protein . Glucose availability is sensed by glucose-specific enzyme II (EIIGlc), the product of ptsG . In the absence of inducer, the glucose permease negatively controls the activity of the antiterminator . The GlcT antiterminator has a modular structure . The isolated N-terminal part contains the RNA-binding protein and acts as a constitutively acting antiterminator . GlcT contains two PTS regulation domains (PRDs) at the C terminus . One (PRD-I) is the target of negative control exerted by EIIGlc . A conserved His residue (His-104 in GlcT) is involved in inactivation of GlcT in the absence of glucose . It was previously proposed that PRD-containing transcriptional antiterminators are phosphorylated and concomitantly inactivated in the absence of the substrate by their corresponding PTS permeases . The results obtained with B . subtilis glucose permease with site-specific mutations suggest, however, that the permease might modulate the phosphorylation reaction without being the phosphate donor.

J Biol Chem, 1998 Oct 16, 273(42), 27347 - 56
The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase . Assignment to family 4 of the glycosylhydrolase superfamily; Thompson J et al.; The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli . The purified protein GlvA (449 residues, Mr = 50,513) is a unique 6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-alpha-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity . 6-Phospho-alpha-glucosidase (EC 3.2.1.122) from B . subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73% . Estimates for the Mr of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence . The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA . The chromogenic and fluorogenic substrates, p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively . Site-directed mutagenesis shows that three acidic residues, Asp41, Glu111, and Glu359, are required for GlvA activity . Asp41 is located at the C terminus of a betaalphabeta fold that may constitute the dinucleotide binding domain of the protein . Glu111 and Glu359 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-alpha-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate . In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer . By comparative sequence alignment with its homologs, the novel 6-phospho-alpha-glucosidase from B . subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.

J Biol Chem, 1998 Oct 16, 273(42), 27146 - 53
RNA structure inhibits the TRAP (trp RNA-binding attenuation protein)-RNA interaction; Xirasagar S et al.; TRAP (trp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides . Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding . The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer . In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP . This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism . Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction . This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy . The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions . We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs . Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli.

Planta, 1998 Oct, 206(3), 476 - 8
Identification of sequence homology between the internal hydrophilic repeated motifs of group 1 late-embryogenesis-abundant proteins in plants and hydrophilic repeats of the general stress protein GsiB of Bacillus subtilis; Stacy RA et al.; Late embryogenesis abundant (LEA) proteins are speculated to protect against water stress in plants . Group 1 LEA proteins are hydrophilic and vary mainly in the numbers of an extremely hydrophilic internal 20-amino-acid motif . This motif is present up to four times in Arabidopsis thaliana and Hordeum vulgare Group 1 proteins and has been described in numerous plant species . However, no similarity has yet been described between Group 1 genes or gene products and those from non-plant species . We report here the striking similarity between the repeated internal motif of Group 1 LEA proteins and a repeated hydrophilic motif present in a stress-related protein (GsiB) from Bacillus subtilis.

Acta Crystallogr D Biol Crystallogr, 1998 Mar 1, 54 ( Pt 2), 215 - 25
Low-resolution structural characterization of the arginine repressor/activator from Bacillus subtilis: a combined X-ray crystallographic and electron microscopical approach; Glykos NM et al.; Attempts to determine the X-ray crystal structure of the intact homohexameric arginine repressor/activator from B . subtilis have so far been unsuccessful . The major problem appears to be the lack of an isomorphous heavy-atom derivative with a manageable number of substitution sites . Here it is shown how electron microscopy of thin three-dimensional crystals, the same as those used for the X-ray crystallographic studies, made it possible (i) to obtain experimental support for some conclusions drawn on the basis of X-ray data alone, (ii) to determine the low-resolution distribution of electron density in several different crystallographic projections, and (iii) to obtain a tentative low-resolution model of the whole hexamer.

Appl Environ Microbiol, 1998 Oct, 64(10), 4015 - 20
A new operation for producing disease-suppressive compost from grass clippings
Nakasaki K, Hiraoka S, Nagata H.
This study evaluated the use of grass clippings discharged from golf courses as the raw material for production of a suppressive compost to control Rhizoctonia large-patch disease in mascarene grass . Bacillus subtilis N4, a mesophilic bacterium with suppressive effects on the pathogenic fungus Rhizoctonia solani AG2-2, was used as an inoculum in a procedure developed with the aim of controlling composting temperatures and inoculation timing . The population density of mesophilic bacteria in the raw material was reduced to around 5 log10 CFU/g (dry weight) of composting material in the self-heating reaction at the initial stage of composting by maintaining a temperature of 80 degreesC for 1 day . The inoculum was applied immediately, and the composting material was maintained at 40 degreesC for 3 days . This served both to highly concentrate the suppressive bacterium and to achieve sporulation . The temperature was then raised to 60 degreesC and maintained, enabling hygienic, high-speed composting while maintaining the population density of the suppressive bacterium as high as 8 log10 CFU/g (dry weight) in the compost . The suppressiveness of compost made in this way was confirmed in a turf grass disease prevention assay.

J Endod, 1998 Aug, 24(8), 561 - 3
Rapid sterilization of gutta-percha cones with glutaraldehyde; Cardoso CL et al.; Five commercially available liquid glutaraldehyde preparations (Glutaron II, Cidex 28, Glutalabor, Banicide, and Anti-G-Plus) were compared for effectiveness in sterilizing gutta-percha cones artificially contaminated with Bacillus subtilis ATCC 6633 spores . Sporicidal activity differed for the various brands of cones, but after 15 min all glutaraldehyde solutions were effective in eliminating the spores . However, three solutions (Cidex 28, Banicide, and Anti-G-Plus) showed sporicidal activity within a shorter time (10 min) . All glutaraldehyde solutions tested may be used in endodontic practice for rapid decontamination of gutta-percha cones, thus contributing to the maintenance of the aseptic chain, an essential factor for successful root canal treatment.

Appl Environ Microbiol, 1998 Oct, 64(10), 4109 - 12
Heat killing of Bacillus subtilis spores in water is not due to oxidative damage; Setlow B et al.; The heat resistance of wild-type spores of Bacillus subtilis or spores (termed alpha-beta-) lacking DNA protective alpha/beta-type small, acid-soluble spore proteins was not altered by anaerobiosis or high concentrations of the free radical scavenging agents ethanethiol and ethanedithiol . Heat-killed wild-type and alpha-beta- spores exhibited no increase in either protein carbonyl content or oxidized bases in DNA . These data strongly suggest that oxidative damage to spore macromolecules does not contribute significantly to spore killing by heat.

J Biol Chem, 1998 Oct 9, 273(41), 26447 - 54
Electrogenic antiport activities of the Gram-positive Tet proteins include a Na+(K+)/K+ mode that mediates net K+ uptake; Guffanti AA et al.; Two Gram-positive Tet proteins, TetA(L) from Bacillus subtilis and TetK from a Staphylococcus aureus plasmid, have previously been suggested to have multiple catalytic modes and roles . These include: tetracycline (Tc)-metal/H+ antiport for both proteins (Yamaguchi, A., Shiina, Y., Fujihira, E., Sawai, T., Noguchi, N., and Sasatsu, M . (1995) FEBS Lett . 365, 193-197; Cheng, J . Guffanti, A . A., Wang, W., Krulwich, T . A., and Bechhofer, D . H . (1996) J . Bacteriol . 178, 2853-2860); Na+(K+)/H+ antiport for both proteins (Cheng et al . (1996)); and an electrical potential-dependent K+ leak mode for TetK and highly truncated segments thereof that can facilitate net K+ uptake (Guay, G . G., Tuckman, M., McNicholas, P., and Rothstein, D . M . (1993) J . Bacteriol . 175, 4927-4929) . Studies of membrane vesicles from Escherichia coli expressing low levels of complete and 3'-truncated versions of tetA(L) or tetK, now show that the full-length versions of both transporters catalyze electrogenic antiport and that demonstration of electrogenicity depends upon use of a low chloride buffer for the assay . The K+ uptake mode, assayed via 86Rb+ uptake, was also catalyzed by both full-length TetA(L) and TetK . This mode does not represent a potential-dependent leak . Such a leak was not demonstrable in energized membrane vesicles . Rather, Rb+ uptake occurred in right-side-out vesicles when the intravesicular space contained either Na+ or K+ but not choline . If an outwardly directed gradient of Na+ or K+ was present, Rb+ uptake occurred without energization in vesicles from cells transformed with a plasmid containing tetA(L) or tetK but not a control plasmid . Experiments in which a comparable exchange was carried out in low chloride buffers to which oxonol was added confirmed that the exchange was electrogenic . Thus, the K+ uptake mode is proposed to be a mode of the electrogenic monovalent cation/H+ antiport activity of TetA(L) and TetK in which K+ takes the place of the external protons . Truncated TetK and TetA(L) failed to catalyze either Tc-metal/H+ or Na+/H+ antiport in energized everted vesicles . Truncated TetK, but not TetA(L), did, however, exhibit modest, electrogenic Na+(K+)/Rb+ exchange as well as a small, potential-dependent leak of Rb+ . The C-terminal halves of the TetA(L) and TetK proteins are thus required both for proton-coupled active transport activities of the multifunctional transporter and, perhaps, for minimizing cation leakiness.

J Biochem (Tokyo), 1998 Oct, 124(4), 790 - 7
Intersubunit structure within heterodimers of medium-chain prenyl diphosphate synthases . Formation of a hybrid-type heptaprenyl diphosphate synthase; Koike-Takeshita A et al.; Among prenyltransferases that catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphate to produce prenyl diphosphates with various chain lengths and stereochemistries, medium-chain prenyl diphosphate synthases are exceptional in that they comprise two dissociable heteromeric protein components . These components exist without binding with each other under physiological conditions, and neither of them has any prenyltransferase activity by itself . In order to elucidate the precise molecular mechanism underlying expression of the catalytic function by such a unique two-component system, we examined the possibility of forming a hybrid between two of the components of three different medium-chain prenyl diphosphate synthases, components I and II of heptaprenyl diphosphate synthase from Bacillus subtilis, components I' and II' of heptaprenyl diphosphate synthase from Bacillus stearothermophilus, and components A and B of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 . As a result, only the hybrid-type combination of component I and component II' gave distinct prenyltransferase activity . The hybrid-type enzyme catalyzed the synthesis of heptaprenyl diphosphate and showed moderate heat stability, which lay between those of the natural enzymes from B . subtilis and B . stearothermophilus . There is no possibility of forming a hybrid between the heptaprenyl and hexaprenyl diphosphate synthases.

Structure, 1998 Sep 15, 6(9), 1129 - 40
A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure; Rizzi M et al.; BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) has a central role in life processes . The ubiquitous enzyme NAD+ synthetase catalyzes a key step in NAD+ biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction . NAD+ synthetase belongs to the amidotransferase family and has been recognized as a member of the family of N-type ATP pyrophosphatases . In order to investigate the mechanism of the reaction carried out by NAD+ synthetase we have determined a high-resolution three-dimensional structure of the Bacillus subtilis homodimeric NAD+ synthetase in complex with the trapped reaction intermediate NAD-adenylate . RESULTS: Two NAD-adenylate molecules and two pyrophosphate (PPi) molecules are observed in the 1.3 A resolution structure of the NAD+ synthetase-NAD-adenylate complex . Structural studies on the NAD+ synthetase-NAD-adenylate adduct and on the cation-binding sites reveal a new deamido-NAD+-binding site located at the subunit interface, locate a binuclear magnesium cluster at the ATP-binding site and, identify two monovalent cation sites, one of which may represent an ammonium-binding site . CONCLUSIONS: Our results suggest that two different catalytic strategies have been adopted by NAD+ synthetase in the two different steps of the reaction . During the adenylation step, no protein residues seem to be located properly to directly participate in catalysis, which is likely to be carried out with the fundamental assistance of an electron-withdrawing trimetallic constellation present in the active site . A different behavior is observed for the second step, in which an ammonium ion is the binding species . In this step, Asp173 is a key residue in both deprotonation of the primarily bound ammonium ion, and stabilization of the tetrahedral transition-state intermediate . Moreover, the structural data suggest that product release can take place only after all substrates are bound to the enzyme, and product release is ultimately controlled by the conformation adopted by two mobile loops.

Mol Cells, 1998 Aug 31, 8(4), 459 - 65
Major identity element of glutamine tRNAs from Bacillus subtilis and Escherichia coli in the reaction with B . subtilis glutamyl-tRNA synthetase; Kim SI et al.; Early investigations revealed that Bacillus subtilis glutamyl-tRNA synthetase {GluRS (bs)} is responsible for aminoacylating both glutamate tRNA {tRNA(Glu) (bs)} and glutamine tRNA {tRNA(Gln) (bs)} with glutamate . The same Bacillus enzyme can also efficiently attach glutamate to one isoacceptor glutamine tRNA {tRNA(Gln) (ec)} of Escherichia coli in vitro but not to tRNA2(Gln) (ec) and tRNA(Glu) (ec) . To characterize identity elements of these glutamine tRNAs in the interaction with GluRS (bs), tRNA2(Gln) (ec), tRNA1(Gln) (ec), three other mutant glutamine tRNAs {tRNA2(Gln) (AU) (C34 --> U34), tRNA2(Gln) (12M) (C34 --> U34, 31A-U39 --> 31U-A39), and tRNA2(Gln) (M21) (64C --> G50 --> 64G-C50, 63U-A51 --> 63A-U51)} originated from tRNA2(Gln) (ec), tRNA(Gln) (bs), and a mutant tRNAM(Gln) (bs) whose U at the 34th position (U34), was replaced to C (C34), were produced in E . coli . All of the E . coli glutamine tRNAs containing U34 such as tRNA1(Gln), tRNA2(Gln) (AU), and tRNA2(Gln) (12M) could be charged with glutamate by GluRS (bs), whereas tRNA2(Gln) (ec) and its T-stem mutant tRNA2(Gln) (M21) containing C34 could not be charged by the same enzyme . The unique change of C34 to U34 of tRNA2(Gln) (ec) acquired glutamate acceptor activity by GluRS (bs) . This result suggests that the U34 is the major identity element of tRNA1(Gln) (ec) in the recognition by GluRS (bs) . The same situation was found in tRNA(Gln) (bs) . The glutamate acceptor activity of tRNA(Gln) (bs) disappeared on replacement of U34 to C34 . To find out whether modified bases in tRNA(Gln) (bs) are involved in the recognition by GluRS (bs), glutamylation of tRNA(Gln) (bs) produced by in vitro transcription was also examined but the in vitro transcript of tRNA(Gln) (bs) could not be charged with glutamic acid by GluRS (bs) . All of these mean that the major recognition element for GluRS (bs) is U at the 34th position of both tRNA(Gln) (bs) and tRNA1(Gln) (ec) as a modified form.

Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 65 - 79
Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31; Tokunaga H et al.; Using part of the dnaK gene from Bacillus subtilis as a probe, a 4 . 4-kbp SacI-BglII fragment of chromosomal DNA of Bacillus brevis, a protein-hypersecreting bacterium, was cloned . Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues . We purified DnaK protein to homogeneity from B . brevis HPD31 harboring a multi-copy dnaK expression plasmid . Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion proteins . DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP . The specific activity of DnaK-ATPase was 7.25x10-3 unit/mg protein (the turnover number against ATP was 0.47 min-1) under our assay conditions . The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPgammaS, but not ADP or AMP . DnaK formed a stable complex with permanently unfolded carboxymethylated alpha-lactalbumin but not with native alpha-lactalbumin, and this complex was dissociated by addition of ATP/Mg . Formation of this complex was inhibited in the presence of inorganic phosphate.

J Bacteriol, 1998 Oct, 180(19), 5129 - 34
hrcA, encoding the repressor of the groEL genes in Streptomyces albus G, is associated with a second dnaJ gene; Grandvalet C et al.; Expression of the principal chaperones of the heat shock stimulon of Streptomyces albus G are under the negative control of different repressors . The dnaK operon is regulated by hspR, the last gene of the operon (dnaK-grpE-dnaJ-hspR) . hsp18, encoding a member of the small heat shock protein family, is regulated by orfY, which is in the opposite orientation upstream of hsp18 . The groES-groEL1 operon and the groEL2 gene are regulated differently . They present tandem copies of the CIRCE element found in the 5' region of many heat shock genes and shown to act in Bacillus subtilis as an operator for a repressor encoded by hrcA (hrc stands for heat regulation at CIRCE) . We report the identification in S . albus of a new heat shock operon containing hrcA and dnaJ homologs . Disruption of hrcA increased the transcription of the groES-groEL1 operon and of the groEL2 gene . These features were lost when the mutant was complemented in trans by an intact copy of hrcA . Despite considerable accumulation of the GroE chaperones in the hrcA mutant, there was no effect on formation of the aerial mycelium and sporulation, indicating that neither hrcA nor the level of groE gene expression is directly involved in the regulation of Streptomyces morphological differentiation.

Biochemistry, 1998 Sep 22, 37(38), 13411 - 20
Two subunits of heptaprenyl diphosphate synthase of Bacillus subtilis form a catalytically active complex; Zhang YW et al.; Heptaprenyl diphosphate synthase of Bacillus subtilis, which participates in the biosynthesis of the side chain of menaquinone-7, is composed of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon {Zhang, Y.-W., et al . (1997) J . Bacteriol . 179, 1417-1419} . This enzyme essentially requires the coexistence of both subunits for its catalysis . Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells . After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses . When a mixture of the two components that had been preincubated in the presence of Mg2+ and farnesyl diphosphate was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually . This fraction contained both components I and II, and it corresponded to a molecular mass that is in accord with the sum of the values of the two components . Cross-linking studies indicate that the two essential subunits, farnesyl diphosphate, and Mg2+ form a ternary complex which seems to represent a catalytically active state of the heptaprenyl diphosphate synthase . On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg2+ . A photoaffinity analogue of farnesyl diphosphate was shown to preferentially label the component I protein, suggesting that component I possesses a specific affinity for the allylic substrate . Furthermore, the photoaffinity labeling of component I significantly increased in the presence of component II . The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic product from soluble substrates.

Biochemistry, 1998 Sep 22, 37(38), 13392 - 9
Cyclophilin and trigger factor from Bacillus subtilis catalyze in vitro protein folding and are necessary for viability under starvation conditions; Gothel SF et al.; Cyclophilin (the product of the ppiB gene) and the trigger factor (the product of the tig gene) are the only cytosolic peptidyl-prolyl cis-trans isomerases that are known in Bacillus subtilis . Both enzymes catalyze the in vitro refolding of ribonuclease T1, a reaction that is limited in rate by a prolyl cis/trans isomerization . The efficiency of cyclophilin as a folding catalyst is only modest with a kcat/KM value of 3.8 x 10(4) M-1 s-1, but the trigger factor shows an almost 40-fold higher specific activity with a kcat/KM value of 1.4 x 10(6) M-1 s-1 . This high catalytic activity originates from the tight binding to the protein substrate as reflected in both the low KM value of 0.5 microM and in the strong inhibition of the trigger factor by unfolded proteins . By use of a protein-folding assay, the concentrations of cyclophilin and the trigger factor in the cytosol of B . subtilis could be determined as 26 and 35 microM, respectively . Together they account for the entire folding activity that is detectable in crude extracts of wild-type B . subtilis cells . The genes encoding cyclophilin and the trigger factor in the B . subtilis chromosome were disrupted individually and simultaneously . Even in combination, these disruptions had no effect on cell viability in rich medium or under several stress conditions, such as heat, osmotic, or oxidative stress . However, in poor medium and, in particular, in the absence of amino acids, the growth of the double mutant strain was strongly decelerated, indicating that the prolyl isomerases become essential for growth under starvation conditions . It is not yet known whether this function relates to the catalysis of the proline-limited folding of essential proteins.

J Biol Chem, 1998 Oct 2, 273(40), 25818 - 24
Contributions of the domains of the Bacillus subtilis response regulator Spo0A to transcription stimulation of the spoIIG operon; Rowe-Magnus DA et al.; Spo0A is a response regulator that controls entry into sporulation by specifically stimulating or repressing transcription of critical developmental genes . Response regulators have at least two domains: an output transcription regulation domain and a receiver domain that inhibits the output domain . Phosphorylation of the receiver domain relieves the inhibition . We examined the in vitro transcription activation mechanism for Spo0A, phosphorylated Spo0A (Spo0A approximately P), and a deletion mutant that consists solely of the C-terminal output domain (Spo0ABD) . Both Spo0A approximately P and Spo0ABD stimulated transcription from the spoIIG promoter 10-fold more efficiently than Spo0A . Spo0A approximately P and Spo0ABD induced DNA denaturation by RNA polymerase in the -10 recognition region, whereas Spo0A did not . DNase I footprint assays revealed that phosphorylation enhanced binding of intact Spo0A to the 0A boxes, while the binding of Spo0ABD was similar to that of Spo0A . Thus, activation of Spo0A by phosphorylation is not primarily due to enhanced DNA binding . The presence of a phosphorylated N terminus increased the stability of the ternary complex at the spoIIG promoter . We propose that the primary effect of phosphorylation is to expose an RNA polymerase interaction domain to promote transcription from PspoIIG.

J Cell Biol, 1998 Sep 21, 142(6), 1595 - 604
The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge; Melby TE et al.; Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing . They are large proteins characterized by an NH2-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain . Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein . Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge . The hinge appears to be quite flexible: the arms can open up to 180 degrees, separating the terminal domains by 100 nm, or close to near 0 degrees, bringing the terminal globular domains together . A surprising observation is that the approximately 300-amino acid-long coiled coils are in an antiparallel arrangement . Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35-45 amino acids long . This antiparallel arrangement produces a symmetrical molecule with both an NH2- and a COOH-terminal domain at each end . The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms . The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.

Am J Med Genet, 1998 Aug 27, 79(1), 22 - 6
Recurrent missense mutation in the protoporphyrinogen oxidase gene underlies variegate porphyria; Frank J et al.; The porphyrias represent a heterogeneous group of disorders of porphyrin or porphyrin-precursor metabolism, resulting from the inherited or acquired dysregulation of one of the eight enzymes in the porphyrin-heme biosynthetic pathway . Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of the penultimate enzyme in the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO) . Recently, VP has been linked to the PPO gene on chromosome 1q22-23, and several disease-causing mutations have been described . In this study, we identified the underlying genetic lesion in two unrelated patients with VP and investigated all available family members by polymerase chain reaction, heteroduplex analysis, automated sequencing, and restriction enzyme digestion . Mutation analyses in both families revealed a G-to-A transition in exon 6 of the PPO gene resulting in the substitution of arginine by histidine at position 168 of the protein (R168H) . This arginine residue is evolutionarily conserved in human, mouse, and Bacillus subtilis, indicating the importance of this residue in PPO function . Our study establishes a recurrent missense mutation as the underlying genetic defect in two unrelated patients with VP and explains the occurrence of the phenotype in their families.

Eur J Biochem, 1998 Aug 1, 255(3), 690 - 7
Translocation of the precursor of alpha-amylase into Bacillus subtilis membrane vesicles; van Wely KH et al.; Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases . Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system . To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease . Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation . This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase . Translocation of the urea-denatured precursor of the Bacillus licheniformis alpha-amylase, preAmyL, and B . subtilis alkaline phosphatase, prePhoB, into the B . subtilis membrane vesicles require the B . subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force . These studies establish an authentic in vitro translocation system for protein secretion in B . subtilis.

J Bacteriol, 1998 Sep, 180(18), 4987 - 90
A region in Bacillus subtilis sigmaH required for Spo0A-dependent promoter activity; Buckner CM et al.; Spo0A activates transcription in Bacillus subtilis from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, sigmaA, and RNA polymerase containing a secondary sigma factor, known as sigmaH . The region of sigmaA near positions 356 to 359 is required for Spo0A-dependent promoter activation, possibly because Spo0A interacts with this region of sigmaA at these promoters . To determine if the amino acids in the corresponding region of sigmaH are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in sigmaH (201 to 210) . Two alanine substitutions in sigmaH, at glutamine 201 (Q201A) and at arginine 205 (R205A), significantly decreased activity from the Spo0A-dependent, sigmaH-dependent promoter spoIIA but did not affect expression from the sigmaH-dependent, Spo0A-independent promoters citGp2 and spoVG . Therefore, promoter activation by Spo0A requires homologous regions in sigmaA and sigmaH . A mutant form of Spo0A, S231F, that suppresses the sporulation defect caused by several amino acid substitutions in sigmaA did not suppress the sporulation defects caused by the Q201A and R205A substitutions in sigmaH . This result and others indicate that different surfaces of Spo0A probably interact with sigmaA and sigmaH RNA polymerases.

DNA Res, 1998 Jun 30, 5(3), 195 - 201
Sequence analysis of the Bacillus subtilis 168 chromosome region between the sspC and odhA loci (184 degrees-180 degrees); Ghim SY et al.; The nucleotide sequence of 45,389 bp in the 184 degrees-180 degrees region of the Bacillus subtilis chromosome, containing the cge cluster, which is controlled by the sporulation regulatory protein GerE, was determined . Fifty-four putative ORFs with putative ribosome-binding sites were recognized . Seven of them correspond to previously characterized genes: cgeB, cgeA, cgeC, cgeD, cgeE, ctpA, and odhA . The deduced products of 25 ORFs were found to display significant similarities to proteins in the data banks . We have identified genes involved in detoxification, cell walls, and in the metabolism of biotins, purines, fatty acids, carbohydrates and amino acids . The remaining 22 ORFs showed no similarity to known proteins . Both an attachment site of the SPbeta prophage and 2 new putative DNA replication terminators were identified in this region.

J Bacteriol, 1998 Sep, 180(18), 4967 - 73
Characterization of dacC, which encodes a new low-molecular-weight penicillin-binding protein in Bacillus subtilis; Pedersen LB et al.; The pbp gene (renamed dacC), identified by the Bacillus subtilis genome sequencing project, encodes a putative 491-residue protein with sequence homology to low-molecular-weight penicillin-binding proteins . Use of a transcriptional dacC-lacZ fusion revealed that dacC expression (i) is initiated at the end of stationary phase; (ii) depends strongly on transcription factor sigmaH; and (iii) appears to be initiated from a promoter located immediately upstream of yoxA, a gene of unknown function located upstream of dacC on the B . subtilis chromosome . A B . subtilis dacC insertional mutant grew and sporulated identically to wild-type cells, and dacC and wild-type spores had the same heat resistance, cortex structure, and germination and outgrowth kinetics . Expression of dacC in Escherichia coli showed that this gene encodes an approximately 59-kDa membrane-associated penicillin-binding protein which is highly toxic when overexpressed.

J Bacteriol, 1998 Sep, 180(18), 4879 - 85
Spore photoproduct lyase from Bacillus subtilis spores is a novel iron-sulfur DNA repair enzyme which shares features with proteins such as class III anaerobic ribonucleotide reductases and pyruvate-formate lyases; Rebeil R et al.; The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP) . An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase . To study the molecular aspects of SP lyase-mediated SP repair, the cloned B . subtilis splB gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus . The engineered six-His-tagged SP lyase expressed from the amyE locus restored UV resistance to spores of a UV-sensitive mutant B . subtilis strain carrying a deletion-insertion mutation which removed the entire splAB operon at its natural locus and was shown to repair SP in vivo during spore germination . The engineered SP lyase was purified both from dormant B . subtilis spores and from an Escherichia coli overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases . SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose . SP lyase activity was dependent upon reducing conditions and addition of S-adenosylmethionine as a cofactor.

J Biotechnol, 1998 Jul 16, 62(3), 177 - 85
Effect of the replication mode of a plasmid on the stability of multimeric endoxylanase genes in Bacillus subtilis; Lee PC et al.; Effect of the replication mode of a plasmid on the stability of tandemly multimerized endoxylanase genes and a gene dose-dependent expression of the endoxylanase were studied in Bacillus subtilis . The structural genes encoding an endoxylanase, carrying its original promoter and ribosomal binding sequence, were tandemly multimerized and cloned into the Escherichia coli-B . subtilis shuttle plasmid, pJH27 delta 88 or pMTL500e, which has a rolling circle-replicon or a theta (theta)-replicon in B . subtilis, respectively . The cloned dimers in pJH27 delta 88, which has a rolling circle-replicon, spontaneously rearranged to monomers in B . subtilis DB104, whereas those in pMTL500e, having a theta (theta)-replicon, were stably maintained . Expression level of the endoxylanase was proportional to the gene dosage in multimers . The endoxylanase activity in the supernatant increased from 80 U ml-1 with pMTL-1x containing a monomer of the gene to 165 U ml-1 with pMTL-4x containing a tetramer . These results indicate that high level expression of the endoxylanase gene can be obtained by tandemly multimerizing the genes in a plasmid with a theta (theta)-replicon.

J Biol Chem, 1998 Sep 11, 273(37), 23849 - 55
Phosphorylation of the Spo0B response regulator phosphotransferase of the phosphorelay initiating development in Bacillus subtilis; Tzeng YL et al.; The initiation of sporulation in Bacillus subtilis is regulated by the phosphorelay, a complex signal transduction system consisting of kinases and response regulators . The key component of a phosphorelay is the phosphotransferase, which recognizes two response regulators and transfers a phosphoryl group between them . In this reaction, the phosphoryl of one response regulator is transferred to a histidine on the phosphotransferase before phosphorylating an aspartate of the second response regulator . The phosphorylated histidine on the Spo0B phosphotransferase was found to be His30 . Site-directed mutation of His30 to alanine destroyed its phosphotransferase activity in vitro and strains constructed with this mutation were unable to sporulate . None of the other 10 histidines of Spo0B was implicated in the phosphotransferase reaction . A structurally vulnerable site, histidine 23, was also identified through the mutational study . The His30 of Spo0B resides in a domain with little sequence homology to functionally equivalent domains in the phosphorelays of other bacterial and yeast systems, suggesting that the two types of phosphotransfer domains evolved convergently.

J Biol Chem, 1998 Sep 11, 273(37), 23812 - 22
The soluble alpha-glycerophosphate oxidase from Enterococcus casseliflavus . Sequence homology with the membrane-associated dehydrogenase and kinetic analysis of the recombinant enzyme; Parsonage D et al.; The soluble flavoprotein alpha-glycerophosphate oxidase from Enterococcus casseliflavus catalyzes the oxidation of a "non-activated" secondary alcohol, in contrast to the flavin-dependent alpha-hydroxy- and alpha-amino acid oxidases . Surprisingly, the alpha-glycerophosphate oxidase sequence is 43% identical to that of the membrane-associated alpha-glycerophosphate dehydrogenase from Bacillus subtilis; only low levels of identity (17-22%) result from comparisons with other FAD-dependent oxidases . The recombinant alpha-glycerophosphate oxidase is fully active and stabilizes a flavin N(5)-sulfite adduct, but only small amounts of intermediate flavin semiquinone are observed during reductive titrations . Direct determination of the redox potential for the FAD/FADH2 couple yields a value of -118 mV; the protein environment raises the flavin potential by 100 mV in order to provide for a productive interaction with the reducing substrate . Steady-state kinetic analysis, using the enzyme-monitored turnover method, indicates that a ping-pong mechanism applies and also allows the determination of the corresponding kinetic constants . In addition, stopped-flow studies of the reductive half-reaction provide for the measurement of the dissociation constant for the enzyme . alpha-glycerophosphate complex and the rate constant for reduction of the enzyme flavin . These and other results demonstrate that this enzyme offers a very promising paradigm for examining the protein determinants for flavin reactivity and mechanism in the energy-yielding metabolism of alpha-glycerophosphate.

Appl Environ Microbiol, 1998 Sep, 64(9), 3220 - 4
Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures; Wuytack EY et al.; We have studied pressure-induced germination of Bacillus subtilis spores at moderate (100 MPa) and high (500 to 600 MPa) pressures . Although we found comparable germination efficiencies under both conditions by using heat sensitivity as a criterion for germination, the sensitivity of pressure-germinated spores to some other agents was found to depend on the pressure used . Spores germinated at 100 MPa were more sensitive to pressure (>200 MPa), UV light, and hydrogen peroxide than were those germinated at 600 MPa . Since small, acid-soluble proteins (SASPs) and dipicolinic acid (DPA) are known to be involved in spore resistance to UV light and hydrogen peroxide, we studied the fate of these compounds during pressure germination . DPA was released upon both low- and high-pressure germination, but SASP degradation, which normally accompanies nutrient-induced germination, occurred upon low-pressure germination but not upon high-pressure germination . These results adequately explain the UV and hydrogen peroxide resistance of spores germinated at 600 MPa . The resistance to pressure inactivation of 600-MPa-germinated spores could also, at least partly, be attributed to alpha/beta-type SASPs, since mutants deficient in alpha/beta-type SASPs were more sensitive to inactivation at 600 MPa . Further, germination at 100 MPa resulted in rapid ATP generation, as is the case in nutrient-induced germination, but no ATP was formed during germination at 600 MPa . These results suggest that spore germination can be initiated by low- and high-pressure treatments but is arrested at an early stage in the latter case . The implications for the use of high pressure as a preservation treatment are discussed.

Mol Microbiol, 1998 Aug, 29(3), 905 - 13
Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis; Chung YS et al.; The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface . We have explored the processing of the ComG proteins and the cellular localization of six of them . All of the proteins were found to be membrane associated . The four proteins with N-terminal sequence motifs typical of type 4 pre-pilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene . The unprocessed forms of ComGC and GD behave like integral membrane proteins . Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein . The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin . ComGG exists in part as a disulphide-cross-linked homodimer in vivo . ComGC was found to possess an intramolecular disulphide bond . The previously identified homodimer form of this protein is not stabilized by disulphide bond formation . ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein . Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.

Mol Microbiol, 1998 Aug, 29(3), 851 - 8
Characterization of polyamine synthesis pathway in Bacillus subtilis 168; Sekowska A et al.; The ubiquitous polyamines fulfil a variety of functions in all three kingdoms of life . However, little is known about the biosynthesis of these compounds in Gram-positive bacteria . We show that, in Bacillus subtilis, there is a single pathway to polyamines, starting from arginine, with agmatine as an intermediate . We first identified the structural gene of arginine decarboxylase, speA (formerly cad), and then described the speE speB operon, directing synthesis of spermidine synthase and agmatinase . This operon is transcribed into two messenger RNAs, a major one for the speE gene and a minor one for both speEand speB . The promoter of the operon was identified upstream from the speE gene by primer extension analysis . Transcription of this operon indicated that the level of agmatinase synthesis is very low, thus allowing a stringent control on the synthesis of putrescine and, therefore, of all polyamines . This is consistent with the level of polyamines measured in the cell.

Mol Microbiol, 1998 Aug, 29(3), 787 - 98
DNA polymerase template switching at specific sites on the phi29 genome causes the in vivo accumulation of subgenomic phi29 DNA molecules; Murthy V et al.; The accumulation of subgenomic phage phi29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants . Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb . Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage phi29 genome . The nature of the 4, 2.3, 2 and 1 kb molecules was studied . The 2 kb molecules were shown to be single-stranded self-complementary strands forming hairpin structures . The other molecules consisted of palindromic linear double-stranded DNA molecules . Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template . Once formed, the subgenomic molecules are then amplified in vivo . Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGg-AACA-3' being a likely candidate for a protein binding site . Database analysis showed that this sequence occurs four times in the phi29 genome . In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once . These five DSEs are all located in the right half of the phi29 genome, and the same sequences are also present in the linear DNA of related B . subtilis phages . Most interestingly, this sequence is also found in the spoOJ gene of the B . subtilis chromosome . Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE . As the same subgenomic phi29 DNA molecules accumulate after infection of B . subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.

Mol Microbiol, 1998 Aug, 29(3), 709 - 18
Inhibition of a naturally occurring rolling-circle replicon in derivatives of the theta-replicating plasmid pIP501; Pujol C et al.; The mechanisms ensuring regulation of DNA replication in genomes containing multiple replicons are poorly understood . In this report, we addressed this question by analysing in Bacillus subtilis the replication of a derivative of the promiscuous plasmid pIP501 that carries a rolling-circle and a theta replicon . Genetic analyses revealed that the rolling-circle replicon is strongly inhibited in the derivative and that inhibition requires three elements involved in theta replication: the replication origin, the initiator RepR protein and strong transcription of the repR gene . Inhibition is, however, independent of DNA synthesis at the theta origin . We conclude that rolling-circle inhibition is caused by an inhibitory signal encoded by the theta replicon and propose that the signal is composed, at least, of the RepR protein bound to its cognate origin.

J Bacteriol, 1998 Sep, 180(17), 4475 - 80
Unique regulation of carbohydrate chemotaxis in Bacillus subtilis by the phosphoenolpyruvate-dependent phosphotransferase system and the methyl-accepting chemotaxis protein McpC; Garrity LF et al.; The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates . The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP) . As for E . coli, a B . subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates . Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus) . An mcpC null mutant showed no response to the stepwise addition or removal of D-glucose or D-mannitol, both of which are PTS substrates . Therefore, in contrast to E . coli PTS carbohydrate chemotaxis, B . subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC . In the case of the PTS substrate D-glucose, the response to negative stimulus is also partially mediated by McpA . Finally, we show that B . subtilis EnzymeI-P has the ability to inhibit B . subtilis CheA autophosphorylation in vitro . We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient . Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble . In the case of D-glucose, a decrease in D-glucose-associated McpA may also contribute to the inhibition of CheA . This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.

J Biol Chem, 1998 Sep 4, 273(36), 23134 - 42
Identification and characterization of the structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168; Paik SH et al.; An antimicrobial peptide produced by Bacillus subtilis 168 was isolated and characterized . It was named sublancin 168, and its behavior during Edman sequence analysis and its NMR spectrum suggested that sublancin is a dehydroalanine-containing lantibiotic . A hybridization probe based on the peptide sequence was used to clone the presublancin gene, which encoded a 56-residue polypeptide consisting of a 19-residue leader segment and a 37-residue mature segment . The mature segment contained one serine, one threonine, and five cysteine residues . Alkylation of mature sublancin showed no free sulfhydryl groups, suggesting that one sulfydryl had formed a beta-methyllanthionine bridge with a dehydrobutyrine derived by posttranslational modification of threonine; with the other four cysteines forming two disulfide bridges . It is unprecedented for a lantibiotic to contain a disulfide bridge . The sublancin leader was similar to known type AII lantibiotics, containing a double-glycine motif that is typically recognized by dual-function transporters . A protein encoded immediately downstream from the sublancin gene possessed features of a dual-function ABC transporter with a proteolytic domain and an ATP-binding domain . The antimicrobial activity spectrum of sublancin was like other lantibiotics, inhibiting Gram-positive bacteria but not Gram-negative bacteria; and like the lantibiotics nisin and subtilin in its ability to inhibit both bacterial spore outgrowth and vegetative growth . Sublancin is an extraordinarily stable lantibiotic, showing no degradation or inactivation after being stored in aqueous solution at room temperature for 2 years . The fact that sublancin is a natural product of B . subtilis 168, for which a great deal of genetic information is available, including the entire sequence of its genome, suggests that sublancin will be an especially good model for studying the potential of lantibiotics as sources of novel biomaterials.

J Bacteriol, 1998 Sep, 180(17), 4760 - 3
Transcriptional activation of the Bacillus subtilis spoIIG promoter by the response regulator Spo0A is independent of the C-terminal domain of the RNA polymerase alpha subunit; Rowe-Magnus DA et al.; In vitro transcription from the spoIIG promoter by Bacillus subtilis RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A . Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.

J Bacteriol, 1998 Sep, 180(17), 4603 - 12
Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores; Atrih A et al.; Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis . The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant L-alanine . We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass . Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion . These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination . The exudate of germinated spores of B . subtilis 168 contains novel muropeptides in addition to those present in spore-associated material . Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides . These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium . There is also evidence for the activity of a glucosaminidase during the germination process . Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan . The spore-specific residue, muramic delta-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.

J Bacteriol, 1998 Sep, 180(17), 4555 - 63
Bacillus subtilis cells lacking penicillin-binding protein 1 require increased levels of divalent cations for growth; Murray T et al.; Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg2+ or Ca2+ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs . Growth of ponA cells in a medium low in Mg2+ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs . The addition of high levels of Mg2+ to growth media eliminated these phenotypes of a ponA mutant . In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium . Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium . Again, Mg2+ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl . These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.

J Bacteriol, 1998 Sep, 180(17), 4547 - 54
Identification of the gene encoding the alternative sigma factor sigmaB from Listeria monocytogenes and its role in osmotolerance; Becker LA et al.; Listeria monocytogenes is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH . Although studies have provided insight into the mechanisms used by L . monocytogenes to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated . In the studies presented here, we have cloned the sigB gene and several rsb genes from L . monocytogenes, encoding homologs of the alternative sigma factor sigmaB and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium Bacillus subtilis . The L . monocytogenes and B . subtilis sigB and rsb genes are similar in sequence and physical organization; however, we observed that the activity of sigmaB in L . monocytogenes was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium . The magnitude of the response was greatest after an osmotic upshift, suggesting a role for sigmaB in coordinating osmotic responses in L . monocytogenes . A null mutation in the sigB gene led to substantial defects in the ability of L . monocytogenes to use betaine and carnitine as osmoprotectants . Subsequent measurements of betaine transport confirmed that the absence of sigmaB reduced the ability of the cells to accumulate betaine . Thus, sigmaB coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of L . monocytogenes under conditions of high osmotic strength.

Mol Microbiol, 1998 Jul, 29(2), 593 - 604
Characterization of the essential cell division gene ftsL(yIID) of Bacillus subtilis and its role in the assembly of the division apparatus; Daniel RA et al.; We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL, of Escherichia coli . Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell division . The filaments show no sign of invagination, indicating that division is blocked at an early stage . FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore-specific sigma factor, sigmaF . Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other division proteins, DivIB and DivIC, at the site of division is prevented . Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable . The instability of one or more components of the division apparatus may be important for the cyclic assembly/disassembly of the division apparatus.

Mol Microbiol, 1998 Jul, 29(2), 505 - 13
ClpC regulates the fate of a sporulation initiation sigma factor, sigmaH protein, in Bacillus subtilis at elevated temperatures; Nanamiya H et al.; Using a strain carrying a clpC-bgaB transcriptional fusion at the amyE locus, we found that the expression of a clpC operon was induced at the end of exponential growth in a sigmaB-independent manner and ceased around T3.5 in the wild type but not in a spo0H mutant . This suggests that some gene product(s) whose expression is dependent on sigmaH function is required for the turn-off of clpC transcription during an early stage of sporulation . A clpC deletion mutant showed a temperature-sensitive sporulation phenotype and exhibited an abnormally large accumulation of sigmaH in the cell at 45 degrees C after T2, at which time the sigmaH level in the wild type had begun to decrease . These results, together with the fact that spo0H transcription in the clpC deletion mutant was similar to that of the wild type, suggested that ClpC may be responsible for the degradation of sigmaH after the accomplishment of its role in sporulation . Moreover, as expected from these results, overproduction of Spo0A was also observed after the initiation of sporulation in the clpC deletion mutant at 45 degrees C.

Mol Microbiol, 1998 Jul, 29(2), 419 - 30
A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products; Jorasch P et al.; We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR . After cloning and expression in E . coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa . Chromatographic analysis of the lipids extracted from the transformed E . coli revealed several new glycolipids . These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry . They were identified as 3-{O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl}-1,2-diacylgl ycerol, 3-{O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl}-1,2-diacylglycerol and 3-{O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl}-1,2-diacylglycerol . The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates . In these assays, one additional glycolipid was formed and tentatively identified as 3-{O-beta-D-glucopyranosyl}-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells . Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase . This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.

Microbiology, 1998 Aug, 144 ( Pt 8), 2125 - 30
The gerC locus of Bacillus subtilis, required for menaquinone biosynthesis, is concerned only indirectly with spore germination; Leatherbarrow AJ et al.; The gerC region of Bacillus subtilis comprises a tricistronic operon, encoding enzymes that catalyse the late stages of menaquinone biosynthesis . The gerC58 mutation is responsible for a severe growth defect; unsuppressed mutant cells grow as very short rods, which sometimes septate aberrantly . Cultures grow only to a low cell density, rapidly lose viability, and never sporulate . Unlinked suppressor mutations can restore near-normal growth . Several independent suppressed isolates were examined; all grew to normal cell length, but they showed, to varying extents, a residual defect in the placement of the cell division septum . The germination properties of the suppressed derivatives varied from normal to significantly slow in germination in all germinants; therefore, the combination of the gerC mutation and different suppressor alleles resulted in spores with very different germination properties . This suggests that any relationship between the gerC gene products and spore germination is indirect . The gerCC58 mutation maps in a gene encoding the catalytic subunit of the heptaprenyldiphosphate synthase, which is responsible for formation of the isoprenoid side chain of menaquinone-7, and it is proposed that the gerCA, gerCB and gerCC genes be renamed hepA, menG and hepB, respectively.

Microbiology, 1998 Aug, 144 ( Pt 8), 2073 - 84
Identification and sequencing of the groE operon and flanking genes of Lawsonia intracellularis: use in phylogeny; Dale CJ et al.; Proliferative enteropathy (PE) is a complex of diseases of commercial importance to the pig industry . The obligate intracellular bacterium Lawsonia intracellularis is consistently associated with PE and pure cultures of this bacterium have been used to reproduce PE in pigs . In this study L . intracellularis bacteria were purified directly from PE-affected tissue . DNA extracted from purified bacteria was used to construct a partial genomic library which was screened using sera from L . intracellularis-immunized rabbits . Two seroreactive recombinant clones were identified, one of which expressed proteins of 10 and 60 kDa . The sequence of the insert from this clone, pISI-2, revealed ORFs with sequence similarity to the groES/EL operon of Escherichia coli, the 505 ribosomal proteins L21 and L27 of E . coli, a GTP-binding protein of Bacillus subtilis and a possible protoporphyrinogen oxidase, HemK, of E . coli . Primers designed from unique sequences from the pISI-2 insert amplified DNA from infected, but not non-infected, porcine ilea; the amplicon sequence obtained from tissue-cultured L . intracellularis was identical to the corresponding sequence in pISI-2, confirming the origin of the clone . The sequence of L . intracellularis GroEL and other GroEL sequences in the databases were used to construct a partial phylogenetic tree . Analysis of the GroEL sequence relationship suggested that L . intracellularis is not significantly related to other organisms whose GroEL sequences are held in the databases and supports previous data from 16S sequence analyses suggesting that L . intracellularis is a member of a novel group of enteric pathogens.

Genetika, 1998 Jun, 34(6), 839 - 42
{Cloning and biochemical identification of the ribR gene in Bacillus subtilis}; Solov'eva IM et al.; The PCR copy of the ribR gene of Bacillus subtilis was subcloned in Escherichia coli cells under the control of the phage T7 inducible promoter . The polypeptide of 26 kDa corresponding to the 690-bp gene is the product of the ribR gene . The protein encoded by the ribR gene is flavokinase, and the riboflavin-reduced form is the substrate for it.

Biochemistry, 1998 Aug 25, 37(34), 11745 - 61
Nuclear magnetic resonance structure of d(GCATATGATAG) . d(CTATCATATGC): a consensus sequence for promoters recognized by sigma K RNA polymerase; Tonelli M et al.; The three-dimensional structure of d(GCATATGATAG).d(CTATCATATGC), from the promoter region of a gene regulating sporulation in Bacillus subtilis mother cells, was determined utilizing two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (2QF-COSY) spectra . To minimize the effect of methods used to obtain restraints and refine structure, several variables were studied . Interproton distance bounds were calculated very conservatively by running the complete relaxation matrix program MARDIGRAS hundreds of times using 2D NOE spectra for exchangeable and for nonexchangeable protons at different mixing times, assuming different overall correlation times and different starting structures . The 435 distance restraints were used with two different structural refinement methods: restrained molecular dynamics (rMD) and restrained Monte Carlo calculations (rMC) . Refinement using different procedures and starting structures resulted in essentially the same structure (<0.8 A rmsd), indicating that the structure is defined by experimental restraints and not the refinement method or variables used . R factors indicate the structures fit the experimental NOE data very well . Some helical parameters, notably large negative X displacement, are characteristic of A-DNA, but others are characteristic of B-DNA . As with TG.CA steps in other duplex DNA sequences studied in our laboratory, the two TG.CA steps have a positive roll, with T6-G7 exhibiting the largest, and consequently a bent helix axis . The converged structure represents a time-averaged structure . However, multiple conformations, especially in deoxyriboses, were evident from vicinal coupling constants obtained from quantitative simulations of 2QF-COSY cross-peaks and from persistent inconsistencies in experimental distances due to nonlinear conformational averaging.

Lett Appl Microbiol, 1998 Jun, 26(6), 417 - 21
A parametric study of the destruction efficiency of Bacillus spores in low pressure oxygen-based plasmas; Hury S et al.; The destruction of Bacillus spores in oxygen-based plasmas sustained in the millitorr pressure range has been studied as functions of various biological and plasma parameters, namely Bacillus species, surface concentration of spores, treatment temperature, and gas composition . In an oxygen plasma, Bacillus stearothermophilus appears less plasma-resistant than the other spores tested . Oxygen, H2O2 and chiefly CO2 plasmas are clearly shown to be much more efficient in destroying Bacillus subtilis spores than pure argon plasma . The bacterial surface concentration on the spore carriers and the treatment temperature also lead to significant variations in the destruction efficiency of spores when using CO2 plasma.

J Appl Microbiol, 1998 Jun, 84(6), 1117 - 24
Antibacterial properties of extracts from selected planktonic freshwater cyanobacteria--a comparative study of bacterial bioassays; Ostensvik O et al.; Aqueous and methanol extracts from five selected cyanobacteria were examined for antibacterial properties in six different bacterial bioassays . All five cyanobacteria revealed antibacterial properties . Methanol extracts made from Tychonema bourrellyi, Aphanizomenon flos-aquae and Cylindrospermopsis raciborskii showed the most pronounced inhibitory effects, Aqueous extracts made from Microcystis aeruginosa and T . bourrellyi possessed evident antibacterial properties . The bacterial bioassays were based on agar diffusion tests and included pour-plate methods commonly used to detect residues of antibacterial substances in food . In addition, a pourplate bioassay with Aeromonas hydrophila was developed and described . Antibacterial effects were observed in five of the six bacterial bioassays . No antibacterial effect was observed in the Micrococcus luteus bioassay . Bioassays based on Aer . hydrophila, Bacillus cereus and B . subtilis grown in Antibiotic Medium 8, pH5.85, seemed to be sensitive and suitable . The MIC value of diluted MeOH extracts made from C . raciborskii and T . bourrellyi against Aer . hydrophila corresponded to 38 mg freeze-dried cyanobacteria . Bacillus subtilis was more sensitive when grown in a culture medium with pH 5.85 than 7.9 . The antibacterial properties of extracts from the cyanobacteria examined differed from defined cyanotoxins and antibacterial substances . The pattern of inhibition in the bacterial bioassays indicated that various antibacterial substances are involved.

Arch Pharm (Weinheim), 1998 Jun, 331(6), 219 - 23
Antimicrobial activity of some 1,2-benzisothiazoles having a benzenesulfonamide moiety; Zani F et al.; Some sulfonamide and sulfonylurea derivatives of unsubstituted and 5-methylsubstituted 1,2-benzisothiazole were studied in vitro for their antimicrobial properties against bacteria and fungi . Compounds 7 and 8 exhibited good antibacterial activity against Gram positive bacteria . A strong synergism was observed when their growth-inhibitory effect was assayed in combination with trimethoprim by using Bacillus subtilis and Staphylococcus aureus as test microorganisms . The antimycotic action of benzenesulfonylurea derivative 9 was very marked for Madurella mycetomatis and dermatophytes Epidermophyton floccosum, Microsporum gypseum and Trichophyton spp. . Structure-activity relations are discussed.

Biochem Biophys Res Commun, 1998 Aug 19, 249(2), 556 - 61
Characterization of ftsZ gene and its protein product from Streptomyces collinus producing kirromycin; Zhulanova E et al.; The FtsZ protein is required for septation and conversion of aerial mycelium into chains of streptomycete spores . We have cloned and sequenced the ftsZ gene from Streptomyces collinus . The ftsZ of S . collinus is not in juxtaposition with ftsA and IpxC as in Escherichia coli or Bacillus subtilis . The gene encodes a polypeptide of 402 amino acid residues with a molecular mass of 41.3 kDa . N-terminus shares a high level of sequence similarity with FtsZ of S . coelicolor and S . griseus, respectively . C-terminal part is variable both in length and sequence . The purified protein binds GTP . Using polyclonal antisera against FtsZ, we have found that the protein is expressed at the beginning of germination of spores and is present in vegetative cells and aerial mycelium, but not in spores .

Structure, 1998 Jun 15, 6(6), 697 - 710
A promiscuous binding surface: crystal structure of the IIA domain of the glucose-specific permease from Mycoplasma capricolum; Huang K et al.; BACKGROUND: The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a bacterial and mycoplasma system responsible for the uptake of some sugars, concomitant with their phosphorylation . The sugar-specific component of the system, enzyme II (EII),consists of three domains, EIIA, EIIB and EIIC . EIIA and ELLB are cytoplasmic and EIIC is an integral membrane protein that contains the sugar-binding site . Phosphoenolpyruvate (PEP) provides the source of the phosphoryl group, which is transferred via several phosphoprotein intermediates, eventually being transferred to the internalized sugar . Along the pathway, EIIA accepts a phosphoryl group from the phosphocarrier protein HPr and transfers it to EIIB . The structure of the glucose-specific EIIA (EIIAglc) from Mycoplasma capricolum reported here facilitates understanding of the nature of the interactions between this protein and its partners . RESULTS: The crystal structure of EIIAglc from M . capricolum has been determined at 2.5 A resolution . two neighboring EIIAglc molecules associate with one another in a front-to-back fashion, such that Glu149 of one molecule forms electrostatic interactions with the active-site histidine residues, His90 and His75, of the other . Glu149 is therefore considered to mimic the interaction that a phosphorylated histidine of a partner protein makes with EIIA . Another interaction, an ion pair between the active-site Asp94 and Lys168 of a neighboring molecule, may be analogous to the interaction between Asp94 of EIIAglc and Arg17 of HPr . Analysis of molecular packing in this crystal, and in the crystals of two other homologous proteins from Escherichia coli and Bacillus subtilis, reveals that in all cases active-site hydrophobic residues are involved in crystal contacts, but in each case a different region of the neighboring molecule is involved . The transition-state complexes of M . capricolum EIIAglc with HPr and EIIBglc have been modeled; in each case, different structural units are shown to interact with EIIAglc . Many of the interactions are hydrophobic with no sequence specificity . The only specific interaction, other than that formed by the phosphoryl group, involves ion pairs between two invariant aspartate residues of EIIAglc and arginine/lysine residues of HPr or EIIBglc . CONCLUSIONS: The non-discriminating nature of the hydrophobic interactions that EIIAglc forms with a variety of partners may be a consequence of the requirement for interaction with a variety of proteins that show no sequence or structural similarity . Nevertheless, specificity is provided by an ion-pair interaction that is enhanced by the apolar nature of the interface.

Bioorg Khim, 1998 Jun, 24(6), 446 - 8
{Structure of teichoic acids from marine microorganisms Bacillus subtilis and Bacillus licheniformis}; Komandrova NA et al.; Teichoic acids from the cell walls of marine bacilli Bacillus subtilis CMM (Collection of Marine Microorganisms) 234 (R-1) and B . licheniformis CMM 454 (1-1G-2) were isolated and characterized . These teichoic acids were found to have identical structures and are composed of the glucose, ribitol, and phosphoric acid residues . On the basis of 13C NMR and 31P NMR spectra of the teichoic acids and the products of their dephosphorylation, we established the following structure for the biopolymer: poly{-->5)-4-O-beta-D-glucopyranosylribitol-(1-phospho}.

Mol Microbiol, 1998 Jul, 29(1), 343 - 57
A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2); Ryding NJ et al.; whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes . DNA complementing whiH mutants was located immediately upstream on hrdB, which encodes the principal sigma factor of S . coelicolor . Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype . Four whiH mutants contained base changes or a frameshift in this gene . The deduced product of whiH related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism . Transcription of whiH was initiated at a single promoter, PwhiH . Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable . Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production . PwhiH was directly dependent on the sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.

Mol Microbiol, 1998 Jul, 29(1), 285 - 96
Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A- and sigma B-dependent stress-responsive promoters; Spiegelhalter F et al.; The opuE gene from Bacillus subtilis encodes a transport system (OpuE) for osmoprotective proline uptake and is expressed from two osmoregulated promoters: opuE P-1 recognized by the vegetative sigma factor A (sigma A and opuE P-2 dependent on the stress-induced transcription factor sigma B (sigma B) . The contributions of these two promoters to osmoregulation of opuE were analysed . Genetic studies using chromosomal opuE-treA operon fusions revealed that opuE transcription is rapidly induced after an osmotic upshock . The strength of opuE expression is proportionally linked to the osmolarity of the growth medium . Deletion analysis of the opuE regulatory region identified a 330 bp DNA segment carrying all sequences required in cis for full and osmoregulated transcription . The proper rotational orientation of the upstream region present within this fragment was essential for the function of both opuE promoters . Mutant opuE-treA fusions with defects in either the sigma A-or the sigma B-dependent promoters revealed different contributions of these sequences to the overall osmoregulation of opuE . opuE P-2 (sigma B) activity increased transiently after an osmotic upshock and did not significantly contribute to the level of opuE expression in cells subjected to long-term osmotic stress . In contrast, transcription initiating from opuE P-1 (sigma A) rose in proportion to the external osmolarity and was maintained at high levels . Moreover, both promoters exhibited a different response to the osmoprotectant glycine betaine in the medium . Our results suggest that at least two different signal transduction pathways operate in B . subtilis to communicate osmotic changes in the environment to the transcription apparatus of the cell.

Mol Microbiol, 1998 Jul, 29(1), 261 - 73
PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication; Petit MA et al.; The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication for helicase . In contrast, in Bacillus subtilis, in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA . It is 40% identical to the Rep and UvrD DNA helicases of E . coli and 61% identical to the PcrA helicase of Staphylococcus aureus . This gene is located at 55 degree on the chromosome and belongs to a putative operon together with a ligase gene (lig) and two unknown genes named pcrB and yerH . As PcrA was essential for cell viability, conditional mutants were constructed . In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited . Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion . To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E . coli . PcrA suppressed the UV sensitivity defect at a uvrD mutant but not its mutator phenotype . Furthermore, it conferred a Rep-phenotype on E . coli . Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.

Mol Microbiol, 1998 Jul, 29(1), 189 - 98
Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors; Bsat N et al.; Fur (ferric uptake regulator) proteins control iron uptake in many Gram-negative bacteria . Although Fur homologues have been identified in Gram-positive bacteria, their roles in gene regulation are unknown . Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG . We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri-siderophore uptake genes is constitutive in the yqkL mutant . Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur . B . subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation . Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor . We demonstrate that a ygaG mutant has the perR phenotype: It is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA . Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of managanous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene . Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.

Mol Microbiol, 1998 Jul, 29(1), 179 - 87
A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition; Moriya S et al.; We have analysed the function of a gene of Bacillus subtilis, the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation . Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null) . Both could form colonies at 23 degree C but not at 37 degree C in the absence of the expression of the Smc protein, indicating that the B . subtilis smc gene was essential for cell growth at higher temperatures . Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23 degree C and in the conditional null mutant grown in low concentrations of IPTG at 37 degree C . In addition, immunofluorescence microscopy showed that subcellular localization of the SpoOJ partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells . These results indicate that the B . subtilis smc gene is essential for chromosome partition . The role of B . subtilis Smc protein in chromosome partition is discussed.

Mol Microbiol, 1998 Jul, 29(1), 85 - 95
A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis; Cervin MA et al.; The SpoOJA and SpoOJB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid-partitioning proteins, respectively, and mutation of spoOJB prevents the expression of stage II genes of sporulation . This phenotype is a consequence of SpoOJA activity in the absence of SpoOJB, and its basis was unknown . In the studies reported here, SpoOJA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor SpoOA and RNA polymerase with the spollG promoter . This repressor-like activity is likely to be the basis for preventing the onset of differentiation in vivo . SpoOJB is known to neutralize SpoOJA activity in vivo and also to interact with a mitotic-like apparatus responsible for chromosome partitioning . These data suggest that SpoOJA and SpoOJB form a regulatory link between chromosome partition and development gene expression.

Mol Microbiol, 1998 Jul, 29(1), 19 - 26
On the birth and fate of bacterial division sites; Bouche JP et al.; Thanks to genetics, to the study of protein-protein interactions and to direct viewing of subcellular structures by the use of immunofluorescence and green fluorescent protein (GFP) fusions, the organization of the constriction apparatus of walled bacteria is gradually coming to light . The tubulin-like protein FtsZ assembles as a ring around the site of constriction and operates as an organizer and activator of septum-shaping proteins . Much less is known about the factors specifying the location of FtsZ rings . Circumstantial evidence favours the presence at future ring positions of fixed elements, the potential division sites (PDS), before FtsZ assembles . FtsZ polymerization is initiated from a point on a PDS, the nucleation site, still to be identified, and proceeds bidirectionally around the cell . We hypothesize that new PDS are specified in a manner that depends on the functioning of an active chromosome partition apparatus . This view is supported by the fact that formation of mid-cell PDS requires initiation of DNA replication, and by recent studies supporting the existence of a specialized partition apparatus in a variety of microorganisms . Although PDS may be specified directly by the partition apparatus, indirect localization linked to compartmentalized gene expression during chromosome segregation is also possible . Once created, PDS are used in a regulated manner, and several mechanisms normally operate to direct constriction to selected PDS at the correct time . One, dedicated to the permanent suppression of polar PDS, rests on the minicell suppression system and involves a protein that is able to discriminate between polar and non-polar sites . Another is involved in asymmetric site selection at the early stages of sporulation in Bacillus subtilis . Finally, a mechanism observed only in certain multi-nucleated cells appears to favour division at non-polar PDS related to the most ancient replication/DNA segregation events.

J Formos Med Assoc, 1998 Jul, 97(7), 445 - 52
Molecular mechanisms of clarithromycin resistance in Helicobacter pylori; Hsieh PF et al.; Combination antibiotic therapy for Helicobacter pylori has now become the standard means of treating peptic ulcer diseases . Clarithromycin is a newly adopted antibiotic for H . pylori eradication . However, resistance to clarithromycin reduces the efficacy of clarithromycin-containing regimens . We explored mechanisms of clarithromycin resistance by evaluating H . pylori for macrolide resistance mechanisms reported in H . pylori and other bacteria . Degenerate polymerase chain reaction analysis of the H . pylori genome failed to yield products homologous to methylase, a drug inactivation enzyme, or efflux pumps . Clarithromycin selection in Escherichia coliNM522, transformed with an expression library that was constructed with genomic DNA from a clarithromycin-resistant strain of H . pylori, revealed six clones that conferred clarithromycin resistance consistently after retransformation . Southern hybridization and DNA sequencing revealed that four of the six clones contained the same locus . Comparison of DNA and amino acid sequences showed that the 1.3-kb DNA fragment had significant homology to the 3-oxoadipate CoA-transferase subunit A (yxjD) and subunit B (yxjE) of Bacillus subtilis . However, the clarithromycin inactivation assay and knockout mutation analysis showed that the gene increased clarithromycin resistance in E . coli, but not in H . pylori . In contrast, sequencing of the 23S rRNA gene in six clarithromycin-resistant H . pylori clinical isolates revealed an A to G transitional mutation at position 2515 of the 23S rRNA gene in all isolates . Natural transformation with the 23S rRNA gene from resistant strains conferred clarithromycin resistance in clarithromycin-sensitive strains . We conclude that the 23S rRNA mutation is sufficient to confer clarithromycin resistance and that it is the major mechanism of clarithromycin resistance in H . pylori.

J Chromatogr B Biomed Sci Appl, 1998 Jun 26, 711(1-2), 319 - 29
On the kinetics of phase separation in aqueous two-phase systems; Salamanca MH et al.; The effect of the tie-line location (phase volume ratio) on the kinetics of phase separation in batch PEG/salt aqueous two-phase systems (ATPS) has been investigated . PEG/sulphate systems with a stability ratio (sr) of 0.34 and 0.37 and relative tie-line lengths in the range 0.1 to 0.6 for a continuous top phase and in the range 0.03 to 0.15 for a continuous bottom phase were used in the batch studies . A continuous settler was designed with three different inlet geometries . Phase separation is much faster when the bottom phase is continuous and in this case the location on the tie-line and the presence or absence of Bacillus subtilis extract makes little difference . When the top phase is continuous the relative sizes of the phases (phase ratio, R . relative distance on tie-line, rd) has an important effect, the larger the top phase (larger R and rd) the slower the phase separation . The presence of Bacillus extract also makes the operation slower which is more marked at the largest values of R (and rd) . At the largest volume ratios (R or rd) three different settling regions have been recognised, a region of coalescence, a region of drops moving to the interphase and a region where drops queue at the interphase to coalesce into the large phase . A modified correlation that takes into account the location on the tie-line and thus volume ratio (R) and relative distance (rd) has been proposed and successfully tested . The behavior of batch and continuous systems in the presence and absence of Bacillus subtilis extract in systems with continuous bottom phase was also studied . The settling velocity was lower in the continuous than in the batch systems, and in both cases the initial rate was lower in the presence of Bacillus extract.

J Bacteriol, 1998 Aug, 180(16), 4309 - 13
Identification and expression of the Bacillus subtilis fructose-1, 6-bisphosphatase gene (fbp); Fujita Y et al.; The Bacillus subtilis fbp gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified as yydE . The fbp gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis . fbp transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript . Interestingly, B . subtilis FBPase exhibited no significant similarity to other FBPases in protein sequence databases.

J Bacteriol, 1998 Aug, 180(16), 4291 - 3
Substitution of an alanine residue for glycine 146 in TMP kinase from Escherichia coli is responsible for bacterial hypersensitivity to bromodeoxyuridine; Tourneux L et al.; The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2'-deoxyuridine were characterized comparatively . The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase . Plasmids carrying the wild-type tmk gene from Escherichia coli or Bacillus subtilis, but not the defective tmk gene, restored the resistance to bromodeoxyuridine of an E . coli mutant strain.

J Bacteriol, 1998 Aug, 180(16), 4243 - 51
A molecular switch controlling competence and motility: competence regulatory factors ComS, MecA, and ComK control sigmaD-dependent gene expression in Bacillus subtilis; Liu J et al.; Bacillus subtilis, like many bacteria, will choose among several response pathways when encountering a stressful environment . Among the processes activated under growth-restricting conditions are sporulation, establishment of motility, and competence development . Recent reports implicate ComK and MecA-ClpC as part of a system that regulates both motility and competence development . MecA, while negatively controlling competence by inhibiting ComK, stimulates sigmaD-dependent transcription of genes that function in motility and autolysin production . Both ComK-dependent and -independent pathways have been proposed for MecA's role in the regulation of motility . Mutations in mecA reduce the transcription of hag . encoding flagellin, and are partially suppressed by comK in both medium promoting motility and medium promoting competence . Reduced sigmaD levels are observed in mecA mutants grown in competence medium, but no change in sigmaD concentration is detected in a comK mutant . The comF operon, transcription of which requires ComK, is located immediately upstream of the operon that contains the flgM gene, encoding the sigmaD-specific antisigma factor . An insertion mutation that disrupts the putative comF-flgM transcription unit confers a phenotype identical to that of the comK mutant with respect to hag-lacZ expression . Expression of a flgM-lacZ operon fusion is reduced in both sigD and comK mutant cells but is abolished in the sigD comK double mutant . Reverse transcription-PCR examination of the comF-flgM transcript indicates that readthrough from comF into the flgM operon is dependent on ComK . ComK negatively controls the transcription of hag by stimulating the transcription of comF-flgM, thereby increasing the production of the FlgM antisigma factor that inhibits sigmaD activity . There likely exists another comK-independent mechanism of hag transcription that requires mecA and possibly affects the sigmaD concentration in cells undergoing competence development.

J Bacteriol, 1998 Aug, 180(16), 4212 - 8
One of two osmC homologs in Bacillus subtilis is part of the sigmaB-dependent general stress regulon; Volker U et al.; In this report we present the identification and analysis of two Bacillus subtilis genes, yklA and ykzA, which are homologous to the partially RpoS-controlled osmC gene from Escherichia coli . The yklA gene is expressed at higher levels in minimal medium than in rich medium and is driven by a putative vegetative promoter . Expression of ykzA is not medium dependent but increases dramatically when cells are exposed to stress and starvation . This stress-induced increase in ykzA expression is absolutely dependent on the alternative sigma factor sigmaB, which controls a large stationary-phase and stress regulon . ykzA is therefore another example of a gene common to the RpoS and sigmaB stress regulons of E . coli and B . subtilis, respectively . The composite complex expression pattern of the two B . subtilis genes is very similar to the expression profile of osmC in E . coli.

J Bacteriol, 1998 Aug, 180(16), 4166 - 70
Suppression of TGA mutations in the Bacillus subtilis spoIIR gene by prfB mutations; Karow ML et al.; An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the spoIIR locus of Bacillus subtilis . Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations . Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the prfB gene encoding protein release factor 2 . These are the first mutations shown to map to the B . subtilis prfB locus . The sequence of the prfB gene was completed, and two revisions of the published sequence were made . The five prfB mutations also resulted in suppression of the catA86-TGA mutation to between 19 and 54% of the expression of catA86(+), compared to the readthrough level of 6% in the prfB+ strain . N-terminal sequencing of suppressed catA86-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the prfB1 mutations was tryptophan.

Microbiology, 1998 Jul, 144 ( Pt 7), 1799 - 805
A transposition-induced mutant of Nostoc ellipsosporum implicates an arginine-biosynthetic gene in the formation of cyanophycin granules and of functional heterocysts and akinetes; Leganes F et al.; In strain NE1 of Tn5-1058-mutagenized Nostoc ellipsosporum, the transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis . The argC reported from Anabaena sp . strain PCC 7120 hybridized to a sequence different from the one interrupted by the transposon in NE1 . The newly identified gene from N . ellipsosporum was denoted argL . The argL mutation renders certain processes in strain NE1 conditionally dependent on provision of L-arginine . Heterocysts and apparent akinetes that formed in the absence of added L-arginine failed to fix dinitrogen or to germinate, respectively, and lacked granules of cyanophycin, composed of copolymers of arginine and aspartic acid . However, apparent akinetes that differentiated upon growth of the mutant in the presence of L-arginine plus nitrate formed cyanophycin granules and could regenerate a new culture.

J Biol Chem, 1998 Aug 14, 273(33), 21217 - 24
SecDF of Bacillus subtilis, a molecular Siamese twin required for the efficient secretion of proteins; Bolhuis A et al.; In the present studies, we show that the SecD and SecF equivalents of the Gram-positive bacterium Bacillus subtilis are jointly present in one polypeptide, denoted SecDF, that is required to maintain a high capacity for protein secretion . Unlike the SecD subunit of the pre-protein translocase of Escherichia coli, SecDF of B . subtilis was not required for the release of a mature secretory protein from the membrane, indicating that SecDF is involved in earlier translocation steps . Strains lacking intact SecDF showed a cold-sensitive phenotype, which was exacerbated by high level production of secretory proteins, indicating that protein translocation in B . subtilis is intrinsically cold-sensitive . Comparison with SecD and SecF proteins from other organisms revealed the presence of 10 conserved regions in SecDF, some of which appear to be important for SecDF function . Interestingly, the SecDF protein of B . subtilis has 12 putative transmembrane domains . Thus, SecDF does not only show sequence similarity but also structural similarity to secondary solute transporters . Our data suggest that SecDF of B . subtilis represents a novel type of the SecD and SecF proteins, which seems to be present in at least two other organisms.

Electrophoresis, 1998 Jun, 19(8-9), 1501 - 5
Two-dimensional gel electrophoresis for proteome projects: the effects of protein hydrophobicity and copy number; Wilkins MR et al.; Two-dimensional (2-D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type . Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two-dimensional gel . The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms . The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2-D gels . Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2-D gel separations of whole cell lysates . A clear hydrophobicity cutoff point was seen, above which current 2-D electrophoresis methods appear not to be useful for protein separation . The effect of cellular protein copy number on a protein's presence on a 2-D gel was investigated by means of a graphical model . This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2-D gel . Considering the current maximum in 2-D gel loading capacity, it was found that 2-D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell . We conclude that further developments of 2-D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.

Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1109 - 14
Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli; Kobayashi K et al.; We cloned and characterized a gene, tgl, encoding transglutaminase in Bacillus subtilis . The tgl gene contained a open reading frame 735-nucleotides long that encoded a 245-residue protein with the molecular weight of 28,300 . The deduced amino acid sequence had little sequence similarity with sequences of other transglutaminases from a Streptoverticillium sp . or from mammals . The -10 and -35 regions of a putative promoter resembled the consensus sequence for the sigma K-dependent promoter . In addition, a sequence similar to the consensus sequence for the GerE binding site was found upstream from this region . These findings suggested that tgl was transcribed in the mother cells during a late stage of sporulation . Evidence for this suggestion was that transglutaminase activity was detected in sporulating cells during the same stage . Transglutaminase activity was detected in Escherichia coli cells transformed with a plasmid for expression of the tgl gene.

Microb Comp Genomics, 1997, 2(2), 103 - 11
Characterization of a family of bacterial response regulator aspartyl-phosphate (RAP) phosphatases; Reizer J et al.; We have characterized a novel family of response regulator aspartyl-phosphate (RAP) phosphatases found exclusively in gram-positive bacteria . The family consists of 15 members, 12 of which are from Bacillus subtilis . The N-terminal domains proved to be more highly conserved than the C-terminal domains, and a signature sequence for the family was derived from the former domains . Phylogenetic analyses revealed clustering patterns showing that all Bacillus proteins are closely related . Most of the Bacillus RAP phosphatase genes are followed by and are translationally coupled to small nonhomologous phosphatase regulator (phr) genes that encode exported peptides with regulatory functions . Most of the paralogous RAP phosphatases of B . subtilis may serve related functions in signal transduction systems . They appear to have arisen by relatively recent gene duplication events that occurred after the divergence of major groups within the gram-positive bacterial kingdom . We suggest that the N-terminal domains of the RAP phosphatases function in catalysis, whereas the C-terminal domains function in regulation.

Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9590 - 5
NADP, corepressor for the Bacillus catabolite control protein CcpA; Kim JH et al.; Expression of the alpha-amylase gene (amyE) of Bacillus subtilis is subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism in Bacillus and other Gram-positive bacteria . To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to the amyE operator, amyO . Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription . HPr-P (Ser-46), proposed as an effector of CcpA, also was tested . In DNase I footprint assays, the affinity of CcpA for amyO was stimulated 2-fold by fructose-1,6-diphosphate (FDP), 1.5-fold by oxidized or reduced forms of NADP, and 10-fold by HPr-P (Ser-46) . However, the triple combinations, CcpA/NADP/HPr-P (Ser-46) and CcpA/FDP/HPr-P (Ser-46) synergistically stimulated DNA-binding affinity by 120- and 300-fold, respectively . NADP added to CcpA specifically stimulated transcription inhibition of the amyE promoter by 120-fold . CcpA combined with HPr (Ser-46) inhibited transcription from the amyE promoter, but it also inhibited several control promoters . FDP did not stimulate transcription inhibition by CcpA nor did the triple combinations . The finding that NADP had little effect on CcpA DNA binding but increased the ability of CcpA to inhibit transcription suggests that catabolite repression is not simply caused by CcpA binding amyO but rather a result of interactions with the transcription machinery enhanced by NADP.

FEBS Lett, 1998 Jul 3, 430(3), 385 - 9
The influence of protein folding on late stages of the secretion of alpha-amylases from Bacillus subtilis; Stephenson K et al.; A derivative of the alpha-amylase from Bacillus licheniformis (AmyL) engineered to give an active enzyme with increased net positive charge is secreted by Bacillus subtilis with a yield that is significantly lower than that of the native enzyme . This reduction in yield is the result of increased proteolysis during or shortly after translocation through the cytoplasmic membrane . When we compared the overall rate of folding of the engineered derivative (AmyLQS50.5) with that of AmyL it exhibited a greater dependency on Ca2+ ions for in vitro folding . When the concentration of Ca2+ in the growth medium was increased, so too did the relative yield of AmyLQS50.5 . We discuss the importance of secretory protein folding at the membrane/cell wall interface with respect to the yield of native and heterologous proteins from B . subtilis.

Appl Environ Microbiol, 1998 Aug, 64(8), 2875 - 81
Influence of a cell-wall-associated protease on production of alpha-amylase by Bacillus subtilis; Stephenson K et al.; AmyL, an extracellular alpha-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth . Nevertheless, when AmyL is produced and secreted by B . subtilis, it is subject to considerable cell-associated proteolysis . Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B . subtilis wprA gene . Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity . Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL . A construct in which wprA is not expressed exhibits an increased yield of alpha-amylase . The potential role of wprA in protein secretion is discussed, together with implications for the use of B . subtilis and related bacteria as hosts for the secretion of heterologous proteins.

Antimicrob Agents Chemother, 1998 Aug, 42(8), 2041 - 7
Acquisition of certain streptomycin-resistant (str) mutations enhances antibiotic production in bacteria; Hosoya Y et al.; Physiological differentiation (including antibiotic production) in microorganisms usually starts when cells encounter adverse environmental conditions and is frequently accompanied by an increase in the accumulation of intracellular ppGpp . We have found that the acquisition of certain streptomycin-resistant (str) mutations enables cells to overproduce antibiotics, demonstrating an increase in productivity 5- to 50-fold greater than that of wild-type strains . The frequency of such antibiotic-overproducing strains among the str mutants was shown to range from 3 to 46%, as examined with several strains of the genera Streptomyces, Bacillus, and Pseudomonas . Analysis of str mutants from Bacillus subtilis Marburg 168 revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12, changing Lys-56 (corresponding to Lys-43 in Escherichia coli) to Asn, Arg, Thr, or Gln . Antibiotic productivity increased in a hierarchical manner depending upon which amino acid residue replaced Lys at this position . The strA1 mutation, a genetic marker frequently used for mapping, had no effect on antibiotic productivity even though it was found to result in an amino acid alteration of Lys-56 to Ile . Gene replacement experiments with the str alleles demonstrated unambiguously that the str mutation is responsible for the antibiotic overproductivity observed . These results offer a rational approach for improving the production of antibiotic (secondary metabolism) from microorganisms.

Nucleic Acids Res, 1998 Aug 15, 26(16), 3806 - 12
The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase; Cervin MA et al.; Initiation of sporulation in Bacillus subtilis is controlled by several regulators which affect activation by phosphorylation of the key response regulator Spo0A or transcription of Spo0A-P-dependent genes . In vivo overexpression of one of these regulators, sinR , results in suppression of transcription from the Spo0A-P-dependent promoters of spo0A , spoIIA , spoIIE and spoIIG and in vitro SinR binds to the promoters of the spoIIA operon and the spo0A gene . In this study we have demonstrated that in vitro SinR directly repressed Spo0A- P-dependent transcription by B.subtilis RNA polymerase from the spoIIG operon promoter . SinR inhibited transcription prior to formation of heparin-resistant complexes but did not displace RNA polymerase from the spoIIG promoter . DNase I protection studies demonstrated that SinR protected a large region of the spoIIG promoter and induced DNase I hypersensitive sites, particularly around the 0A boxes, at the same positions as those induced by zinc . Since binding of zinc induces bends in the DNA, we concluded that SinR binding also altered the conformation of the spoIIG promoter . We propose that SinR-induced conformational changes in Spo0A-dependent promoters prevent activation of trans-cription by Spo0A-P.

Nucleic Acids Res, 1998 Aug 15, 26(16), 3717 - 23
Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P; Odell L et al.; The ribozyme from bacterial ribonuclease P recognizes two structural modules in a tRNA substrate: the T stem-loop and the acceptor stem . These two modules are connected through a helical linker . The T stem-loop binds at a surface confined in a folding domain away from the active site . Substrates for the Bacillus subtilis RNase P RNA were previously selected in vitro that are shown to bind comparably well or better than a tRNA substrate . Chemical modification of P RNA-substrate complexes with dimethylsulfate and kethoxal was performed to determine how the P RNA recognizes three in vitro selected substrates . All three substrates bind at the surface known to interact with the T stem-loop of tRNA . Similar to a tRNA, the secondary structure of these substrates contains a helix around the cleavage site and a hairpin loop at the corresponding position of the T stem-loop . Unlike a tRNA, these two structural modules are connected through a non-helical linker . The two structural modules in the tRNA and in the selected substrates bind to two different domains in P RNA . The properties of substrate recognition exhibited by this ribozyme may be exploited to isolate new ribozyme-substrate pairs with interactive structural modules.

J Biol Chem, 1998 Aug 7, 273(32), 20494 - 503
trp RNA-binding attenuation protein-mediated long distance RNA refolding regulates translation of trpE in Bacillus subtilis; Du H et al.; Expression of the trpEDCFBA operon is regulated at both the transcriptional and translational levels by the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis . When cells contain sufficient levels of tryptophan to activate TRAP, the protein binds to trp operon transcripts as they are being synthesized, most often causing transcription termination . However, termination is never 100% efficient, and transcripts that escape termination are subject to translational control . We determined that TRAP-mediated translational control of trpE can occur via a novel RNA conformational switch mechanism . When TRAP binds to the 5'-untranslated leader segment of a trp operon read-through transcript, it can disrupt a large secondary structure containing a portion of the TRAP binding target . This promotes refolding of the RNA such that the trpE Shine-Dalgarno sequence, located more than 100 nucleotides downstream from the TRAP binding site, becomes sequestered in a stable RNA hairpin . Results from cell-free translation, ribosome toeprint, and RNA structure mapping experiments demonstrate that formation of this structure reduces TrpE synthesis by blocking ribosome access to the trpE ribosome binding site . The role of the Shine-Dalgarno blocking hairpin in controlling translation of trpE was confirmed by examining the effect of multiple nucleotide substitutions that abolish the structure without altering the Shine-Dalgarno sequence itself . The possibility of protein-mediated RNA refolding as a general mechanism in controlling gene expression is discussed.

Anal Biochem, 1998 Jul 15, 261(1), 1 - 7
Development of a magnetic microplate chemifluorimmunoassay for rapid detection of bacteria and toxin in blood; Yu H et al.; A magnetic microplate chemifluorimmunoassay (MMCIA) is described using an immunomagnetic separation and a fluorescent microplate technique for rapid detection of low-level Escherichia coli O157:H7, Bacillus subtilis var . niger spores, and Staphylococcal enterotoxin type B from whole blood . In general, the MMCIA has at least several-fold more sensitivity than the conventional enzyme-linked immunosorbent assay . In addition, the assay sensitivities using direct fluorochrome label as the reporter, or alkaline phosphatase (AP) with various assay substrates, such as pNPP and AttoPhos, were assessed .

J Bacteriol, 1998 Aug, 180(15), 4007 - 10
Role of Pho-P in transcriptional regulation of genes involved in cell wall anionic polymer biosynthesis in Bacillus subtilis; Qi Y et al.; tagA, tagD, and tuaA operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in Bacillus subtilis . Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited . Expression of these genes is controlled by PhoP-PhoR, a two-component system . It has been proposed that Pho-P plays a key role in the activation of tuaA and the repression of tagA and tagD . In this study, we demonstrated the role of Pho-P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system . The results indicate that PhoP approximately P is sufficient to repress the transcription of the tagA and tagD promoters and also to activate the transcription of the tuaA promoter.

J Bacteriol, 1998 Aug, 180(15), 3765 - 70
Promoter recognition by Bacillus subtilis sigmaW: autoregulation and partial overlap with the sigmaX regulon; Huang X et al.; The Bacillus subtilis genome encodes at least 17 distinct sigma factors, including seven members of the extracytoplasmic function (ECF) subfamily . We have investigated the expression and regulation of the ECF sigma factor encoded by the sigW gene . A sigmaW-dependent promoter (PW) precedes sigW, demonstrating that this transcription factor is positively autoregulated . Expression of sigW is regulated by both growth phase and medium composition . Maximal expression is attained in early-stationary-phase cells grown in rich medium . We previously reported that sigW mutants have elevated transcription of some sigmaX-controlled genes, and we now report that the converse is also true: in a sigX mutant, PW is derepressed during logarithmic growth . Thus, these two regulons are mutually antagonistic . Reconstituted sigmaW holoenzyme faithfully recognizes the PW preceding sigW but does not recognize the PX promoter preceding the sigX gene . Autoregulation of sigX is also highly specific: sigmaX holoenzyme initiates transcription from PX but recognizes PW poorly if at all . In contrast, several promoters that are at least partially under sigmaX control are active with both the sigmaX and sigmaW holoenzymes in vitro . This finding supports the suggestion that the sigmaW and sigmaX regulons overlap . Sequence comparisons suggest that promoters recognized by these two sigma factors have similar -35 elements but are distinguished by different base preferences at two key positions within the -10 element.

Extremophiles, 1997 Feb, 1(1), 22 - 8
Diverse genes of alkaliphilic Bacillus firmus OF4 that complement K+-uptake-deficient Escherichia coli include an ftsH homologue; Ito M et al.; Seven clones isolated from libraries of DNA from alkaliphilic Bacillus firmus OF4 restored the growth of a K+-uptake-deficient Escherichia coli mutant on only 10 mM K+ . None of the clones contained genes with apparent homology to known K+ transport systems in other organisms . Based on sequence homologies, the newly isolated alkaliphile loci included: ftsH; a dipeptide transport system; a gerC locus with hydrophobic open reading frames not found in the comparable locus of Bacillus subtilis; a sugar phosphotransferase enzyme; and a capBC homologue . The ftsH gene provided a new and striking example of a recognized property of extracellular and external regions of polytopic alkaliphile proteins: a significant paucity of basic amino acid residues relative to neutrophile counterparts . The alkaliphile ftsH gene was able to complement a mutant of E . coli with a temperature-sensitive ftsH gene product.

Mol Microbiol, 1998 Jun, 28(6), 1187 - 97
PhoP-P and RNA polymerase sigmaA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP-P activator sites within the coding region stimulate transcription in vitro; Qi Y et al.; The Bacillus subtilis pstS operon and phoA gene are members of the Pho regulon that is controlled by PhoR, a histidine kinase, and PhoP, a response regulator . Footprinting analysis showed that phosphorylated PhoP extended the PhoP protected region in pstS and phoA promoters, and also bound to a separate site within the coding region of each gene . Our previous in vivo studies have shown that, in contrast to other Pho regulon promoters that are not expressed in either phoP or phoR mutants, a low-level induction from the pstS promoter (25% of parent strain) can be detected in a phoR mutant . In this study, by using an in vitro transcription system, we demonstrate that (i) only phosphorylated PhoP is a transcriptional activator of the pstS operon and of the phoA gene; (ii) phosphorylated PhoP and RNA polymerase sigmaA holoenzyme are sufficient for in vitro transcription of the pstS promoter and the phoA promoter; (iii) the activation of the pstS promoter requires lower concentrations of phosphorylated PhoP than does the phoA promoter for transcription; and (iv) PhoP binding sites in both the pstS promoter core binding region and in the 5' coding region of the gene, which have been identified by footprinting analysis, are important for the transcription of the pstS promoter in vitro.

DNA Res, 1998 Apr 30, 5(2), 121 - 6
An 8 kb nucleotide sequence at the 3' flanking region of the sspC gene (184 degrees) on the Bacillus subtilis 168 chromosome containing an intein and an intron; Ghim SY et al.; As part of the Bacillus subtilis genome sequencing project, we determined the complete nucleotide sequence of an 8000-bp fragment downstream of the sspC gene (184 degrees) of the B . subtilis 168 chromosome . The sequence analysis shows that the sspC gene is located inside of the SP beta region, which differs from the current genetic map of B . subtilis 168 . This region contains 12 putative ORFs (yojQ through yojZ and sspC) . A homology search for the deduced products of the ORFs shows significant similarities to enzymes involved in deoxyribonucleotide metabolism: ribonucleotide reductase (Nrd) E, NrdF, thioredoxin and dUTPase . Interestingly, this DNA fragment includes two split genes, yojP containing conserved motifs of an intein and yojQ and yojS with an 808-bp intervening sequence for a putative intron structure . In addition, the yojR gene includes a putative new DNA replication terminator.

FEBS Lett, 1998 Jun 23, 430(1-2), 28 - 36
The complete genome of Bacillus subtilis: from sequence annotation to data management and analysis; Moszer I; The completion of the entire 4.2-Mb genome sequence of the gram-positive bacterium Bacillus subtilis has been a milestone for biological studies on this model organism . This paper describes bioinformatics work related to this joint European and Japanese project: methods and strategies for gene annotation and detection of sequencing errors, using an integrated cooperative computer environment (Imagene); construction of a specialized database for data management and a WWW server for data retrieval (SubtiList); DNA sequence analysis, yielding striking results on oligonucleotide bias, repeated sequences, and codon usage, all landmarks of evolutionary events shaping the B . subtilis genome.

J Biol Chem, 1998 Jul 31, 273(31), 19542 - 7
Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor; Oguro A et al.; Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family . It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA . In the precursor, the 5'- and 3'-flanking regions form a stable double-stranded structure based on their complementary sequence . This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNase III . The B . subtilis enzyme that has similar activity to Escherichia coli RNase III has been purified and is designated Bs-RNase III . Recently, B . subtilis rncS has been shown to encode Bs-RNase III (Wang, W., and Bechhofer, D . H . (1997) J . Bacteriol . 179, 7379-7385) . We show here that Bs-RNase III and the purified His-tagged product of rncS cleave pre-scRNA at both 5'- and 3'-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3'-site is less efficient . The 5'-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3'-end . Bs-RNase III cleavage yields a two-base 3'-overhang, which is consistent with the manner in which E . coli RNase III cleaves . We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant . It is concluded that Bs-RNase III is an enzyme that processes pre-scRNA.

Biochem J, 1998 Aug 1, 333 ( Pt 3), 565 - 71
4-Aminobutyrate (GABA) transporters from the amine-polyamine-choline superfamily: substrate specificity and ligand recognition profile of the 4-aminobutyrate permease from Bacillus subtilis; Brechtel CE et al.; A previous study {Ferson, Wray and Fisher (1996) Mol . Microbiol . 22, 693-701} has shown that transposon-mediated disruption of a protein 47% identical to the Escherichia coli GABA (4-aminobutyrate) transporter abolishes the ability of nitrogen-limited culture conditions to induce expression of a GABA transport activity in Bacillus subtilis . Here it is demonstrated directly that the B . subtilis GABA permease (gabP) gene can complement the transport defect in the gabP-negative E . coli strain . Unexpectedly, the ligand-recognition profile of the B . subtilis GabP was found to differ substantially from that of the highly homologous E . coli GabP . Unlike the E . coli GabP, the B . subtilis GabP: (i) exhibits approx . equal preference for the 3-carbon (beta-alanine, Km=9.6 microM) and the 4-carbon (GABA, Km=37 microM) amino acids, and (ii) resists inhibition by bulky, conformationally constrained compounds (e.g . nipecotic acid, guvacine), which are active against GABA transporters from brain . The present study shows additionally that the B . subtilis GabP can translocate several open-chain GABA analogues (3-aminobutyrate, 3-aminopropanoate, cis-4-aminobutenoate) across the membrane via counterflow against {3H}GABA . Thus, consistent with the idea that the ligand-recognition domain of the B . subtilis GabP is less spacious than that of the close homologue from E . coli, the former exhibits more stringent requirements than the latter for substrate recognition and translocation . These distinct functional characteristics of the E . coli and B . subtilis GABA transporters provide a basis by which to identify ligand-recognition domains within the amine-polyamine-choline transporter superfamily.

Biochim Biophys Acta, 1998 Jul 28, 1386(1), 211 - 9
Purification and biochemical analysis of WprA, a 52-kDa serine protease secreted by B . subtilis as an active complex with its 23-kDa propeptide; Babe LM et al.; The Gram-positive bacterium Bacillus subtilis produces numerous proteases that are secreted to the extracellular milieu, and as strains are generated which lack the more prominent proteases, minor ones become detectable . We have isolated a 52-kDa secreted protease from the protease-deficient strain WB600 . It is encoded by the wprA gene which encompasses a signal sequence, a 46-kDa propeptide further processed to 23 kDa, and the 52-kDa mature protease . The 52-kDa and 23-kDa polypeptides were previously detected in cell-wall preparations of a wild-type strain . We have co-purified these proteins from culture supernatant, and confirmed the same N-termini and molecular weights as the membrane-bound species . The WprA protease domain has 28.5% identity to subtilisin A, and like other subtilisins, it displays a broad substrate specificity . WprA and subtilisin A have similar pH profiles, showing optimal activity near pH 7.5 for substrates with Met, Gln, or Lys residues at P1 . Using a substrate with Asp at P1, another peak of activity was observed for WprA at pH 5 and at pH 6 for subtilisin A . The pH dependence of some bacterial proteases in their interaction with substrates and inhibitors may be biologically relevant.

Protein Expr Purif, 1998 Jul, 13(2), 155 - 62
Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli; Roy S et al.; We have designed a novel coupled transcriptional construct wherein Escherichia coli uracil DNA glycosylase (UDG) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (Ptrc) . Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo . The system has also been exploited for purification of large amounts of Ugi . While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide corresponding to UDG . We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins . Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed .

Can J Microbiol, 1998 Apr, 44(4), 378 - 81
Overproduction of the Bacillus subtilis glutamyl-tRNA synthetase in its host and its toxicity to Escherichia coli; Pelchat M et al.; The Bacillus subtilis glutamyl-tRNA synthetase (GluRS), encoded by the gltX gene, aminoacylates its homologous tRNA(Glu) and tRNA(Gln) with glutamate . This gene was cloned with its sigma A promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and B . subtilis . Transformation of B . subtilis with this recombinant plasmid (pMP411) led to a 30-fold increase of glutamyl-tRNA synthetase specific activity in crude extracts . Transformation of E . coli with this plasmid gave no recombinants, but transformation with plasmids bearing an altered gltX was successful . These results indicate that the presence of B . subtilis glutamyl-tRNA synthetase is lethal for E . coli, probably because this enzyme glutamylates tRNA1(Gln) in vivo as it does in vitro.

Nucleic Acids Res, 1998 Aug 1, 26(15), 3584 - 90
The -16 region of Bacillus subtilis and other gram-positive bacterial promoters; Voskuil MI et al.; The Bacillus subtilis alpha-amylase promoter amy P contains an essential TGTG motif (-16 region) upstream of the -10 region . Mutations of this region significantly reduced in vitro promoter strength . A -15 G-->C transversion eliminated transcription from amy P by both B.subtilis and Escherichia coli RNA polymerase (RNAP) . A second alpha-amylase promoter ( amy P2) also required the -16 region for function . To determine conserved sequences in promoters containing -16 region elements, sequences of 64 B.subtilis promoters with the second TG motif of the -16 region were aligned and analyzed . Unlike the E.coli class of 'extended -10 promoters', with a similar TG motif but lacking a -35 region, the -16 region promoters contain highly conserved -35 regions . They also contain conserved A n and T n tracts upstream of the -35 region . In addition, we analyzed all available gram-positive bacterial promoter compilations to determine the generality of the -16 region . From this analysis, the -16 region TRTG motif (R = purine) appears to be a basic element found in a large portion of gram-positive bacterial promoters and is, in the case of at least the alpha-amylase promoters, necessary for transcription by the major form of B.subtilis and E.coli RNAP.

J Exp Med, 1998 Jul 20, 188(2), 305 - 15
Mechanism of gram-positive shock: identification of peptidoglycan and lipoteichoic acid moieties essential in the induction of nitric oxide synthase, shock, and multiple organ failure; Kengatharan KM et al.; The incidence of septic shock caused by gram-positive bacteria has risen markedly in the last few years . It is largely unclear how gram-positive bacteria (which do not contain endotoxin) cause shock and multiple organ failure . We have discovered recently that two cell wall fragments of the pathogenic gram-positive bacterium Staphylococcus aureus, lipoteichoic acid (LTA) and peptidoglycan (PepG), synergize to cause the induction of nitric oxide (NO) formation, shock, and organ injury in the rat . We report here that a specific fragment of PepG, N-acetylglucosamine-beta-{1--> 4}-N-acetylmuramyl-L-alanine-D-isoglutamine, is the moiety within the PepG polymer responsible for the synergism with LTA (or the cytokine interferon gamma) to induce NO formation in the murine macrophage cell line J774.2 . However, this moiety is also present in the PepG of the nonpathogenic bacterium Bacillus subtilis . We have discovered subsequently that S . aureus LTA synergizes with PepG from either bacterium to cause enhanced NO formation, shock, and organ injury in the rat, whereas the LTA from B . subtilis does not synergize with PepG of either bacterium . Thus, we propose that the structure of LTA determines the ability of a particular bacterium to cause shock and multiple organ failure (pathogenicity), while PepG acts to amplify any response induced by LTA.

Mol Gen Genet, 1998 Jun, 258(5), 538 - 45
The stability of mRNA from the gsiB gene of Bacillus subtilis is dependent on the presence of a strong ribosome binding site; Jurgen B et al.; In Bacillus subtilis IS58 starved of glucose or exposed to heat shock, ethanol or salt stress, the sigmaB-dependent general stress protein GsiB is accumulated to a higher level than other general stress proteins . This high-level accumulation of GsiB can at least partially be attributed to the remarkably long half-life (approximately 20 min) of the gsiB mRNA . Analysis of different gsiB-lacZ fusions revealed that this stability is not determined by sequences at the 3' end of the transcript but rather by sequences upstream of the translational start codon . Site-directed mutagenesis established that a strong ribosome binding site was crucial for the increased stability of the gsiB mRNA . A comparison of the sequences upstream of the translational start codons of three general stress genes, gsiB, gspA and ctc, revealed a direct correlation between mRNA stability and the strength of their translational signals.

Tuber Lung Dis, 1997, 78(1), 3 - 12
Mycobacterium tuberculosis sigF is part of a gene cluster with similarities to the Bacillus subtilis sigF and sigB operons; DeMaio J et al.; The Mycobacterium tuberculosis (MTB) SigF alternate sigma factor has been shown to have significant homology to the Bacillus subtilis (BSU) stress-response sigma factor, SigB, as well as to the BSU developmental sigma factor, SigF . In this study we report that like both the BSU sigB and sigF genes, MTB sigF is preceded by an open reading frame (usfX) encoding a protein with significant homology to the previously described BSU anti-sigma factors, RsbW and SpollAB . Sequence analysis suggests that the usfX and sigF genes appear to be cotranscribed and translationally coupled . A second open reading frame called usfY precedes usfX, but has no significant homologues and may not be contranscribed with the usfX and sigF . The sigF gene has been overexpressed in Escherichia coli, purified, and used to raise polyclonal antibodies . Immunoblotting demonstrates that MTB SigF is antigenically closer to BSU SigB than to BSU SigF . Fusion of the MTB sigF gene to the MTB hsp60 promoter has demonstrated that inappropriate overexpression of sigF is lethal for the slow-grower Mycobacterium bovis bacille Calmette-Guerin (BCG), but not for the rapid-grower Mycobacterium smegmatis which lacks a sigF homologue . Hence, sigF, encoding an MTB stress response, stationary phase transcription factor, is preceded by an antisigma factor homologue and is incompatible with growth when constitutively overexpressed in BCG.

Biofactors, 1998, 7(4), 337 - 44
Ribonucleotide reductase in Bacillus subtilis--evidence for a Mn-dependent enzyme; Mohamed SF et al.; The reduction of 2'-ribonucleotides to 2'-deoxyribonucleotides, a unique step in DNA formation, is catalyzed by ribonucleotide reductase (RRase), an allosterically regulated, cell cycle-dependent enzyme . This work reports a reversible impairment of DNA formation and ribonucleotide reduction upon manganese depletion in Bacillus subtilis demonstrated through in vivo labeling with necleic acid precursors and enzyme assays with ether-permeabilized cells . No deoxyadenosylcobalamin-dependent reduction of ribonucleotides was detected in the cytosol, and the properties of a partially purified enzyme fraction, i.e., sensitivity towards EDTA and hydroxyurea (HU), indicated a metal-dependent type of RRase . The enzyme was enriched by gel filtration on Superose 12 from glycerol- or fumarate-grown cells and submitted to Q-band electron paramagnetic resonance (EPR) spectroscopy for further characterization of the metal center . A distinct Mn(II) signal was obtained in both preparations characteristic of a protein-bound mangaenese in a mononuclear metal center with axial symmetry . The intensity of this Mn signal was not affected by addition of the radical scavenger HU (10 mM) but reduced in the presence of 2.5 mM EDTA . On the basis of these results, we suggest that Bacillus subtilis has a Mn-dependent ribonucleotide reductase.

Biochemistry, 1998 Jul 14, 37(28), 10126 - 33
Recognition of the 5' leader and the acceptor stem of a pre-tRNA substrate by the ribozyme from Bacillus subtilis RNase P; Loria A et al.; The catalysis by the ribozyme from bacterial RNase P involves specific interactions with the structure of the tRNA substrate . Recognition of the T stem-loop by this ribozyme occurs in a groove-like structure dictated by the tertiary folding of tRNA {Loria, A., and Pan, T . (1997) Biochemistry 36, 6317} . Effects of 2'-OH --> 2'-H modifications within the acceptor stem and the 5' leader on substrate binding and catalysis are determined using a tRNAPhe substrate that is significantly cleaved at more than one site . In all but one case, the 2'-deoxy substitution has little effect on binding for cleavage at the correct and incorrect sites . Substitution of the 2'-OH group at the correct site, however, decreases the cleavage chemistry by more than 3.4 kcal/mol for cleavage at both the correct and incorrect sites . Substitutions of the 2'-OH groups at the incorrect sites have no effect for cleavage at the incorrect and correct sites . Truncation of the 5' leader results in differential effects on cleavage at different sites . These observations lead to a model in which cleavage at the correct and incorrect sites involves formation of different ribozyme-substrate complexes depending on binding of specific nucleotides in the 5' leader . Binding of the T stem-loop of tRNA and the 2'-OH group at the correct cleavage site is common for all ES complexes . An A/U-rich 5' leader significantly promotes formation of the ES complex and accelerates the cleavage chemistry over those of a C/G-rich 5' leader, but only moderately enhances cleavage at the correct site over cleavage at the incorrect sites . Since cleavage at different sites requires formation of different ES complexes, cleavage site selection can occur at the level of the ES complex and at the chemical step.

Biochemistry, 1998 Jul 14, 37(28), 9991 - 8
Electron transfer kinetics during the reduction and turnover of the cytochrome caa3 complex from Bacillus subtilis; Assempour M et al.; The cytochrome caa3 complex from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory enzyme complexes . The key difference in the cytochrome caa3 complex lies in the addition of a domain, homologous with mitochondrial cytochrome c, that is fused to the C-terminal end of its subunit II . Measurements of steady-state and transient reduction kinetics have been carried out on the cytochrome caa3 complex . Reduction of the cyanide-bound enzyme with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) supports a sequence of electron transfer in which cytochromec is reduced initially, and this is followed by rapid internal electron transfer from cytochrome c to CuA and from CuA to cytochrome a . Steady-state kinetics with exogenous cytochrome c as the substrate demonstrates the capability of the cytochrome caa3 complex to act as a cytochrome c oxidase . The cytochrome c from B . subtilis is the most efficient cytochrome c of those tested . Steady-state kinetics with ascorbate-TMPD as the reductant, in the absence of exogenous cytochrome c, reveals a biphasic pattern even though only a single, covalent cytochrome c interaction site is present . The two-phase kinetics are characterized by a low activity phase associated with a high apparent affinity for TMPD and a high activity phase with a low affinity for TMPD . This pattern is observed over a wide range of ionic strengths and enzyme concentrations, and with both purified and membrane extract forms of cytochrome caa3 . It is proposed that the biphasic steady-state kinetics of this oxidase, and other members of the cytochrome oxidase superfamily, do not result directly from different interactions with cytochrome c but are due to a change in the redox kinetics within the centers of the conventional oxidase unit itself . Our results will be related to models that account for the biphasic steady-state kinetics exhibited by cytochrome oxidase.

Mol Microbiol, 1998 Jun, 28(5), 981 - 90
A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: implications for co-ordination of chromosome segregation and cell division; Sharpe ME et al.; The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome . Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites . Soon after duplication, sister oriC/Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 microm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle . The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication . It could also explain how segregation can proceed accurately in the absence of cell division . The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.

Mol Microbiol, 1998 Jun, 28(5), 931 - 43
The Bacillus SpoIIGA protein is targeted to sites of spore septum formation in a SpoIIE-independent manner; Fawcett P et al.; The process of bacterial cell division involves the assembly of a complex of proteins at the site of septation that probably provides both the structural and the cytokinetic functions required for elaboration and closure of the septal annulus . During sporulation in Bacillus subtilis, this complex of proteins is modified by the inclusion of a sporulation-specific protein, SpoIIE, which plays a direct role in gene regulation and also has a genetically separable role in determining the gross structural properties of the specialized sporulation septum . We demonstrate by both green fluorescent protein (GFP) fusions and indirect immunofluorescence microscopy that SpoIIGA, a protein required for proteolytic cleavage of pro-sigmaE, is also targeted to the sporulation septum . Septal localization of SpoIIGA-GFP occurred even in the structurally abnormal septum formed by a SpoIIE null mutant . We also report the isolation of a spoIIGA homologue from Bacillus megaterium, a species in which the cells are significantly larger than those of B . subtilis . We have exploited the physical dimensions of the B . megaterium sporangium, in conjunction with wide-field deconvolution microscopy, to construct three-dimensional projections of sporulating cells . These projections indicate that SpoIIGA-GFP is initially localized in an annulus at the septal periphery and is only later localized uniformly throughout the septa . Localization was also detected in a B . subtilis spo0H null strain that fails to construct a spore septum . We propose that SpoIIGA is sequestered in the septum by an interaction with components of the septation machinery and that this interaction begins before the construction of the asymmetric septum.

Mol Microbiol, 1998 Jun, 28(5), 883 - 92
Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis; Webb CD et al.; We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle . The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor . We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle . This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J . The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1) . The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region . These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.

Mikrobiologiia, 1998 Mar-Apr, 67(2), 165 - 9
{Effect of Bacillus intermedius RNAases with various catalytic activity on multiplication of Escherichia coli and Bacillus subtilis}; Kipenskaia LV et al.; The effect of the Bacillus intermedius ribonuclease and its mutant forms derived by site-specific mutagenesis on the growth of gram-positive and gram-negative bacteria was studied . Both catalytically active and catalytically inactive (mutant) ribonucleases stimulated bacterial growth, the extent of stimulation correlating with the catalytic activity of the enzymes . It was suggested that the biological activity of exogenous ribonucleases is mainly due to their catalytic activity.

Gene, 1998 May 28, 212(1), 137 - 46
The Streptomyces coelicolor sporulation-specific sigma WhiG form of RNA polymerase transcribes a gene encoding a ProX-like protein that is dispensable for sporulation; Tan H et al.; In the non-motile mycelial organism Streptomyces coelicolor A3(2), the sporulation gene whiG encodes a protein that closely resembles RNA polymerase sigma factors such as sigma D of Bacillus subtilis, which mainly control motility and chemotaxis genes . Here, we show that the whiG gene product, purified from an Escherichia coli strain carrying an expression construct, could activate E . coli core RNA polymerase in vitro to transcribe a sigma D-dependent motility-related promoter from B . subtilis . Such RNA polymerase holoenzyme preparations could also transcribe from an S . coelicolor promoter, PTH4, previously shown to require an intact whiG gene for in-vivo transcription . The in-vivo dependence on whiG was therefore shown to be direct . Unusually, the initiation of PTH4 transcription in vitro depended on the provision of appropriate dinucleotides . The whiG-dependent PTH4 transcription unit consisted of a single gene, orfTH4 . Sequence comparisons suggested that the gene product was a member of a small group of proteins that include the B . subtilis and E . coli ProX proteins . Though none of these proteins shared more than about 30% of extended primary sequence identity, they had similar size and hydropathy profiles, and could be aligned end to end to reveal a mosaic of similarities . The ProX proteins of B . subtilis and E . coli are implicated in glycine betaine transport in response to hyperosmotic stress . However, disruption of orfTH4 did not cause any obvious phenotypic changes in growth or development on media of varying osmotic strengths.

J Bacteriol, 1998 Jul, 180(14), 3578 - 83
A region in the Bacillus subtilis transcription factor Spo0A that is important for spoIIG promoter activation; Buckner CM et al.; Spo0A is a DNA binding protein in Bacillus subtilis required for the activation of spoIIG and other promoters at the onset of endospore formation . Activation of some of these promoters may involve interaction of Spo0A and the sigmaA subunit of RNA polymerase . Previous studies identified two single-amino-acid substitutions in sigmaA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo . Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in sigmaA . We also found that the S231F substitution partially restored use of the spoIIG promoter by the sigmaA H359R RNA polymerase in vitro . Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229 . This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding . Moreover, the alanine-substituted Spo0A protein activated the spoIIA promoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, sigmaH-directed transcription . These and other results suggest that the region of Spo0A near position 229 is involved in sigmaA-dependent promoter activation.

Microbiology, 1998 May, 144 ( Pt 5), 1349 - 58
Antibacterial action of dipeptides containing an inhibitor of glucosamine-6-phosphate isomerase; Chmara H et al.; Several dipeptides, containing the N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) moiety linked to protein and non-protein amino acids, exhibited a strong growth-inhibitory and bactericidal effect against Bacillus subtilis . FMDP-dipeptides were efficiently transported into bacterial cells by a di-tripeptide permease and subsequently cleaved by intracellular Mn2+/Co2+-dependent peptidases . Cleavage rates {0.1-5.6 micromol min-1 (mg protein)-1} were about two orders of magnitude lower than transport rates {40-200 micromol min-1 (mg dry wt)-1} . The released FMDP inactivated glucosamine-6-phosphate (GlcN-6-P) isomerase, an enzyme catalysing the first committed step in a biosynthetic pathway leading to amino sugar-nucleotide precursors of bacterial peptidoglycan . Inhibition of GlcN-6-P isomerase precluded peptidoglycan biosynthesis and resulted in a strong bacteriolytic effect . Results of the studies on consequences of GlcN-6-P isomerase inhibition upon the action of FMDP-dipeptides provided evidence demonstrating that the lack of endogenous GlcN-6-P could be a reason for the triggering of bacterial autolysis . Peptides containing the inhibitors of GlcN-6-P isomerase are one of the very few antimicrobial agents known that exhibit both bactericidal and fungicidal effects.

J Bacteriol, 1998 Jul, 180(14), 3730 - 3
General stress transcription factor sigmaB and sporulation transcription factor sigmaH each contribute to survival of Bacillus subtilis under extreme growth conditions; Gaidenko TA et al.; The general stress response of the bacterium Bacillus subtilis is controlled by the sigmaB transcription factor . Here we show that loss of sigmaB reduces stationary-phase viability 10-fold in either alkaline or acidic media and reduces cell yield in media containing ethanol . We further show that loss of the developmental transcription factor sigmaH also has a marked effect on stationary-phase viability under these conditions and that this effect is independent from the simple loss of sporulation ability.

J Bacteriol, 1998 Jul, 180(14), 3697 - 703
Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis; Inaoka T et al.; Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores . The Sod activity in vegetative cells was maximal at stationary phase . Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod . The specific activity of purified Sod was approximately 2, 600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer . The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method . DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp . Several putative promoter sites were located in the upstream region of sodA . The deduced amino acid sequence showed high homology with other bacterial manganese Sods . Conserved regions in bacterial manganese Sod could also be seen . The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B . subtilis sodA.

J Bacteriol, 1998 Jul, 180(14), 3671 - 80
Isolation and characterization of Bacillus subtilis sigB operon mutations that suppress the loss of the negative regulator RsbX; Smirnova N et al.; sigmaB, a transcription factor that controls the Bacillus subtilis general stress response regulon, is activated by either a drop in intracellular ATP or exposure to environmental stress . RsbX, one of seven sigmaB regulators (Rsb proteins) whose genes are cotranscribed with sigmaB, is a negative regulator in the stress-dependent activation pathway . To better define the interactions that take place among the Rsb proteins, we analyzed sigB operon mutations which suppress the high-level sigmaB activity that normally accompanies the loss of RsbX . Each of these mutations was in one of three genes (rsbT, -U, and -V) which encode positive regulators of sigmaB, and they all defined amino acid changes which either compromised the activities of the mutant Rsbs or affected their ability to accumulate . sigmaB activity remained inducible by ethanol in several of the RsbX- suppressor strains . This finding supports the notion that RsbX is not needed as the target for sigmaB activation by at least some stresses . sigmaB activity in several RsbX- strains with suppressor mutations in rsbT or -U was high during growth and underwent a continued, rather than a transient, increase following stress . Thus, RsbX is likely responsible for maintaining low sigmaB activity during balanced growth and for reestablishing sigmaB activity at prestress levels following induction . Although RsbX likely participates in limiting the sigmaB induction response, a second mechanism for curtailing unrestricted sigmaB activation was suggested by the sigmaB induction profile in two suppressor strains with mutations in rsbV . sigmaB activity in these mutants was stress inducible but transient, even in the absence of RsbX.

J Bacteriol, 1998 Jul, 180(14), 3650 - 6
General stress transcription factor sigmaB and its role in acid tolerance and virulence of Listeria monocytogenes; Wiedmann M et al.; The gene encoding the general stress transcription factor sigmaB in the gram-positive bacterium Listeria monocytogenes was isolated with degenerate PCR primers followed by inverse PCR amplification . Evidence for gene identification includes the following: (i) phylogenetic analyses of reported amino acid sequences for sigmaB and the closely related sigmaF proteins grouped L . monocytogenes sigmaB in the same cluster with the sigmaB proteins from Bacillus subtilis and Staphylococcus aureus, (ii) the gene order in the 2, 668-bp portion of the L . monocytogenes sigB operon is rsbU-rsbV-rsbW-sigB-rsbX and is therefore identical to the order of the last five genes of the B . subtilis sigB operon, and (iii) an L . monocytogenes sigmaB mutant had reduced resistance to acid stress in comparison with its isogenic parent strain . The sigB mutant was further characterized in mouse models of listeriosis by determining recovery rates of the wild-type and mutant strains from livers and spleens following intragastric or intraperitoneal infection . Our results suggest that sigmaB-directed genes do not appear to be essential for the spread of L . monocytogenes to mouse liver or spleen at 2 and 4 days following intragastric or intraperitoneal infection.

J Bacteriol, 1998 Jul, 180(14), 3584 - 91
Spo0A mutants of Bacillus subtilis with sigma factor-specific defects in transcription activation; Hatt JK et al.; The transcription factor Spo0A of Bacillus subtilis has the unique ability to activate transcription from promoters that require different forms of RNA polymerase holoenzyme . One class of Spo0A-activated promoter, which includes spoIIEp, is recognized by RNA polymerase associated with the primary sigma factor, sigma A (sigmaA); the second, which includes spoIIAp, is recognized by RNA polymerase associated with an early-sporulation sigma factor, sigma H (sigmaH) . Evidence suggests that Spo0A probably interacts directly with RNA polymerase to activate transcription from these promoters . To identify residues of Spo0A that may be involved in transcriptional activation, we used PCR mutagenesis of the entire spo0A gene and designed a screen using two distinguishable reporter fusions, spoIIE-gus and spoIIA-lacZ . Here we report the identification and characterization of five mutants of Spo0A that are specifically defective in activation of sigmaA-dependent promoters while maintaining activation of sigmaH-dependent promoters . These five mutants identify a 14-amino-acid segment of Spo0A, from residue 227 to residue 240, that is required for transcriptional activation of sigmaA-dependent promoters . This region may define a surface or domain of Spo0A that makes direct contacts with sigmaA-associated holoenzyme.

J Bacteriol, 1998 Jul, 180(14), 3548 - 55
Dual promoters are responsible for transcription initiation of the fla/che operon in Bacillus subtilis; Estacio W et al.; The fla/che region contains more than 30 genes required for flagellar synthesis and chemotaxis in Bacillus subtilis, including the gene for the flagellum-specific sigmaD factor, sigD . Sequence and primer extension data demonstrate that a PA promoter immediately upstream of flgB, henceforth referred to as the fla/che PA, and the PD-3 promoter are active in vivo . Transcription from the PD-3 element is dependent on sigmaD activity and is regulated by the flagellum-specific negative regulator, FlgM . In a strain containing a deletion of fla/che PA (PADelta), sigmaD protein was not detected, demonstrating that the fla/che PA is necessary for wild-type expression of the sigD gene . Thus, sigD is part of the >26-kb fla/che operon . Consistent with a lack of detectable sigmaD protein, the PADelta strain grows as long filaments and does not express a sigmaD-dependent hag::lacZ reporter construct . These phenotypes are indicative of a lack of sigD expression or complete inhibition of sigmaD activity by FlgM . However, sigmaD activity is found in a double mutant containing the PADelta and a null mutation in flgM . The double mutant no longer grows as long filaments, and expression of hag::lacZ is partially restored . These data demonstrate that a low level of sigmaD activity does exist in the PADelta mutant but can be detected only in the presence of a null mutation in flgM . Therefore, normal expression of sigD may also involve another promoter(s) within the fla/che operon.

Virology, 1998 Jul 5, 246(2), 329 - 40
Genes and regulatory sites of the "host-takeover module" in the terminal redundancy of Bacillus subtilis bacteriophage SPO1; Stewart CR et al.; Early in infection of Bacillus subtilis by bacteriophage SPO1, the synthesis of most host-specific macromolecules is replaced by the corresponding phage-specific biosyntheses . It is believed that this subversion of the host biosynthetic machinery is accomplished primarily by a cluster of early genes in the SPO1 terminal redundancy . Here we analyze the nucleotide sequence of this 11.5-kb "host-takeover module," which appears to be designed for particularly efficient expression . Promoters, ribosome-binding sites, and codon usage statistics all show characteristics known to be associated with efficient function in B . subtilis . The promoters and ribosome-binding sites have additional conserved features which are not characteristic of their host counterparts and which may be important for competition with host genes for the cellular biosynthetic machinery . The module includes 24 genes, tightly packed into 12 operons driven by the previously identified early promoters PE1 to PE12 . The genes are smaller than average, with half of them having fewer than 100 codons . Most of their inferred products show little similarity to known proteins, although zinc finger, trans-membrane, and RNA polymerase-binding domains were identified . Transcription-termination and RNase III cleavage sites were found at appropriate locations.

J Biol Chem, 1998 Jul 10, 273(28), 17326 - 32
Identification of protein-protein contacts between alpha/beta-type small, acid-soluble spore proteins of Bacillus species bound to DNA; Hayes CS et al.; Small, acid-soluble spore proteins (SASP) of the alpha/beta-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA . Significant protein cross-linking was not detected in the absence of DNA . In all four alpha/beta-type SASP examined, the amino donor in the EDC induced amide cross-links was the alpha-amino group of the protein . However, the carboxylate containing amino acid residues involved in cross-linking varied . In SASP-A and SASP-C of Bacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation . In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins . These data indicate that, although there are likely to be subtle structural differences between different alpha/beta-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state.

Biochemistry, 1998 Jun 30, 37(26), 9409 - 16
The protein component of Bacillus subtilis ribonuclease P increases catalytic efficiency by enhancing interactions with the 5' leader sequence of pre-tRNAAsp; Crary SM et al.; Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the formation of the mature 5' end of tRNA . To investigate the role of the protein component in enhancing the affinity of Bacillus subtilis RNase P for substrate (Kurz, J . C., Niranjanakumari, S., Fierke, C . A . (1998) Biochemistry 37, 2393), the kinetics and thermodynamics of binding and cleavage were analyzed for pre-tRNAAsp substrates containing 5' leader sequences of varying lengths (1-33 nucleotides) . These data demonstrate that the cleavage rate constant catalyzed by the holoenzyme is not dependent on the leader length; however, the association rate constant for substrate binding to holoenzyme increases as the length of the leader increases, and this is reflected in enhanced substrate affinity of up to 4 kcal/mol . In particular, the protein component of RNase P stabilizes interactions with nucleotides at -2 and -5 in the 5' leader sequence of the pre-tRNA substrate . A 1 nucleotide leader decreases substrate affinity >/=15-fold compared to tRNAAsp due to ground-state destabilization of the enzyme-substrate complex . This destabilization is overcome by increasing the length of the leader to 2 nucleotides due to P RNA-pre-tRNA contacts that are stabilized by the P protein . The affinity of RNase P holoenzyme (but not RNA alone) for pre-tRNAAsp is further enhanced with a substrate containing a 5 nucleotide leader . These data indicate that novel direct or indirect interactions occur between the 5' leader sequence of pre-tRNAAsp and the protein component of RNase P.

Mol Gen Genet, 1998 May, 258(4), 385 - 8
The UP element of the promoter for the flagellin gene, hag, stimulates transcription from both SigD- and SigA-dependent promoters in Bacillus subtilis; Caramori T et al.; DNA sequences upstream (UP element) of the core promoter (-10, -35 region) of the Bacillus subtilis flagellin gene hag stimulate transcription in vivo and in vitro . We constructed a number of hybrids, placing the UP element of hagp upstream of the core of one SigD-dependent (fliDp) and two SigA-dependent (tmsp, vegp) B . subtilis promoters . The hybrid promoters were fused to a lacZ reporter gene and their activity tested in vivo . The presence of the UP module enhanced transcription at both types of promoters . We conclude that the hagp UP sequence can act as a promoter module independently of the core sequence.

Vet Microbiol, 1998 Feb 28, 60(2-4), 215 - 25
Effect of Bacillus subtilis spore administration on activation of macrophages and natural killer cells in mice; Kosaka T et al.; The effect of Bacillus subtilis (strain A102) spores on the activation of murine macrophages and natural killer cells (NK) was examined . The macrophage activity and NK activity were enhanced by oral administration of A102 spores, and slightly enhanced by oral administration of culture supernatant . There was no difference in the results of macrophage activity and NK activity using other live or dead spores . The NK activity and macrophage activity were increased with increments of concentration up to 0.1 g per mouse, and both activities were decreased at concentration of more than 0.15 g per mouse . The NK activity was increased 1 and 2 days after oral administration of A102 spores, and the activity level 2 days after administration was about 3-fold higher than the level prior to treatment . Macrophage activity was also increased from 1 to 3 days after oral administration of A102 spores, and the activity level 3 days after administration was about 3-fold higher than the level prior to treatment . The induction of interferons at 1 day after oral administration in mouse serum was 5-fold higher than that in controls . These findings indicate that oral administration of A102 gave rise to the induction of interferons, and it is likely that macrophages and NK cells were activated by interferons.

J Biochem (Tokyo), 1998 Jul, 124(1), 98 - 104
Feedback loops involving Spo0A and AbrB in in vitro transcription of the genes involved in the initiation of sporulation in Bacillus subtilis; Fujita M et al.; Through mainly in vivo studies, the initiation of sporulation in Bacillus subtilis has been shown to depend on the phosphorylation of the Spo0A transcription factor mediated by the multicomponent phosphorelay via KinAB (C), Spo0F, Spo0B, and Spo0A in this order . RNA polymerase containing sigmaA (EsigmaA) or sigmaH (EsigmaH) transcribes the genes of the phosphorelay components . Phosphorylated Spo0A is also involved in their expression and is required for the induction of sigmaH by repressing its repressor gene abrB . We have examined the effects of phosphorylated Spo0A (Spo0A-P) and AbrB on in vitro transcription of the genes involved in the Spo0A phosphorylation and initiation of sporulation . Spo0A-P repressed EsigmaA-dependent transcription of the kinC and EsigmaH-dependent transcription of spo0A and kinA . EsigmaH-dependent transcription of spo0F was stimulated by Spo0A-P at low concentrations but was repressed by higher amounts of Spo0A-P . On the other hand, AbrB repressed EsigmaA-dependent transcription of spo0H (sigmaH gene), kinC, and abrB, although its effect was not strong . With the present results providing in vitro evidence for the roles of Spo0A-P and AbrB as transcriptional regulators, and other results described in the literature, the positive and negative feedback loops controlling the temporal expression of early sporulation genes are discussed.

J Biochem (Tokyo), 1998 Jul, 124(1), 89 - 97
Promoter selectivity of the Bacillus subtilis RNA polymerase sigmaA and sigmaH holoenzymes; Fujita M et al.; The sigmaH of Bacillus subtilis directs transcription of a large number of early sporulation genes, whereas the principal sigma factor, sigmaA, is essential for the transcription of the genes for vegetative growth and early sporulation . We have purified sigmaA and sigmaH proteins, and characterized their properties . The genes encoding sigmaA or sigmaH were separately cloned into an expression vector under the control of T7 promoter . Both proteins were overproduced in Escherichia coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride . Antigenicities and N-terminal amino acid sequences of the overproduced proteins were used to identify both proteins . Unlike sigmaA protein, sigmaH protein showed a DNA-binding ability . To compare the promoter selectivity of the sigmaA protein with that of the sigmaH protein, transcription in vitro of 16 promoters was performed using RNA polymerase holoenzymes reconstituted from a purified core enzyme with either sigmaH or sigmaA . These holoenzymes correctly recognized each of the cognate promoters; sigmaH-RNA polymerase recognized sigmaH promoters but not sigmaA promoters, and vice versa . A competition experiment for core RNA polymerase using sigmaA and sigmaH revealed that sigmaA had a stronger affinity . We propose that the predicted replacement of a sigma subunit in a holoenzyme from sigmaA to sigmaH in vivo at late logarithmic growth phase may require an additional factor, or the modification of a core enzyme or sigma factor.

Mol Microbiol, 1998 May, 28(4), 787 - 802
Stress induction of the Bacillus subtilis clpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance; Gerth U et al.; The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced . The amount of clpP-specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin . Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by sigmaA and sigmaB transcriptional factors of the B . subtilis RNA polymerase respectively . Transcription initiation occurred predominantly at the putative sigmaA-dependent promoter in exponentially growing cells and was induced under stress conditions . After exposure to stress, initiation of transcription also increased at the sigmaB-dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter-controlled subgroup of class III general stress genes in B . subtilis . In a sigB mutant strain, clpP remained heat and stress inducible at the sigmaA-dependent promoter . BgaB-reporter gene fusions, carrying either the sigmaA- or the sigmaB-dependent promoter, showed a higher bgaB induction at the sigmaA-dependent promoter, whereas a significantly lower level of induction was measured at the sigmaB-dependent promoter . The sigmaA-dependent promoter appeared to be crucial for the heat-inducible transcription of clpP . A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites . Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type . Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation . Comparison of two-dimensional protein gels from wild-type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern . Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.

J Bacteriol, 1998 Jul, 180(13), 3483 - 5
The fnr gene of Bacillus licheniformis and the cysteine ligands of the C-terminal FeS cluster; Klinger A et al.; In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated . Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues . Unlike in Fnr from B . subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center . Transfer of the B . licheniformis gene to an fnr mutant of B . subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth.

J Bacteriol, 1998 Jul, 180(13), 3470 - 3
tetA(L) mutants of a tetracycline-sensitive strain of Bacillus subtilis with the polynucleotide phosphorylase gene deleted; Bechhofer DH et al.; A Bacillus subtilis strain with the polynucleotide phosphorylase gene deleted was sensitive for growth in the presence of tetracycline . This strain was used to select for tetracycline-resistant mutants . A point mutation in the tetA(L) promoter and a spontaneously occurring tetA(L) gene copy number mutant were characterized.

J Bacteriol, 1998 Jul, 180(13), 3405 - 9
Genetic recombination in Bacillus subtilis 168: effects of recU and recS mutations on DNA repair and homologous recombination; Fernandez S et al.; Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions . The yppB:cat and ypbC:cat null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec+ B . subtilis strain . The yppB gene complemented the defect of the recG40 strain . yppB and ypbC and their respective null alleles were termed "recU" and "recU1" (recU:cat) and "recS" and "recS1" (recS:cat), respectively . The recU and recS mutations were introduced into rec-deficient strains representative of the alpha (recF), beta (addA5 addB72), gamma (recH342), and epsilon (recG40) epistatic groups . The recU mutation did not modify the sensitivity of recH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells . The recS mutation did not modify the sensitivity of addAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recU cells . The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells . Intramolecular recombination was blocked in recS recF, recS addAB, and recS recU cells . RecU and RecS have no functional counterparts in Escherichia coli . Altogether, these data indicate that the recU and recS proteins are required for DNA repair and intramolecular recombination and that the recF (alpha epistatic group), addAB (beta), recH (gamma), recU (epsilon), and recS genes provide overlapping activities that compensate for the effects of single mutation . We tentatively placed recS within a new group, termed "zeta".

J Bacteriol, 1998 Jul, 180(13), 3360 - 7
Replication terminator protein-based replication fork-arrest systems in various Bacillus species; Griffiths AA et al.; The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region . A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another . In the present work, RTP homologs in four species closely related to B . subtilis (B . atrophaeus, B . amyloliquefaciens, B . mojavensis, and B . vallismortis) have been identified and characterized . An RTP homolog could not be identified in another closely related species, B . licheniformis . The nucleotide and amino acid changes from B . subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species . The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B . licheniformis . Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP . The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning . By coincidence, the single change (E30K) found in the B . mojavensis RTP corresponds exactly to that purposefully introduced by others into B . subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism . The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B . subtilis in vivo . Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B . subtilis 168 genome sequence to bring the total to nine.

J Bacteriol, 1998 Jul, 180(13), 3304 - 11
Anaerobic regulation of Bacillus subtilis Krebs cycle genes; Nakano MM et al.; Krebs cycle enzyme activity in Bacillus subtilis was examined under aerobic and anaerobic conditions . Citrate synthase and aconitase activities in cells grown anaerobically in the presence of nitrate were reduced by as much as 10- and 30-fold, respectively, from levels observed under aerobic culture conditions . The maximum level of isocitrate dehydrogenase activity during anaerobic growth was only twofold lower than that in aerobic cultures . These reductions in activity under conditions of anaerobiosis were found to be primarily the result of reduced Krebs cycle gene transcription . This repression was not dependent on either the fnr or resDE gene products, which have been shown to regulate expression of other B . subtilis genes in response to anaerobic conditions . Additionally, catabolite control proteins CcpA and CcpB were not responsible for the repression . A dyad symmetry element located between positions -73 and -59 relative to the transcription start site of the aconitase gene (citB) promoter was previously shown to be a target of catabolite repression and the binding site for a putative negative regulator during aerobic growth . The deletion of the upstream arm of the dyad symmetry region abolished the citB repression observed during anaerobic growth . Furthermore, neither citZ or citB was repressed in an anaerobically grown citB mutant, an effect that was very likely the result of citrate accumulation . These results suggest that catabolite repression and anaerobic repression of citZ and citB are regulated by a common mechanism that does not involve CcpA, CcpB, Fnr, or ResDE.

J Bacteriol, 1998 Jul, 180(13), 3285 - 94
A complex pattern of traveling stripes is produced by swimming cells of Bacillus subtilis; Mendelson NH et al.; Motile cells of Bacillus subtilis inadvertently escaped from the surface of an agar disk that was surrounded by a fluid growth medium and formed a migrating population in the fluid . When viewed from above, the population appeared as a cloud advancing unidirectionally into the fresh medium . The cell population became spontaneously organized into a series of stripes in a region behind the advancing cloud front . The number of stripes increased progressively until a saturation value of stripe density per unit area was reached . New stripes arose at a fixed distance behind the cloud front and also between stripes . The spacing between stripes underwent changes with time as stripes migrated towards and away from the cloud front . The global pattern appeared to be stretched by the advancing cloud front . At a time corresponding to approximately two cell doublings after pattern formation, the pattern decayed, suggesting that there is a maximum number of cells that can be maintained within the pattern . Stripes appear to consist of high concentrations of cells organized in sinking columns that are part of a bioconvection system . Their behavior reveals an interplay between bacterial swimming, bioconvection-driven fluid motion, and cell concentration . A mathematical model that reproduces the development and dynamics of the stripe pattern has been developed.

J Bacteriol, 1998 Jul, 180(13), 3276 - 84
Establishment of prespore-specific gene expression in Bacillus subtilis: localization of SpoIIE phosphatase and initiation of compartment-specific proteolysis; Lewis PJ et al.; Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in Bacillus subtilis . Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum . Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein . The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor sigma E and the other dependent on the prespore-specific sigma factor sigma F.

J Mol Biol, 1998 Jun 19, 279(4), 773 - 93
Derivation of the three-dimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis; Massire C et al.; The secondary structure of bacterial RNase P RNA, a ribozyme responsible for the maturation of the 5' end of tRNAs, is well established on the basis of sequence comparison analysis . RNase P RNA secondary structures fall into two types, A and B, which share a common core formed by the assembly of two main folding domains, but differ in their peripheral elements.A revised alignment of 137 available sequences reveals new covariations allowing for the refinement of both types of secondary structures . Phylogenetic evidence is thus provided for the extension of stems P11, P14, P19, P10.1 and P15.1 through further canonical base-pairs or GAellipsisGA mismatches . These refinements led in turn to a new organization of the catalytic core, with coaxial stackings of helices P2 and P19 as well as P1 and P4 . New inter-domain tertiary interactions involve loop L9 and helix P1 and loop L8 with helix P4 . These features were incorporated into atomic-scale 3D models of RNase P RNA for representatives of each structural type, namely Escherichia coli and Bacillus subtilis . In each model, the juxtaposition of the core helices creates a cradle onto which the pre-tRNA substrate binds with most evolutionarily conserved residues converging towards the cleavage site . The inner cores of both types are stabilized similarly, albeit by different peripheral elements, emphasizing the modular and hierarchical organisation of the architecture of RNase P RNAs . Similarities are thus apparent between the type A modules, P16/P17/P6 and P13/P14, and their type B analogs, P5.1/P15.1 and P10 . 1/P10.1a, respectively . Other noteworthy features of these models include compactness and good agreement with published crosslinking data .

Microbiology, 1998 Jun, 144 ( Pt 6), 1593 - 600
The yvsA-yvqA (293 degrees-289 degrees) region of the Bacillus subtilis chromosome containing genes involved in metal ion uptake and a putative sigma factor; Wipat A et al.; The region between yvsA (293 degrees) and yvqA (289 degrees) of the Bacillus subtilis chromosome has been sequenced within the framework of the B . subtilis 168 international sequencing programme . A primary analysis of the 42 ORFs identified in this 43 kb region is presented . The region included a high proportion of genes that did not show homology with genes in other bacteria . The identified ORFs showed homology to proteins involved in the transport of metal ions, two-component signal transducers, ATP-binding-cassette-type transporters and a sigma factor.

Arch Microbiol, 1998 Jul, 170(1), 38 - 42
Two malate dehydrogenases in Methanobacterium thermoautotrophicum; Thompson H et al.; Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized . One was specific for NAD+ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD+ and NADP+ as coenzyme and catalyzed essentially only the reduction of oxalacetate . Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M . thermoautotrophicum (strain DeltaH) . Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related . The NAD+-specific malate dehydrogenase showed high sequence similarity to L-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)+-using malate dehyrogenase showed high sequence similarity to L-lactate dehydrogenase from Thermotoga maritima and L-malate dehydrogenase from Bacillus subtilis . A function of the two malate dehydrogenases in NADPH:NAD+ transhydrogenation is discussed.

J Mol Biol, 1998 May 29, 279(1), 165 - 73
Identification of target promoters for the Bacillus subtilis sigma X factor using a consensus-directed search; Huang X et al.; The promoter selectivity of RNA polymerase (RNAP) can be altered by the association with alternative sigma subunits . Bacillus subtilis hosts a multitude of sigma factors, several of which coordinate the complex developmental program culminating in endospore formation . Genome sequencing has revealed an unanticipated seven new sigma factors of the highly divergent extracytoplasmic function (ECF) sub-family . Virtually nothing is known regarding either the promoter selectivity or the target genes for these newly identified sigma factors . We have used saturation mutagenesis to define a promoter consensus for recognition by one such ECF sigma factor, sigma X . The resulting consensus sequence was used to identify candidate sigma X target sites . Three newly identified sigma X-dependent promoters precede genes encoding regulatory proteins: an AbrB homolog (Abh), a putative response regulator aspartate phosphatase (RapD), and a regulator of autolysin expression (LytR) . sigma X also contributes to the expression of CsbB, a putative membrane-bound glucosyl transferase that is partially controlled by the sigma B stress response sigma factor . Since LytR modulates the expression of the major autolytic amidase and CsbB may function in peptidoglycan synthesis or modification, we suggest that sigma X participates in the regulation of peptidoglycan synthesis and turnover.

Biotechnology (N Y), 1995 Jan, 13(1), 67 - 71
Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant; Lereclus D et al.; The spo0A gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation . It was previously shown that the B . thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spo0A mutant of B . subtilis . In this paper we describe the construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins . The spo0A gene of Bt was cloned and identified by its ability to transform a B . subtilis spo0A mutant to prototrophy . Its nucleotide sequence is homologous to the B . subtilis gene . The spo0A gene was replaced in the Bt genome with a disrupted copy to give an Spo- strain unable to initiate sporulation . When the cryIIIA gene was cloned in the Bt spo0A mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell . These encapsulated toxins were highly active against coleopteran larvae . We anticipate that the cryIIIA expression system and the Bt spo0A mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.

Arch Biochem Biophys, 1998 Jun 1, 354(1), 47 - 56
Purification, properties, and multiple forms of a manganese-activated inorganic pyrophosphatase from Bacillus subtilis; Kuhn NJ et al.; The hydrolysis of magnesium pyrophosphate by inorganic pyrophosphatase from Bacillus subtilis required its specific, time-dependent, and prior activation by Mn2+ ions . This was reversed when Mn2+ ions were removed with EDTA . Free Mn2+ ions were not required for catalysis . Pyrophosphatase purified to near homogeneity gave a single main band of apparent M(r) 36,000 by SDS-PAGE, but of M(r) 34,000 by matrix-assisted laser desorption ionization-mass spectrometry . The native enzyme equilibrated at pH 7 between three distinct molecular forms . Exposure to Mn2+ generated a catalytically active trimer of specific activity about 5000 mumol pyrophosphate hydrolyzed/min/mg protein . Exposure to EDTA generated two catalytically inactive forms, a dimer at low ionic strength and a separate form, of uncharacterized multimeric nature, at molar concentrations of Na2SO4 or Li2SO4 . The latter form was an intermediate in the dimer-trimer transition caused by addition or removal of manganese ions . Mn2+ reacted with this "intermediate" form, apparently by reversible association with two noninteracting binding sites of Kd approximately 0.005 and 0.35 microM, respectively . The properties of this enzyme may account in part for the unusual manganese requirements of B . subtilis and related species.

J Biol Chem, 1998 May 15, 273(20), 12234 - 8
Mutational analysis of invariant arginines in the IIAB(Man) subunit of the Escherichia coli phosphotransferase system; Gutknecht R et al.; The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate . It consists of the transmembrane IIC(Man)-IID(Man) complex and the cytoplasmic IIAB(Man) subunit . IIAB(Man) has two flexibly linked domains, IIA(Man) and IIB(Man), each containing a phosphorylation site (His-10 and His-175) . Phosphoryl groups are transferred from the phosphoryl carrier protein phospho-HPr to His-10, hence to His-175 and finally to the 6' OH of the transported hexose . Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions . IIB(Man) contains five arginines which are invariant in the homologous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis . The IIA domains have no conserved arginines . The five arginines were replaced by Lys or Gln one at a time, and the mutants were analyzed for transport and phosphorylation activity . All five IIB mutants can still be phosphorylated at His-175 by the IIA domain . R172Q is completely inactive with respect to glucose phosphotransferase (phosphoryltransfer from His-175 to the 6' OH of Glc) and hexose transport activity . R168Q has no hexose transport and strongly reduced phosphotransferase activity . R204K has no transport but almost normal phosphotransferase activity . R304Q has only slightly reduced transport activity . R190K behaves like wild-type IIAB(Man) . Arg-168, Arg-172, and Arg-304 are part of the hydrogen bonding network on the surface of IIB, which contains the active site His-175 and the interface with the IIA domain (Schauder, S., Nunn, R.S., Lanz, R., Erni, B . and Schirmer, T . (1998) J . Mol . Biol . 276, 591-602) (Protein Data Bank accession code 1BLE) . Arg-204 is at the putative interface between IIB(Man) and the IIC(Man)-IID(Man) complex.

Pharmazie, 1998 May, 53(5), 300 - 2
Synthesis, lipophilicity and antimicrobial properties of some O-(5-aryl-1,2,4-triazol-3-yl-ethyl)benzaldoximes and O-(5-aryl-1,3,4-oxydiazol-2-yl-ethyl)benzaldoximes; Papakonstantinou-Garoufalias S et al.; The synthesis of benzaldehyde oximethers with suitably substituted 1,2,4-triazoles and 1,3,4-oxadiazoles from the corresponding benzaldoximes is described . The lipophilicity of the compounds was measured as well as their antimicrobial and antifungal activity in vitro . Certain compounds showed relatively significant activity against Bacillus subtilis.

Chem Biol Interact, 1998 May 1, 113(1), 15 - 25
Induction of a SOS repair system in lysogenic bacteria by zearalenone and its prevention by vitamin E; Ghedira-Chekir L et al.; Zearalenone (Zen) is an oestrogenic mycotoxin produced by several Fusarium species in cereals . It induces modifications of haematological parameters in rats with cytotoxicity and inhibition of macromolecular synthesis (nucleic acids and protein) . Zen and its metabolites have oestrogenic and anabolic activities and interact with human oestrogen receptors . Zen and its metabolites showed a positive DNA damaging effect in recombination tests with Bacillus subtilis . It induces sister chromatid exchange and chromosomal aberration in CHO cells . Zen was found to be capable of inducing DNA-adduct formation in mouse liver . The genotoxicity of Zen was questionable until the last decade when increasing data tended to show this toxin to be genotoxic in vivo . However the mechanism of its genotoxicity and mutagenicity has not been completely clarified . The present investigations were designed to show whether Zen induces an SOS-DNA repair response in lysogenic bacteria which have an integrated lambda-bacteriophage in their genome . Zen was found to be genotoxic in the bacterial systems from a concentration of 1.50 mM and it was also bactericidal (IC50 = 1.45 mM) . In addition vitamin E (6.0-12.0 mM) added 1 h prior to the toxin proved to prevent both the genotoxic and bactericidal effects of Zen . This vitamin could be active both as an antioxidant and as a radical scavenger . The specificity of this prevention is probably due to the similarity of structure between vitamin E and Zen.

Biochemistry, 1998 Jun 9, 37(23), 8481 - 9
Implication of His68 in the substrate site of Bacillus subtilis adenylosuccinate lyase by mutagenesis and affinity labeling with 2-{(4-bromo-2,3-dioxobutyl)thio}adenosine 5'-monophosphate; Lee TT et al.; Adenylosuccinate lyase of Bacillus subtilis is inactivated by 2-{(4-bromo-2,3-dioxobutyl)thio}adenosine 5'-monophosphate (2-BDB-TAMP) at pH 7.0 . As the reagent concentration is increased, a maximum rate constant is approached, indicative of reversible enzyme-reagent complex formation (KR = 68 +/- 9 microM) prior to irreversible modification (kmax = 0.081 +/- 0.004 min-1) . Complete inactivation occurs concomitant with about 1 mol of 2-BDB-{14C}TAMP incorporated/mol of enzyme subunit . Adenylosuccinate, or a combination of AMP and fumarate, decreases the inactivation rate and reduces incorporation of {14C} reagent, whereas either AMP or fumarate alone is much less effective . These observations suggest that 2-BDB-TAMP attacks the adenylosuccinate binding site . Proteolytic digestion of inactivated enzyme, followed by purification of the digest by HPLC, yields the radioactive peptide Ile62-Ala72, in which Arg67 and His68 are the most likely targets . Thus 2-BDB-TAMP reacts with adenylosuccinate lyase at a site distinct from the His141 attacked by 6-BDB-TAMP (Lee, Worby, Dixon, and Colman (1997) J . Biol . Chem . 272, 458-465) . Site-directed mutagenesis was used to construct mutant enzymes with replacements for both Arg67 and His68, and either Arg67 or His68 . The R67M mutant enzyme has almost the same specific activity as the wild-type enzyme under standard assay conditions, whereas the single mutant H68Q and double mutant R67M-H68Q enzymes exhibit specific activities that are decreased more than 100-fold . These results indicate that while Arg67 and His68 may both be in the region of the substrate site, only His68 is important for the catalytic activity of B . subtilis adenylosuccinate lyase . A role is proposed for His68 as a general acid-base catalyst.

J Biochem (Tokyo), 1998 May, 123(5), 853 - 8
Effect of B . subtilis TRNA(Trp) on readthrough rate at an opal UGA codon; Matsugi J et al.; Bacillus subtilis has been thought to have a high readthrough rate at the UGA stop codon because no opal suppressor tRNA has been isolated so far {Lovett et al . (1991) J . Bacteriol . 173, 1810-1812} . To examine whether a tRNATrp which we have characterized {Matsugi et al . (1992) Nucleic Acids Res . 20, 3514} has the ability to read the UGA codon, in vitro translation was performed with a synthetic mRNA containing a test codon, UGA, UAG, UAA, or UGG, in a reading frame . Addition of Trp-tRNATrp to the system significantly increased the readthrough rate only in the case of UGA . This suggests that this tRNATrp has a dual recognition pattern in B . subtilis, i.e., for the canonical tryptophan codon and for readthrough at the UGA stop codon.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3090 - 6
Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis; Niranjanakumari S et al.; Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules . The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity . To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids . The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry . Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C . The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.

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