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 repre