Electrophoresis, 1997 Aug, 18(8), 1451 - 63 First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis; Antelmann H et al.; Data on the identification of proteins of Bacillus subtilis on two-dimensional (2-D) gels as well as their regulation are summarized and the identification of 56 protein spots is included . The pattern of proteins synthesized in Bacillus subtilis during exponential growth, during starvation for glucose or phosphate, or after the imposition of stresses like heat shock, salt- and ethanol stress as well as oxidative stress was analyzed . N-terminal sequencing of protein spots allowed the identification of 93 proteins on 2-D gels, which are required for the synthesis of amino acids and nucleotides, the generation of ATP, for glycolyses, the pentose phosphate cycle, the citric acid cycle as well as for adaptation to a variety of stress conditions . A computer-aided analysis of the 2-D gels was used to monitor the synthesis profile of more than 130 protein spots . Proteins performing housekeeping functions during exponential growth displayed a reduced synthesis rate during stress and starvation, whereas spots induced during stress and starvation were classified as specific stress proteins induced by a single stimulus or a group of related stimuli, or as general stress proteins induced by a variety of entirely different stimuli . The analysis of mutants in global regulators was initiated in order to establish a response regulation map for B . subtilis . These investigations demonstrated that the alternative sigma factor sigma B is involved in the regulation of almost all of the general stress proteins and that the phoPR two-component system is required for the induction of a large part but not all of the proteins induced by phosphate starvation. Mutat Res, 1997 Aug, 384(2), 121 - 34 Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway . II: Host constitutive expression, repair DNA synthesis, and in vitro activity; Radany EH et al.; In the Bacillus subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, sometimes followed by productive cell infection . Previous studies have shown that ultraviolet (UV)-irradiation of the competent host cells, or cotransfection of UV-irradiated heterologous DNA, can increase the efficiency of transfection in some cases; these latter two phenomena have been called transfection enhancement (TE) . In an accompanying paper, we show that TE is apparently confined to the B . subtilis phages that contain hydroxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-irradiated DNA that mediates TE is specific, and different than the pyrimidine dimer, thymine glycol, uracil, or HMU . We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DNA . Based on this DNA base and nucleolytic specificity, we hypothesized that TE reflects the incidental action of a host DNA repair system on transfecting HMU phage DNA . In continuing these studies, we show here that duplex infecting HMU phage DNA is apparently inactivated by this same putative repair system when phage protein synthesis is blocked . We find, too, that this inactivation of infecting HMU phage DNA can be inhibited by UV-irradiated DNA, and that this process has a similar DNA base specificity as for TE . The survival of infecting HMU phage DNA is dependent on host DNA polymerase activity . We can detect specific DNA synthesis consistent with formation of repair patches when inactivation of infecting HMU phage DNA is ongoing, but not when it is inhibited by the presence of UV DNA or by allowing phage gene expression . Each of these results is consistent with the hypothesis that TE reflects the action of a novel DNA repair pathway . We show that a candidate TE-associated enzymatic activity can be detected in cell free extracts of uninfected, but not HMU phage-infected, B . subtilis cells . Correspondingly, the extracts of phage-infected cells appear to contain a diffusible factor that acts as an inhibitor of this host enzyme. Mutat Res, 1997 Aug, 384(2), 107 - 20 Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway . I: DNA base and nucleolytic specificity; Radany EH et al.; Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium . In the B . subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection . Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells . Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE) . We propose here that some transfecting B . subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA . In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA . We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes . Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process . In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E . coli thymine glycol DNA N-glycosylase . This photoproduct is derivable from thymine or HMU . The implications of these results are discussed. FEMS Microbiol Lett, 1997 Sep 1, 154(1), 139 - 44 Histidine 109 in peptidyl-prolyl cis-trans isomerase of Bacillus subtilis plays an important role in catalysis and in cyclosporin A binding; Achenbach TV et al.; The cyclophilin of Bacillus subtilis has a moderate affinity to cyclosporin A (IC50: 120 nM) and low catalytic activity (Kcat/ Km: 1.1 microM-1 s-1) when compared to other ubiquitous peptidyl-prolyl cis-trans isomerases (PPIases) . The active site residues V52, H90 and H109, which are not conserved within other peptidyl-prolyl cis-trans isomerases, were found to play an important role in cyclosporin A binding and catalytic activity . In this work we report on double mutations of these residues, which greatly improved cyclosporin A affinity and catalytic activity . The H90N/H109W mutation displayed an IC50 value of 46 nM whereas the V52M/H109F mutation exhibited over 18-fold higher catalytic activity than that detected for wild-type PPIase . The mutations H109W and H109F of the B . subtilis PPIase showed no change in cyclosporin A affinity and catalytic activity between pH 6 and 8 . In contrast, wild-type PPIase (H109) showed up to 10-fold reduction below pH 7.5, both in cyclosporin A affinity and in catalytic activity . These findings clearly underline the importance of the unique H109 residue in the B . subtilis enzyme. FEMS Microbiol Lett, 1997 Sep 1, 154(1), 23 - 8 Characterization of a novel endo-levanase and its gene from Bacillus sp . L7; Miasnikov AN; A novel endo-levanase producing bacterium belonging to the Bacillus family has been isolated from soil . The enzyme was characterized and found to have no exo-beta-fructofuranosidase activity . The endo-levanase gene was cloned and sequenced . Homology searches have shown that the C-terminal domain of the enzyme is homologous to a number of known beta-fructofuranosidases, including exo-levanase from Bacillus subtilis and yeast invertases . The N-terminal region of the endo-levanase which is homologous to the C-terminal sequence of the B . subtilis levanase appears to be a levan-binding domain. Arch Microbiol, 1997 Oct, 168(4), 321 - 7 The bifunctional enzyme chitosanase-cellulase produced by the gram-negative microorganism Myxobacter sp . AL-1 is highly similar to Bacillus subtilis endoglucanases; Pedraza-Reyes M et al.; The gram-negative bacterium Myxobacter sp . AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth . Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa . Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-beta-D-cellobioside and 4-methylumbelliferyl-beta-D-cellotrioside . These enzymatic preparations also showed a greater capacity at 70 degrees C than at 42 degrees C to degrade chitosan oligomers of a minimum size of six units . Conversely, the beta-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42 degrees C than at 70 degrees C . The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography . Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis. Arch Microbiol, 1997 Oct, 168(4), 282 - 9 Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine; Boch J et al.; Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis . The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway . We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7&phi;10 control . The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose . Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits . The enzyme was specific for glycine betaine aldehyde and could use both NAD+ and NADP+ as cofactors, but NAD+ was strongly preferred . A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed Km values of 125 microM and 143 microM for its substrates glycine betaine aldehyde and NAD+, respectively . Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions . Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained . B . subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro . The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte . The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B . subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress. Prikl Biokhim Mikrobiol, 1997 May-Jun, 33(3), 321 - 4 {Stimulation of growth and spore formation of Bacillus subtilis by optimization of carbohydrate nutrition during submerged cultivation}; Osadchaia AI et al.; A wide range of carbohydrate sources for the growth of industrial Bacillus subtilis strains were studied . The growth was mainly accelerated 2.7 and 2.5-fold by adding green syrup and glucose in a combination with xylose, respectively, in comparison to the traditional glucose . The revealed nutritional demands of these bacteria allowed us to select an optimal composition of the medium using a method of experimental design . Conditions essential for the maximum production of cells and spores were determined, and certain similarities and strain-specific features of this process were found for the strains studied . Mathematical models describing growth and spore formation during submerged cultivation of Bacillus subtilis were also proposed in this work . The antagonistic activity possessed by the strains studied was shown to be unchanged through batch cultivation . We demonstrated the possibility of direct regulation of Bacillus subtilis growth and spore formation by maintaining the concentration of carbon and nitrogen sources and other components of growth media at a certain level. Microbiology, 1997 Mar, 143(Pt 3), 999 - 1017 Specific and general stress proteins in Bacillus subtilis--a two-deimensional protein electrophoresis study; Bernhardt J et al.; A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B . subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose of phosphate starvation . The data provided evidence that the induction of a least 42 general stress proteins absolutely required the alternative sigma factor sigmaB . However, at least seven stress proteins, among them ClpC, ClpP, Sod, AhpC and AhpF, remained stress-inducible in a sigB mutant . Such a detailed analysis also premitted the description of subgroups of general stress proteins which are subject to additional regulatory circuits, indicating a very thorough fine-tuning of this complex response . The relative synthesis rate of the general stress proteins constituted up to 40% of the total protein synthesis of stressed cells and thereby emphasizes the importance of the stress regulon . Besides the induction of these general or rather unspecific stress proteins, the induction of stress-specific proteins is shown and discussed. J Bacteriol, 1997 Sep, 179(18), 5878 - 83 A mutation in the ftsK gene of Escherichia coli affects cell-cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA; Diez AA et al.; An insertional mutation in ftsK, encoding an Escherichia coli product similar to the sporulation protein SpoIIIE of Bacillus subtilis, results in uspA overexpression in stationary phase and impairs cell division . The ftsK1::cat insertion mutant forms chains which are the result of inhibited cell-cell separation, while chromosome synthesis and partitioning appear to be normal as judged by flow cytometry and electron and light microscopy in combination with DNA staining . The cells of the chains are attached to each other by a small envelope structure, and unlike in a spoIIIE mutant of B . subtilis, there is no DNA trapped in the division plane . In addition, plasmids harboring a truncated ftsK allele lacking the last 195 bp of the gene cause chain formation in wild-type cells . While the mutant cells grow at essentially the same rate as the parent in complex and defined minimal media, they are sensitive to stresses . Specifically, the mutant failed to grow at elevated salt concentrations and survived stationary phase poorly . The phenotypes of the ftsK1::cat mutant are complemented by the 3' end (spoIIIE-like half) of the ftsK locus . In contrast, the 5' end of the ftsK locus reported to complement ftsK44(Ts) phenotypes does not complement the phenotypes of the ftsK1::cat mutant. Biochemistry, 1997 Sep 2, 36(35), 10718 - 26 Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides; Chen S et al.; De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase . An important feature of this inhibition is the fact that certain synergistic nucleotide pairs give more than additive inhibition . The physiological importance of synergism is in amplifying regulation by the adenine and guanine nucleotide end products of de novo synthesis . Using a new method to quantitate synergism, ADP plus GMP were confirmed {Meyer, E., and Switzer, R . L . (1978) J . Biol . Chem . 254, 5397-5402} to give strong synergistic inhibition of Bacillus subtilis glutamine PRPP amidotransferase . An X-ray structure of the ternary enzyme.ADP.GMP complex established that ADP binds to the allosteric A site and GMP to the catalytic C site . GMP increased the binding affinity of ADP for the A site by approximately 20-fold . Synergism results from a specific nucleotide-nucleotide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site . Furthermore, synergism is enhanced by the competition between nucleotide inhibitor and PRPP substrate for the C site . Purine base specificity results from a backbone carbonyl interaction of Lys305' with the 6-NH2 group of adenine in the A site and a Ser347 Ogamma interaction with the 2-NH2 group of guanine in the C site . Steric considerations favor binding of the nucleoside diphosphate to the A site . Site-directed replacements of key residues increased the nucleotide concentrations needed for 50% inhibition and in some cases perturbed synergism . Mutations in either of the nucleotide sites perturbed function at both sites, supporting the important role of synergism. J Bacteriol, 1997 Sep, 179(17), 5636 - 8 Isolation and characterization of the lacA gene encoding beta-galactosidase in Bacillus subtilis and a regulator gene, lacR; Daniel RA et al.; We have isolated transposon insertions in the lacA gene encoding an endogenous beta-galactosidase of Bacillus subtilis . Upstream of the putative operon containing lacA is a negative regulator, lacR, which encodes a product related to a family of regulators that includes the lactose repressor, lacI, of Escherichia coli . New strains with insertions in the lacA gene should be of use in studies using lacZ fusions in B . subtilis. J Bacteriol, 1997 Sep, 179(17), 5628 - 31 Properties of the phosphorylation reaction catalyzed by SpoIIAB that help to regulate sporulation of Bacillus subtilis; Najafi SM et al.; Phosphorylation of SpoIIAA on Ser-58 catalyzed by SpoIIAB is important in the regulation of sporulation of Bacillus subtilis . Nucleotide binding experiments showed that the affinity of SpoIIAB for ATP was greatly increased in the presence of SpoIIAA or a mutant SpoIIAA in which Ser-58 had been changed to alanine . Study of the phosphorylation reaction showed that the Km for ATP and the Ki for ADP were both about 1 microM . The kinetics of phosphorylation of SpoIIAA by SpoIIAB were biphasic, comprising a rapid phase (leading to phosphorylation of 1 mol of SpoIIAA/mol of SpoIIAB) followed by a slower, steady-state phase . In the steady state, the rate-determining step proved to be the dissociation of a SpoIIAB-ADP complex . The rate of this dissociation was not affected significantly by changes in the concentration of ATP. J Bacteriol, 1997 Sep, 179(17), 5605 - 8 Activation of the Bacillus subtilis spoIIG promoter requires interaction of Spo0A and the sigma subunit of RNA polymerase; Schyns G et al.; Bacillus subtilis Spo0A activates transcription from both sigmaA- and sigmaH-dependent promoters . Baldus et al . (2) identified two amino acid substitutions in the carboxyl terminus of sigmaA, K356E and H359R, that specifically impaired Spo0A-activated transcription in vivo . To test the model in which the K356E and H359R substitutions in sigmaA interfere with the interaction of Spo0A and sigmaA, we examined the effects of alanine substitutions at these positions in sigmaA on sigmaA's ability to direct transcription in vivo and in vitro . We found that alanine substitutions at these positions specifically reduced expression from the sigmaA-dependent, Spo0A-dependent promoters, spoIIG and spoIIE, in vivo . Furthermore, we found that stimulation of spoIIG promoter activity by Spo0A in vitro was reduced by the single substitutions H359A and H359R in sigmaA. J Bacteriol, 1997 Sep, 179(17), 5534 - 42 SpoVM, a small protein essential to development in Bacillus subtilis, interacts with the ATP-dependent protease FtsH; Cutting S et al.; The spoVM gene encodes a 26-amino-acid polypeptide that is essential for spore formation in Bacillus subtilis . A transposon insertion within the spoVM open reading frame has been shown to encode a chimeric protein which is biologically inactive and produces a phenotype identical to that of a deletion and insertion mutation . A genetic approach was used to identify possible interacting proteins, and the membrane-bound FtsH protease was identified . Mutations in ftsH suppressed the sporulation defect of certain spoVM mutants but not others . However, production of the mother cell sigma factors, sigmaE and sigmaK, was abnormal in the suppressed strains, and mutations in either spoVM or ftsH alone impaired sigma factor production and sporulation gene expression . Using FtsH purified from Escherichia coli, we demonstrated that in vitro (i) SpoVM inhibits FtsH protease activity and (ii) SpoVM is a substrate for the FtsH protease . We propose that during sporulation, SpoVM serves as a competitive inhibitor of FtsH activity . This interaction appears to be important for completion of the prespore engulfment step of sporulation, based on the phenotype of certain spoVM ftsH double mutants. J Bacteriol, 1997 Sep, 179(17), 5494 - 501 Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H; Wray LV Jr et al.; Expression of urease, which is encoded by the ureABC operon, is regulated in response to nitrogen availability in Bacillus subtilis . Three ureABC promoters were identified in primer extension experiments and by examination of beta-galactosidase expression from ure-lacZ fusions . P1, a low-level constitutive promoter, lies immediately upstream of ureA . The P2 promoter is transcribed by the E sigmaH form of RNA polymerase and initiates transcription 270 bp upstream of the ureA start codon . The transcriptional start site for the sigmaA-dependent P3 promoter is located 839 bp upstream of the ureA start codon . To identify transcription factors that control ureABC expression, regulation of the P2 and P3 promoters was examined in wild-type and mutant strains . During rapid growth in minimal medium containing glucose and amino acids, CodY represses expression of the P2 and P3 promoters 30- and 60-fold, respectively . TnrA activates expression of the P3 promoter 10-fold in nitrogen-limited cells, while GlnR represses transcription from the P3 promoter 55-fold during growth on excess nitrogen . Expression of the ureABC operon increases 10-fold at the end of exponential growth in nutrient sporulation medium . This elevation in expression results from the relief of CodY-mediated repression during exponential growth and increased sigmaH-dependent transcription during stationary phase. J Bacteriol, 1997 Sep, 179(17), 5448 - 57 An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport; Belitsky BR et al.; The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J . B . Ward, Jr., and S . A . Zahler, J . Bacteriol . 116:727-735, 1973) . In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport . The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon . Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins. J Bacteriol, 1997 Sep, 179(17), 5436 - 41 Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp . strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity; Lorenz WW et al.; The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp . strain JW/SL YS485 . axe1 is located 22 nucleotides 3' of the xylB sequence . The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1 . The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases . Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli . Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities . Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase . Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum . Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis . Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid. J Virol, 1997 Sep, 71(9), 6765 - 76 Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation; Zhang Y et al.; The human immunodeficiency virus (HIV) Pr55Gag precursor proteins direct virus particle assembly . While Gag-Gag protein interactions which affect HIV assembly occur in the capsid (CA) domain of Pr55Gag, the nucleocapsid (NC) domain, which functions in viral RNA encapsidation, also appears to participate in virus assembly . In order to dissect the roles of the NC domain and the p6 domain, the C-terminal Gag protein domain, we examined the effects of NC and p6 mutations on virus assembly and RNA encapsidation . In our experimental system, the p6 domain did not appear to affect virus release efficiency but p6 deletions and truncations reduced the specificity of genomic HIV-1 RNA encapsidation . Mutations in the nucleocapsid region reduced particle release, especially when the p2 interdomain peptide or the amino-terminal portion of the NC region was mutated, and NC mutations also reduced both the specificity and the efficiency of HIV-1 RNA encapsidation . These results implicated a linkage between RNA encapsidation and virus particle assembly or release . However, we found that the mutant ApoMTRB, in which the nucleocapsid and p6 domains of HIV-1 Pr55Gag were replaced with the Bacillus subtilis MtrB protein domain, released particles efficiently but packaged no detectable RNA . These results suggest that, for the purposes of virus-like particle assembly and release, NC can be replaced by a protein that does not appear to encapsidate RNA. Nucleic Acids Res, 1997 Sep 1, 25(17), 3490 - 6 Contacts between Bacillus subtilis catabolite regulatory protein CcpA and amyO target site; Kim JH et al.; Catabolite control protein A (CcpA) is a global regulatory protein involved in catabolite repression and glucose activation in Gram-positive bacteria . cis -Acting DNA sequences, catabolite response elements ( cre s), involved in this regulatory system contain a 14 base pair (bp) region of dyad symmetry . CcpA, a repressor of the Lac I family, has been shown to bind specifically to cre s . To better understand cre recognition by CcpA, we have focused on the interaction between CcpA and the amyE cre , called amyO, which is located at the transcription start site of the alpha-amylase gene . DNA-protein complexes were probed with dimethylsulfate (DMS) and N -ethylnitrosourea (EtNU) to identify guanines and phosphates that participate in complex formation . Interaction between amyO and CcpA visualized through methylation protection and interference showed that CcpA contacts guanine residues at the outer bounds of amyO with higher affinity than near the dyad axis . From ethylation interference studies, it was found that CcpA contacts three phosphate groups at each end of amyO, and one or two phosphate groups near the dyad axis . Exonuclease III protection revealed that CcpA protects a 26 bp region centered around the dyad axis of amyO . The isolated N-terminal fragment still specifically bound to the sequence resembling the half sites of the amyO sequence . Considering these findings and the helical structure of B-DNA, our results suggest that each of the two monomers of the CcpA molecule contact the major groove in each half of the region of dyad symmetry and that the contacts are on the same face of the DNA helix, which is typical of bacterial repressor-operator interactions . However, the absence of strong contacts near the dyad axis by CcpA is in contrast to the situation with the gal repressor, another member of the Lac I family of repressors. Biochemistry, 1997 Aug 19, 36(33), 10015 - 25 High-resolution NMR structure and backbone dynamics of the Bacillus subtilis response regulator, Spo0F: implications for phosphorylation and molecular recognition; Feher VA et al.; NMR has been employed for structural and dynamic studies of the bacterial response regulator, Spo0F . This 124-residue protein is an essential component of the sporulation phosphorelay signal transduction pathway in Bacillus subtilis . Three-dimensional 1H, 15N, and 13C experiments have been used to obtain full side chain assignments and the 1511 distance, 121 dihedral angle, and 80 hydrogen bonding restraints required for generating a family of structures (14 restraints per residue) . The structures give a well-defined (alpha/beta)5 fold for residues 4-120 with average rms deviations of 0.59 A for backbone heavy atoms and 1.02 A for all heavy atoms . Analyses of backbone 15N relaxation measurements demonstrate relative rigidity in most regions of regular secondary structure with a generalized order parameter (S2) of 0.9 +/- 0.05 and a rotational correlation time (taum) of 7.0 +/- 0.5 ns . Loop regions near the site of phosphorylation have higher than average rms deviation values and T1/T2 ratios suggesting significant internal motion or chemical exchange at these sites . Additionally, multiple conformers are observed for the beta4-alpha4 loop and beta-strand 5 region . These conformers may be related to structural changes associated with phosphorylation and also indicative of the propensity this recognition surface has for differential protein interactions . Comparison of Spo0F structural features to those of other response regulators reveals subtle differences in the orientations of secondary structure in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation . These may be important in providing specificity for protein-protein interactions and for determining the lifetimes of the phosphorylated state. FEMS Microbiol Lett, 1997 Aug 15, 153(2), 405 - 9 The NAD synthetase NadE (OutB) of Bacillus subtilis is a sigma B-dependent general stress protein; Antelmann H et al.; The identification of sigma B-dependent general stress proteins is a useful strategy to understand the physiological role of the unspecific stress response in Bacillus subtilis . By N-terminal sequencing of B . subtilis stress proteins Gsp38 was identified as the NAD-synthetase (NadE) . NadE was previously characterized as spore outgrowth factor B (OutB) conferring a temperature-sensitive spore outgrowth defective phenotype . Transcriptional studies showed that nadE is strongly induced in response to heat, ethanol and salt stress or after starvation for glucose in a sigma B-dependent manner . Two promoters are involved in transcriptional initiation, the sigma A-dependent upstream promoter contributes to the basal level during growth, whereas the sigma B-dependent downstream promoter is induced after different stress conditions. FEMS Microbiol Lett, 1997 Aug 15, 153(2), 247 - 54 Replication fork arrest and termination of chromosome replication in Bacillus subtilis; Wake RG; Sporulation in Bacillus subtilis provided the first evidence for the presence of sequence-specific replication fork arrest (Ter) sites in the terminus region of the bacterial chromosome . These sites, when complexed with the replication terminator protein (RTP), block movement of a replication fork in a polar manner . The Ter sites are organized into two opposed groups which force the approaching forks to meet and fuse within a restricted terminus region . While the precise advantage provided to the cell through the presence of the so-called replication fork trap is not patently obvious, the same situation appears to have evolved independently in Escherichia coli . The molecular mechanism by which the RTP-Ter complex of B . subtilis (or the analogous but apparently unrelated complex in E . coli) functions is currently unresolved and subject to intense investigation . Replication fork arrest in B . subtilis, requiring RTP, also occurs under conditions of the stringent response at so-called STer sites that lie close to and on both sides of oriC . These sites are yet to be identified and characterized . How they are induced to function under stringent conditions is of considerable interest, and could provide vital clues about the mechanism of fork arrest by RTP-terminator complexes in general. J Biol Chem, 1997 Aug 8, 272(32), 19863 - 9 The trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis binds to unstacked trp leader RNA; Baumann C et al.; TRAP (trp RNA-binding attenuation protein) is a tryptophan-activated RNA-binding protein that regulates expression of the trp biosynthetic genes by binding to a series of GAG and UAG trinucleotide repeats generally separated by two or three spacer nucleotides . Previously, we showed that TRAP contains 11 identical subunits arranged in a symmetrical ring . Based on this structure, we proposed a model for the TRAP.RNA interaction where the RNA wraps around the protein with each repeat of the RNA contacting one or a combination of two adjacent subunits of the TRAP oligomer . Here, we have shown that RNAs selected in vitro based on their ability to bind tryptophan-activated TRAP contain multiple G/UAG repeats and show a strong bias for pyrimidines as the spacer nucleotides between these repeats . The affinity of the TRAP.RNA interaction displays a nonlinear temperature dependence, increasing between 5 degrees C and 47 degrees C and then decreasing from 47 degrees C to 67 degrees C . Differential scanning calorimetry and circular dichroism spectroscopy demonstrate that TRAP is highly thermostable with few detectable changes in the structure between 25 degrees C and 70 degrees C, suggesting that the temperature dependence of this interaction reflects changes in the RNA . Results from circular dichroism and UV absorbance spectroscopy support this hypothesis, demonstrating that trp leader RNA becomes unstacked upon binding TRAP . We propose that the bias toward pyrimidines in the spacer nucleotides of the in vitro selected RNAs represents the inability of Us and Cs to form stable base stacking interactions, which allows the flexibility needed for the RNA to wrap around the TRAP oligomer. Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8895 - 900 Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase; Lermontova I et al.; Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids . We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy . The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase . One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues . Both deduced protein sequences share 27.2% identical amino acid residues . The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction . The second enzyme was targeted to mitochondria without any detectable reduction in size . Localization of both enzymes in subcellular fractions was immunologically confirmed . Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth . The mature plastidal and the mitochondrial isoenzyme were overexpressed in E . coli . Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide . The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria. Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8612 - 7 A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay; Perego M; The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators . The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit . The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity . This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene . The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease . This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor . The processing events may, in turn, be controlled by a regulatory hierarchy . Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B . subtilis, suggesting that the use of this mechanism may be widespread in signal transduction. Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8439 - 44 The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression; Galinier A et al.; Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system . In this study, we report the discovery of a new B . subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids . Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh . Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate . We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage . In a B . subtilis ptsH1 mutant strain, synthesis of beta-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR . Additional disruption of the crh gene caused almost complete relief from CCR . In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of beta-xylosidase was also completely relieved from glucose repression . These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46. Lett Appl Microbiol, 1997 Aug, 25(2), 153 - 6 Influence of sporulation medium and divalent ions on the heat resistance of Alicyclobacillus acidoterrestris spores; Yamazaki K et al.; The influence of divalent cations on the heat resistance of spores of the thermoacidophilic spoilage bacterium Alicyclobacillus acidoterrestris was studied . The heat resistance of A . acidoterrestris spores was not affected by the presence of the different divalent cations (Ca2+, Mg2+, Ba2+, Mn2+ and Sr2+) in the sporulation medium, and by the demineralization or remineralization . And the Ca and Mn contents in A . acidoterrestris spores were scarcely changed by these treatments . However, the heat resistance of Bacillus subtilis spores was affected with the changes of Ca content in the spores . The Ca contents in A . acidoterrestris spores of the different forms were greater than the DPA content . In contrast, the DPA content in the B . subtilis spores was greater than the Ca content . These findings suggest that the presence of constant amount of Ca-DPA and a stronger binding characteristic of divalent ions, especially Ca and Mn, is reflected in the specific heat resistance of A . acidoterrestris spores. Microbiology, 1997 Aug, 143 ( Pt 8), 2775 - 82 A 32 kb nucleotide sequence from the region of the lincomycin-resistance gene (22 degrees-25 degrees) of the Bacillus subtilis chromosome and identification of the site of the lin-2 mutation; Kumano M et al.; A 32 kb nucleotide sequence in the region of the lincomycin-resistance gene, located from 22 degrees to 25 degrees on the Bacillus subtilis chromosome, was determined . Among 32 putative ORFs identified, four {lipA for lipase, natA, natB and yzaE (renamed yccK)} have already been reported, although the functions of NatA, NatB and YccK remain to be characterized . Six putative products were found to exhibit significant similarity to known proteins in the databases, namely L-asparaginase precursor, protein aspartate phosphatase, alpha-glucosidase, two tellurite-resistance proteins and a hypothetical protein from B . subtilis . The region of the tellurite-resistance gene, consisting of seven ORFs, seems to correspond to an operon . The products of 14 ORFs exhibited considerable or limited similarity to known proteins . The sequenced region seems to be rich in membrane proteins, since at least 16 gene products appeared to contain membrane-spanning domains . The site of the lin-2 mutation (two nucleotide replacements) was mapped and identified by sequencing . This site is located between a putative promoter and the SD sequence of ImrA (yccB) {a putative repressor of the lmr operon, which consists of lmrA and lmrB (yccA)} . LmrB is a homologue of proteins involved in drug-export systems and seems likely to be the protein responsible for resistance to lincomycin. Microbiology, 1997 Aug, 143 ( Pt 8), 2763 - 7 Sequence and analysis of a 31 kb segment of the Bacillus subtilis chromosome in the area of the rrnH and rrnG operons; Liu H et al.; A 31141 bp continuous nucleotide sequence in the region from trnl to pNEXT52 in the Bacillus subtilis 168 genome was determined . In the region, there were 22 ORFs, two complete rRNA operons, and five tRNA genes . It was deduced that the function of one of the ORFs was similar to that of a sigma factor belonging to the ECF (extra-cytoplasmic functions) subfamily . The gene cluster feuA, B, C reported previously for other strains of B . subtilis was also found in strain 168 and located in this region. Microbiology, 1997 Aug, 143 ( Pt 8), 2753 - 61 Mutational analysis of the early forespore/mother-cell signalling pathway in Bacillus subtilis; Londono-Vallejo JA; Intercellular communication is a crucial phenomenon during spore development in Bacillus subtilis . It couples the establishment of a compartment-specific genetic program to the transcriptional activity of a sigma factor in the other compartment . It also keeps sigma factor activation in register with the morphological process . This study used directed mutagenesis to analyse the pathway that couples sigma E activation in the mother-cell to activation of sigma F in the forespore following asymmetric septation . Targets for mutagenesis in SpoIIGA (the receptor) were chosen based on the predicted topology of the protein when associated with the cell membrane . The results showed that a residue near the N terminus (D6), predicted to be exposed outside the cell, is required for receptor activity, whereas the major extracellular loop (between membrane domains IV and V) is dispensable for function . In contrast, mutations in SpoIIR (the signal) that partially blocked protein release (but not membrane translocation) had no effect on signal transduction . These results do not rule out the possibility that uncharacterized molecules intervene in the signalling pathway that establishes the mother-cell-specific developmental program during the early stage of sporulation. Microbiology, 1997 Aug, 143 ( Pt 8), 2743 - 51 Spo0A represses transcription of the cry toxin genes in Bacillus thuringiensis; Poncet S et al.; The DNA regions upstream from the genes encoding polypeptides of Bacillus thuringiensis subsp . israelensis larvicidal crystals (cry4A, cry4B, cry11A) contain sequences with similarities to the spo0A box of Bacillus subtilis (or '0A' box) and the promoter recognized by the sigma H-associated RNA polymerase of B . subtilis . Expression of cry-lacZ transcriptional fusions was analysed in various B . thuringiensis genetic backgrounds . The early transcription of the toxin genes was not sporulation-dependent, whereas the late-stage expression at t4-6 was sigma E-dependent . Primer extension analysis confirmed that the cry4- and cry11-type toxin genes were weakly transcribed during the transition phase; expression analysis of a cry11A'-lacZ transcriptional fusion in B . subtilis sporulation mutants confirmed the involvement of the sigma H-RNA polymerase . Primer extension analysis showed that in B . thuringiensis subsp . israelensis, the cry4A and cry11A gene transcription observed at the end of the growth stage was turned off at the beginning of the sporulation phase . The DNA region located upstream from the cry11A gene promoter including the putative '0A' box was deleted . This led to a derepression of the expression of the cry11A operon . These results suggest that the cry4A, cry4B and cry11A toxin genes of B . thuringiensis subsp . Israelensis are transcribed during the transition phase by the RNA polymerase associated with the sigma H factor and are subject to Spo0A repression. Biotechniques, 1997 Aug, 23(2), 304 - 10 Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization; Shafikhani S et al.; We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency . This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution . We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity . The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing . The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture . We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G . We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations . These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity. Proteins, 1997 Aug, 28(4), 590 - 4 Crystallization of the RNA-binding domain of the transcriptional antiterminator protein SacY from Bacillus subtilis; Manival X et al.; SacY is the antiterminator protein involved in the induction by sucrose of the expression of the levansucrase gene (sacB) of Bacillus subtilis . In the presence of sucrose, SacY is activated and prevents premature termination of transcription by binding to a RNA-antiterminator (RAT) sequence partially overlapping with the terminator sequence . SacY consists of a RNA-binding N-terminal domain, SacY(1-55), and a regulatory domain, SacY(56-280), sensitive to the sucrose concentration . SacY(1-55) is in itself capable of binding to the RAT sequence and preventing termination independently of the sucrose concentration . In this paper we describe the overexpression, the purification, and the crystallization of SacY(1-55) . We obtained six different crystal forms, some of them diffracting to high resolution (> 1.5 A) . Self rotation function calculations indicated the presence of a dimer in the asymmetric unit, which is in agreement with a proposed oligomeric state in solution as observed by high-resolution NMR measurements . The crystallization of some site-directed cysteine mutants opens the way of solving the structure by multiple isomorphous replacement. J Bacteriol, 1997 Aug, 179(16), 4970 - 6 The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis; Ge Z et al.; The Helicobacter pylori pss gene, coding for phosphatidylserine synthase (PSS), was cloned and sequenced in this study . A polypeptide of 237 amino acids was deduced from the PSS sequence . H . pylori PSS exhibits significant amino acid sequence identity with the PSS proteins found in the archaebacterium Methanococcus jannaschii, the gram-positive bacterium Bacillus subtilis, and the yeast Saccharomyces cerevisiae but none with its Escherichia coli counterpart . Expression of the putative pss gene in maxicells gave rise to a product of approximately 26 kDa, which is in agreement with the predicted molecular mass of 26,617 Da . A manganese-dependent PSS activity was found in the membrane fractions of the E . coli cells overexpressing the H . pylori pss gene product . This result indicates that this enzyme is a membrane-bound protein, a conclusion which is supported by the fact that the PSS protein contains several local hydrophobic segments which could form transmembrane helices . The pss gene was inactivated with a chloramphenicol acetyltransferase cassette on the plasmid . However, an isogenic pss gene-disrupted mutant of H . pylori UA802 could not be obtained, suggesting that this enzyme plays an essential role in the growth of this organism. FEMS Microbiol Lett, 1997 Aug 1, 153(1), 237 - 43 Optimisation of the BgaB reporter system: determination of transcriptional regulation of stress responsive genes in Bacillus subtilis; Schrogel O et al.; We constructed and characterised a new system to determine transcriptional regulation of genes in Bacillus subtilis . The system is based on the B . stearothermophilus-derived beta-galactosidase BgaB . In contrast to the systems described up to now the BgaB protein is not degraded in response to environmental stresses . We optimised buffer conditions for BgaB assays and developed a protocol which allows measurement of BgaB activity without background problems . To test the system we determined induction of the B . subtilis clpC gene in response to stress . Induction of this gene in response to stress occurred as described. FEMS Microbiol Lett, 1997 Aug 1, 153(1), 135 - 9 Neomycin- and spectinomycin-resistance replacement vectors for Bacillus subtilis; Chary VK et al.; A plasmid is described for Bacillus subtilis that facilitates replacement of the widely used neomycin resistance gene (neo) with a spectinomycin resistance (spcE) gene . A second plasmid is described that facilitates replacement of spcS, associated with mini-Tn10 mutagenesis in B . subtilis, with neo . These plasmids can also function as integrative vectors for B . subtilis . They expand the scope of strain construction and gene analysis in B . subtilis. FEMS Microbiol Lett, 1997 Aug 1, 153(1), 63 - 9 Analysis of non-polar deletion mutations in the genes of the spo0K (opp) operon of Bacillus subtilis; LeDeaux JR et al.; The spo0K (opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides, development of genetic competence, and initiation of sporulation . We made in-frame, non-polar deletion mutations in each of the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of competence genes . spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport . Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon. J Mol Biol, 1997 Aug 1, 270(5), 696 - 710 Alanine-scanning mutagenesis of Bacillus subtilis trp RNA-binding attenuation protein (TRAP) reveals residues involved in tryptophan binding and RNA binding; Yang M et al.; In Bacillus subtilis, expression of the trp genes is negatively regulated by an RNA binding protein called TRAP (trp RNA-binding Attenuation Protein), which is activated to bind RNA by binding l-tryptophan . TRAP contains 11 identical subunits assembled in a symmetric ring . We have used alanine-scanning mutagenesis to analyze the functions of surface amino acid residues of TRAP . The in vivo regulatory activity of each mutant TRAP was analyzed in a B . subtilis reporter strain containing a trpE'-'lacZ fusion . Mutant TRAP proteins with defective in vivo regulatory activities were characterized in vitro by measuring their tryptophan binding and RNA binding activities . Most of the mutant proteins with altered tryptophan binding, either affinity or cooperativity, contained substituted residues located on two loops formed by residues 25 to 33 and residues 49 to 52, as well as on the beta-strand and beta-turn contiguous with these loops . Substitution of three residues (Lys37, Lys56 and Arg58) with alanine resulted in significant decreases in the RNA binding activity of TRAP without altering tryptophan binding . Structural analysis shows that these three residues are directly aligned on the outer edge of TRAP . Further mutagenic analysis of these three residues revealed that only lysine or arginine residues at positions 37 or 58 allow proper TRAP function, whereas at position 56, only lysine is functional . Residue Asn20 is the only other residue in TRAP that is located on the line formed by residues 37, 56 and 58, and virtually any amino acid residue is functional at position 20 . We propose that RNA wraps around TRAP by interacting with residues Lys37, Lys56 and Arg58. J Bacteriol, 1997 Aug, 179(15), 4888 - 93 Bacillus subtilis Pro-sigmaE fusion protein localizes to the forespore septum and fails to be processed when synthesized in the forespore; Ju J et al.; Endospore formation in Bacillus subtilis begins with an asymmetric cell division that partitions the bacterium into mother cell and forespore compartments . Mother cell-specific gene expression is initiated by sigmaE, a transcription factor that is active only in the mother cell but which existed as an inactive precursor (pro-sigmaE) in the predivisional cell . Activation of pro-sigmaE involves the removal of 27 amino acids from its amino terminus . A chimera of pro-sigmaE and the green fluorescent protein (GFP) was expressed from either the normal sigE promoter (P(spoIIG)), which places pro-sigmaE::GFP in both mother cell and forespore compartments, or the forespore-specific promoter (P(dacF)), which produces pro-sigmaE::GFP only in the forespore compartment . The pro-sigmaE::GFP expressed from P(spoIIG), but not P(dacF), was converted to a lower-molecular-weight form by a mechanism dependent on gene products (SpoIIGA and sigmaF) that are essential for normal pro-sigmaE processing . This finding is consistent with the pro-sigmaE processing reaction occurring only in the mother cell compartment . In processing-deficient cells, pro-sigmaE::GFP was found to accumulate at the septal membrane, a location where its processing apparatus would be susceptible to triggering from the adjoining forespore. J Bacteriol, 1997 Aug, 179(15), 4725 - 32 Deletion of the Bacillus subtilis isocitrate dehydrogenase gene causes a block at stage I of sporulation; Jin S et al.; A Bacillus subtilis mutant with a deletion of citC, the gene encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, had a greatly reduced ability to sporulate . Analysis of expression of lacZ fusions to various sporulation gene promoters revealed that in the citC mutant development is probably blocked between stage 0 and stage II . That is, genes expressed very early in sporulation, under the direct control of the Spo0A transcription factor, were induced normally in the citC mutant . However, genes expressed after asymmetric septation (stage II) in wild-type cells were not induced in the citC mutant . Analysis of cell morphology by thin-section electron microscopy and immunofluorescence microscopy showed that the mutant formed axial chromosomal filaments and accumulated rings of FtsZ protein at potential polar division sites but failed to form asymmetric division septa, indicating that sporulation is blocked at stage I . The growth and sporulation defects of the B . subtilis citC mutant were fully overcome by introduction and expression of the Escherichia coli icd gene, encoding an isocitrate dehydrogenase similar to the enzyme from B . subtilis. Gene, 1997 Jul 31, 194(2), 191 - 9 Cloning and sequencing of a 35.7 kb in the 70 degree-73 degree region of the Bacillus subtilis genome reveal genes for a new two-component system, three spore germination proteins, an iron uptake system and a general stress response protein; Yamamoto H et al.; In the framework of the international project aimed at sequencing the Bacillus subtilis (Bs) genome, a 35.7-kb chromosome segment around the pel locus has been cloned and sequenced . This region (35,745 bp; 70 degrees-73 degrees of the genetic map) contains two partial and 38 complete orfs . A homology search for the products deduced from the 39 orfs revealed that 26 of them exhibit significant similarity to known proteins, e.g . germination proteins, sodium-alanine symporter, PTS system, methionine amino peptidase, 2-oxoglutarate/malate translocater, pectate lyase, general stress response protein, RNA helicase, iron uptake and two-component systems. Nucleic Acids Res, 1997 Jul 15, 25(14), 2766 - 72 Purification and characterization of the RecF protein from Bacillus subtilis 168; Ayora S et al.; Genetic evidence suggests that the Bacillus subtilis recF gene product is involved in DNA repair and recombination . The RecF protein was overproduced and purified . NH2-terminal protein sequence analysis of RecF was consistent with the deduced amino acid sequence of the recF gene . The RecF protein (predicted molecular mass 42.3 kDa) bound single- and double-stranded DNA in a filter binding and in a gel retarding assay . The RecF-ssDNA or -dsDNA complex formation proceeds in the absence of nucleotide cofactors . RecF-ssDNA interaction is markedly stimulated by divalent cations . The apparent equilibrium constants of the RecF-DNA complexes are approximately 110-130 nM for both ssDNA and dsDNA . The binding reaction shows no cooperativity . The RecF protein does not physically interact with the RecR protein . Under our experimental conditions an ATPase activity was not associated with the purified RecF protein or with the RecF and RecR proteins. J Mol Biol, 1997 Jul 4, 270(1), 50 - 64 The replisome organizer (G38P) of Bacillus subtilis bacteriophage SPP1 forms specialized nucleoprotein complexes with two discrete distant regions of the SPP1 genome; Missich R et al.; Initiation of Bacillus subtilis bacteriophage SPP1 DNA replication requires the products of genes 38, 39 and 40 (G38P, G39P and G40P) . G38P specifically binds two discrete regions, which are 32.1 kb apart in a linear map of the SPP1 genome . One of these target sites, which maps at the left end of the phage genome, within gene 38, was shown to function as an origin of replication and was therefore termed left origin (oriL) . The other site, which lies within a non-coding segment in the late transcribed region on the right end of the genome, was termed oriR . Both sites contain two types of repeated elements (termed Box AB and A + T-rich region) . The K(app) for the G38P-oriL DNA and G38P-oriR DNA complexes was estimated to be 1 nM and 4 nM, respectively . G38P binds to the distant oriL and oriR sites cooperatively . DNase I footprinting experiments showed protection by G38P in Box AB, but not in the A + T-rich region . Electron microscopy analysis showed that G38P forms a higher-order nucleoprotein structure with the SPP1 oriL and oriR sites through protein-protein interaction . G38P binding at its cognate sites does not seem to modify the length of the DNA, but to bend it . These results suggest that G38P forms a nucleoprotein complex on the regions where the SPP1 replication origins were previously predicted. J Biol Chem, 1997 Jul 4, 272(27), 17230 - 7 Multiple phosphorylation of SacY, a Bacillus subtilis transcriptional antiterminator negatively controlled by the phosphotransferase system; Tortosa P et al.; The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene . SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, the B . subtilis sucrose-specific PTS-permease . Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases . However, data reported herein show direct phosphorylation of SacY by HPr(His approximately P) with no requirement for SacX . Experiments were carried out to determine the phosphorylatable residues in SacY . In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators . Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined . In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines . Nevertheless, in vivo studies using cells bearing a sacB'-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity. Mol Microbiol, 1997 Jul, 25(2), 411 - 21 Analysis of cis-acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene; Rollins SM et al.; The Bacillus subtilis tyrS gene belongs to the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes, which are regulated by a common mechanism of transcriptional antitermination . Each gene is induced by specific amino acid limitation; the uncharged cognate tRNA is the effector inducing transcription of the full-length message . The leader regions of the genes in this family share a number of conserved primary sequence and secondary structural elements, the functions of which are unknown . In this study, we examine these regions and report the effects of mutations in several of these elements . In addition, two alternative basepairings in the F box region were found to be necessary for tyrS antitermination. Mol Microbiol, 1997 Jul, 25(2), 275 - 83 The membrane-bound cell division protein DivIB is localized to the division site in Bacillus subtilis; Harry EJ et al.; The cell division gene divIB of Bacillus subtilis is essential for the normal rate of growth and division . The gene product, DivIB, is a membrane-bound protein in which the bulk of the protein (at the C-terminal end) is on the exterior surface of the cell membrane . DivIB is involved in the early stages of septum formation, but its exact role in cell division is unknown . To gain more information about the mode of action of DivIB in septum formation, we determined the location of DivIB within the cell membrane using immunofluorescence . This immunolocalization approach established that DivIB becomes localized to the division site before visible septation and remains localized to this site throughout the division process . Various DivIB immunostaining patterns were observed in immunofluorescence experiments and, together with cell length and nucleoid distance measurements, have allowed us to propose two models to describe DivIB localization during the cell cycle. Appl Microbiol Biotechnol, 1997 Jul, 48(1), 27 - 33 Purification and characterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051; application to the recovery of bioactive peptides from fusion proteins by sequence-specific digestion; Okamoto H et al.; Screening cultures of nonpathogenic microorganisms led us to a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051, which we purified and named BSase . The nucleotide sequence encoding BSase, with a molecular mass of 23,894 Da, completely agreed with that of the mpr gene, which had been reported by Rufo Jr . and Sloma et al . to encode a metalloprotease {J Bacteriol (1990) 172: 1019-1023 and 1024-1029 respectively} . However, enzymatic characterization revealed it to have the catalytic triad of a serine protease and not the consensus sequence of a metalloprotease, and it was inhibited by diisopropylfluorophosphate . We therefore consider BSase (mpr) to be a serine protease . In the alignment of the acidic-amino-acid-specific proteases, the proteases from bacilli have a highly conserved histidine residue, which is most important in the histidine triad in the proteases from streptomycetes . Furthermore, Ca2+ was necessary for its activity and stability . BSase cleaved the C-terminal glutamic acid with high specificity and was very stable over a wide pH range . On the basis of these properties, we tried to retrieve a bioactive peptide from a fusion protein by sequence-specific digestion, and succeeded in obtaining the bioactive peptide . BSase was found to be very useful as a tool for selective cleavage. Biomed Instrum Technol, 1997 Jul-Aug, 31(4), 387 - 90 The use of two or more microorganisms versus one microorganism in the carrier materials for biological indicators; Shintani H; Specification for the preparation of a biological indicator (BI) using two or more microorganisms in the carrier material were deleted from the current ISO 11138 series and Working Draft 14161 because it was assumed that the resistances of the individual microorganisms would be affected by interference from the other microorganisms . This assumption is speculative only, and has not been supported by experimental evidence . To test its validity, the author carried out an experiment to determine the resistances of Bacillus subtilis and Bacillus stearothermophilus when used alone and together in BI carrier material . In total concentrations of 10(6) cfu/0.1 mL, the organisms were applied alone and together to filter paper and dried . After incorporation into the BI, the three preparations (B . subtilis alone, B . stearothermophilus alone, and both together) were subjected to ethylene oxide sterilization or to moist-heat sterilization using the procedures described in ISO 11138-2 or ISO 11138-3, respectively . Resistances were measured in terms of decimal reduction times (D values) . The D values of the preparations were determined using the survival-curve method and the limited Spearman-Karber method in conjunction with a BI evaluator resistometer . The D values of the preparations did not differ significantly with either sterilization method, providing experimental evidence that, at least under these conditions, the presence of a second microorganism in the carrier material did not interfere with the resistance of the original microorganism. Vet Res, 1997 Jul-Aug, 28(4), 385 - 95 Safety of lysosubtilin per os in mice, rabbits and calves; Biziulevichius GA et al.; Lysosubtilin is a broad-spectrum preparation of lytic enzymes from Bacillus subtilis designed for veterinary medicine as an alternative to common antibiotics . The safety of lysosubtilin was studied in acute and subchronic toxicity tests in mice, rabbits, and calves . No sign of toxicosis was observed when lysosubtilin was administered as a single dose per os to white mice in doses reaching 2 x 10(6) U/kg of weight . Subcutaneous LD50 of lysosubtilin in mice was equal to 5 x 10(5) U/kg of weight . In order to determine subchronic toxicity in mice and rabbits the doses of lysosubtilin used in veterinary practice (2 x 10(4) U/kg of weight) were increased up to ten times (2, 8, 20.10(4) U/kg of weight) and when experimenting with calves they were increased up to two times (4.10(4) U/kg of weight) while extending the administration period by three times . In the three animal species, given in increased doses twice a day per os for 30 days, lysosubtilin did not cause any evident signs of clinical toxicosis . The blood and sera indices of rabbits and calves were within the normal range . In calves the changes were insignificant . The ANOVA test, however, revealed statistically significant differences in hemoglobin, WBC, protein, and glucose value changes between rabbit groups which were given different doses of lysosubtilin . A significant increase in daily weight gain (approximately 100 g, P < 0.001), influenced by lysosubtilin, was observed in calves . No deviations from the normal status were observed in the organs and tissues obtained during the post-mortem examination of rabbits and calves slaughtered 1 and 15 days after the end of lysosubtilin administration . Veterinary-sanitary examination of the slaughtered animals and laboratory tests of their meat showed normal commercial and sanitary qualities. Biol Pharm Bull, 1997 Jul, 20(7), 820 - 2 Molecular cloning and sequence analysis of the dnaA gene of Staphylococcus aureus; Katayama H et al.; The dnaA gene of Staphylococcus aureus has now been cloned and sequenced . The deduced amino acid sequence of Staphylococcus aureus DnaA protein is 62% and 39% identical to those of the DnaA protein from Bacillus subtilis and Escherichia coli, respectively . Expression of the dnaA gene of Staphylococcus aureus in Escherichia coli caused lethality in an oriC-dependent manner. Lett Appl Microbiol, 1997 Jul, 25(1), 24 - 9 Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus; Lincoln SP et al.; The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium . Both muramidase (EC.3.2.1.17) and N-acetyl-beta-D-glucosaminidase (beta-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A . bisporus, and in cultures fruiting in sterile and non-sterile compost . A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A . bisporus . A colorimetric assay was used to measure beta-GlcNAcase . Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B . subtilis cells . Muramidase activity was highest in axenic compost cultures . Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost. Yeast, 1997 Jul, 13(9), 819 - 28 Isolation of three contiguous genes, ACR1, ACR2 and ACR3, involved in resistance to arsenic compounds in the yeast Saccharomyces cerevisiae; Bobrowicz P et al.; A 4.2 kb region from Saccharomyces cerevisiae chromosome XVI was isolated as a yeast fragment conferring resistance to 7 mM-sodium arsenite (NaAsO2), when put on a multicopy plasmid . Homology searches revealed a cluster of three new open reading frames named ACR1, ACR2 and ACR3 . The hypothetical product of the ACR1 gene is similar to the transcriptional regulatory proteins, encoded by YAP1, and YAP2 genes from S . cerevisiae . Disruption of the ACR1 gene conduces to an arsenite and arsenate hypersensitivity phenotype . The ACR2 gene is indispensable for arsenate but not for arsenite resistance . The hypothetical product of the ACR3 gene shows high similarity to the hypothetical membrane protein encoded by Bacillus subtilis ORF1 of the skin element and weak similarity to the ArsB membrane protein of the Staphylococcus aureus arsenical-resistance operon . Overexpression of the ACR3 gene confers an arsenite- but not an arsenate-resistance phenotype . The presence of ACR3 together with ACR2 on a multicopy plasmid expands the resistance phenotype into arsenate . These findings suggest that all three novel genes: ACR1, ACR2 and ACR3 are involved in the arsenical-resistance phenomenon in S . cerevisiae. J Bacteriol, 1997 Jul, 179(14), 4591 - 8 Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilis; Yoshida KI et al.; Previous determination of the nucleotide sequence of the iol region of the Bacillus subtilis genome allowed us to predict the structure of the iol operon for myo-inositol catabolism, consisting of 10 iol genes (iolA to iouJ); iolG corresponds to idh, encoding myo-inositol 2-dehydrogenase (Idh) . Primer extension analysis suggested that an inositol-inducible promoter for the iol operon (iol promoter) might be a promoter-like sequence in the 5' region of iolA, which is probably recognized by sigmaA . S1 nuclease analysis implied that a rho-independent terminator-like structure in the 3' region of iolJ might be a terminator for iol transcription . Disruption of the iol promoter prevented synthesis of the iol transcript as well as that of Idh, implying that the iol operon is most probably transcribed as an 11.5-kb mRNA containing the 10 iol genes . Immediately upstream of the iol operon, two genes (iolR and iolS) with divergent orientations to the iol operon were found . Disruption of iolR (but not iolS) caused constitutive synthesis of the iol transcript and Idh, indicating that the iolR gene encodes a transcription-negative regulator (presumably a repressor) for the iol operon . Northern and S1 nuclease analyses revealed that the iolRS genes were cotranscribed from another inositol-inducible promoter, which is probably recognized by sigmaA . The promoter assignments of the iol and iolRS operons were confirmed in vivo with a lacZ fusion integrated into the amyE locus. J Bacteriol, 1997 Jul, 179(14), 4523 - 9 Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis; Schiott T et al.; Cytochromes of the c type contain covalently bound heme . In bacteria, they are located on the outside of the cytoplasmic membrane . Cytochrome c synthesis involves export of heme and apocytochrome across the cytoplasmic membrane followed by ligation of heme to the polypeptide . Using radioactive protoheme IX produced in Escherichia coli, we show that Bacillus subtilis can use heme from the growth medium for cytochrome c synthesis . The B . subtilis ccdA gene encodes a 26-kDa integral membrane protein which is required for cytochrome c synthesis (T . Schiott et al., J . Bacteriol . 179:1962-1973, 1997) . In this work, we analyzed the stage at which cytochrome c synthesis is blocked in a ccdA deletion mutant . The following steps were found to be normal in the mutant: (i) transcription and translation of cytochrome c structural genes, (ii) translocation of apocytochrome across the cytoplasmic membrane, and (iii) heme transport from the cytoplasm to cytochrome polypeptide on the outer side of the cytoplasmic membrane . It is concluded that CcdA is required for a late step in the cytochrome c synthesis pathway. Appl Environ Microbiol, 1997 Jul, 63(7), 2814 - 20 Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different gram-positive bacteria; Meens J et al.; Heterologous protein secretion was studied in the gram-positive bacteria Bacillus subtilis and Staphylococcus carnosus by using the Escherichia coli outer membrane protein OmpA as a model protein . The OmpA protein was found to be translocated across the plasma membrane of both microorganisms . However, the majority of the translocated OmpA was similarly degraded in B . subtilis and S . carnosus despite the fact that the latter organism does not secrete soluble exoproteases into the culture medium . The finding that purified OmpA, which was added externally to the culture medium of growing S . carnosus cells, remained intact indicates that newly synthesized and exported OmpA is degraded by one or more cell-associated proteases rather than by a soluble exoprotease . Fusion of the mature part of OmpA to the pre-pro part of a lipase from Staphylococcus hyicus allowed the efficient release of the corresponding propeptide-OmpA hybrid protein into the supernatant and completely prevented the cell-associated proteolytic degradation of the mature OmpA, most likely reflecting an important function of the propeptide during secretion of its natural mature lipase moiety . The relevance of our findings for the biotechnological use of gram-positive bacteria as host organisms for the secretory production of heterologous proteins is discussed. Nucleic Acids Res, 1997 Jul 1, 25(13), 2603 - 9 sigma factor mutations affecting the sequence-selective interaction of RNA polymerase with -10 region single-stranded DNA; Huang X et al.; Thesigmasubunit of RNA polymerase determines promoter recognition and catalyzes DNA strand separation . The -35 promoter region is recognized by a helix-turn-helix motif in region 4, while the -10 region is specified, at least in part, by an amphipathic helix in region 2 . We have proposed that conserved aromatic residues insigmaregion 2.3 interact with the non-template strand of the -10 element to drive open complex formation . We now report that Bacillus subtilis sigmaA holoenzyme, but neither core nor sigmaA alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus sequence . UV irradiation of holoenzyme-ssDNA complexes efficiently crosslinks sigmaA to DNA and protease mapping supports a primary contact site in or near region 2 . Several mutations in sigmaA region 2.3, shown previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the efficiency of photocrosslinking . These results support a model in which these aromatic amino acids are juxtaposed to ssDNA, consistent with their demonstrated role in stabilizing the open complex. FEBS Lett, 1997 Jun 30, 410(2-3), 351 - 5 Escherichia coli ccm in-frame deletion mutants can produce periplasmic cytochrome b but not cytochrome c; Throne-Holst M et al.; Escherichia coli CcmA, CcmB and CcmC polypeptides are required for cytochrome c synthesis and are thought to constitute the subunits of an ABC-type transporter as judged from sequence data . Using a periplasmic reporter system based on Bacillus subtilis cytochrome c-550 and E . coli cytochrome b-562 we show that the synthesis of the b-type cytochrome in the periplasm is normal in E . coli ccmA and ccmC in-frame deletion mutants . Mutants deleted for ccmF or ccmG encoding a component of a putative cytochrome c-heme lyase and a membrane bound thioredoxin-like protein, respectively, have the same phenotype . The ccm mutants produce cytochrome c-550 polypeptide, but not holocytochrome c . Taken together the results demonstrate that heme can be transported to the periplasm by a ccm-independent mechanism. Biochim Biophys Acta, 1997 Jun 27, 1357(2), 215 - 24 Expression of a bacterial endo (1-4)-beta-glucanase gene in mammalian cells and post translational modification of the gene product; Zhang JX et al.; An endo (1-4)-beta-glucanase gene C6.5 from Bacillus subtilis has been expressed in Chinese hamster ovary (CHO) cells and pancreatic 266-6 cells . The fusion gene, stably transfected into CHO cells consisted of the mouse Amy-2.2 signal peptide coding sequence and the endoglucanase gene C6.5 transcribed from the early SV40 promoter/enhancer, using the dihydrofolate reductase gene as a selective marker . The gene construct transfected into pancreatic 266-6 cells consisted of the mouse Amy-2.2 promoter/enhancer and signal peptide coding sequence and the same C6.5 sequences using the xanthine-guanine phosphoribosyl transferase gene (gpt) as the selective marker . The stably transfected CHO cells synthesized endoglucanase at 1.1 U/mg cell protein in a 72 h culture, with 89% of the activity secreted into the culture fluid in a glycosylated form of 66 kDa as compared with the unglycosylated 53 kDa form expressed in E . coli . Glycosylation did not change the specific activity, protease resistance, or cellulose binding of the endoglucanase as compared to the unglycosylated form of the enzyme from E . coli . The level of expression in the stably transfected pancreatic cells was substantially lower at 0.3 mU/mg cell protein with all detectable activity present in the culture fluid . The secreted enzyme from pancreatic cells was glycosylated with a mass similar to that secreted from CHO cells. J Biol Chem, 1997 Jun 27, 272(26), 16335 - 42 Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis; Jorgensen S et al.; A gene encoding a highly thermostable extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus was identified . The gene was cloned, sequenced, and expressed in Escherichia coli and Bacillus subtilis . The gene is 1383 base pairs long and encodes a protein of 461 amino acids . The open reading frame of the gene was verified by microsequencing of the recombinant purified enzyme . The deduced amino acid sequence is 25 amino acids longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the membrane . The recombinant alpha-amylase was biochemically characterized and shows an activity optimum at pH 4.5, whereas the optimun temperature for enzymatic activity is close to 100 degrees C . alpha-Amylase shows sequence homology to the other known alpha-amylases and belongs to family 13 of glycosyl hydrolases . This extracellular alpha-amylase is not homologous to the subcellular alpha-amylase previously isolated from the same organism. Biochim Biophys Acta, 1997 Jun 20, 1340(1), 97 - 104 Isolated Bacillus subtilis HemY has coproporphyrinogen III to coproporphyrin III oxidase activity; Hansson M et al.; Oxidation of coproporphyrinogen III to coproporphyrin III is found in extracts of Escherichia coli cells containing the Bacillus subtilis HemY protein (M . Hansson and L . Hederstedt, J . Bacteriol . 176, 5962-5970) . We have analysed whether this activity is due to the heterologous expression system, since it in vivo would lead to disruption of the heme biosynthetic pathway . B . subtilis hemY was fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed from plasmids in both E . coli and B . subtilis . The His6-tagged HemY protein extracted from membranes using non-ionic detergent was purified by Ni2+ affinity chromatography . Isolated HemY fusion protein synthesised in E . coli and B . subtilis oxidised coproporphyrinogen III to coproporphyrin III . No direct formation of protoporphyrin IX from coproporphyrinogen III could be detected . Our results suggest that the coproporphyrinogen III to coproporphyrin III activity of HemY is either avoided in B . subtilis in vivo or that coproporphyrin III is a heme biosynthetic intermediate in this bacterium. Yeast, 1997 Jun 15, 13(7), 655 - 72 DNA sequencing and analysis of 130 kb from yeast chromosome XV; Voss H et al.; We have determined the nucleotide sequence of 129,524 bases of yeast (Saccharomyces cerevisiae) chromosome XV . Sequence analysis revealed the presence of 59 non-overlapping open reading frames (ORFs) of length > 300 bp, three tRNA genes, four delta elements and one Ty-element . Among the 21 previously known yeast genes (36% of all ORFs in this fragment) were nucleoporin (NUP1), ras protein (RAS1), RNA polymerase III (RPC1) and elongation factor 2 (EF2) . Further, 31 ORFs (53% of the total) were found to be homologous to known protein or DNA sequences, or sequence patterns . For seven ORFs (11% of the total) no homology was found . Among the most interesting protein identification in this DNA fragment are an inositol polyphosphatase, the second gene of this type found in yeast (homologous to the human OCRL gene involved in Lowe's syndrome), a new ADP ribosylation factor of the arf6 subfamily, the first protein containing three C2 domains, and an ORF similar to a Bacillus subtilis cell-cycle related protein . For each ORF detailed sequence analysis was carried out, with a full consideration of its biological function and pointing out key regions of interest for further functional analysis. J Biol Chem, 1997 Jun 13, 272(24), 15161 - 6 Identification of functional conserved residues of CTP:glycerol-3-phosphate cytidylyltransferase . Role of histidines in the conserved HXGH in catalysis; Park YS et al.; The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTP:phosphocholine cytidylyltransferases of several organisms . To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine . The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases . The kcat values for H14A and H17A were decreased by factors of 5 x 10(-5) and 4 x 10(-4), respectively, with no significant change in Km values . Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyl-tRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 x 10(-3) . Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with kcat values decreasing by factors of 0.14-4 x 10(-4) . The Km values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6-130-fold higher than that of the wild-type enzyme . Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact . One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not . These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases, i.e . to stabilize a pentacoordinate transition state. Gene, 1997 Jun 11, 192(1), 191 - 8 Binding and transport of transforming DNA by Bacillus subtilis: the role of type-IV pilin-like proteins--a review; Dubnau D; The pathway for binding, processing and transport of transforming DNA into competent cells of Bacillus subtilis is described . The known proteins involved in mediating these processes are reviewed in turn, including several that resemble proteins required for type-IV pilus assembly and function, and those involved in protein secretion . Based on the phenotypes of null mutations in the cognate genes for these proteins, on similarities to other proteins and on membrane localization and topology data, proposals are made for the roles of the individual proteins in the transformation process . A dynamic model is suggested for the presentation of transforming DNA to the transport apparatus. Genetika, 1997 Jun, 33(6), 752 - 6 {Divergence between "early" genes from defective phages of various strains of soil bacilli, close to Bacillus subtilis 168}; Lotareva OV et al.; The extent of xre gene divergence was studied in nine soil bacillus strains with different degrees of relationship to Bacillus subtilis 168 . This gene product is a repressor of defective phages . Bac . subtilis 168 recipient strains were transformed by DNA from these bacillus strains for the xhi-1479 marker and ten markers of amino acid and nitrous bases metabolism . The efficiency of soil strain DNA hybridization with Bac . subtilis 168 DNA was assessed . Eight strains were close to Bac . subtilis 168 with respect to the efficiency of heterotransformation for all markers and hybridization, and one strain (1621) strongly differed from other strains with regard to these parameters . As determined by the degree of differences in heterotransformation for all markers, the nucleotide sequence of the xre gene diverged in the evolution process at a rate similar to that of the nucleotide sequences of the housekeeping genes . All examined genes were shown to have similar selective value. Genetika, 1997 Jun, 33(6), 739 - 43 {Cloning of ribR, an additional regulatory gene of the Bacillus subtilis riboflavin operon}; Solov'eva IM et al.; A 13.0-kb EcoRI fragment of Bacillus subtilis DNA carrying an additional regulatory ribR gene of riboflavin operon was cloned on the basis of resistance to 7, 8-dimethyl-10 (O-methylacetoxym)-isoalloxasin . The cloned fragment was trans-dominant with regard to ribC constitutive mutations that block the overproduction of riboflavin but inactive relative to constitutive mutations in the rib(O) regulatory region. J Antibiot (Tokyo), 1997 Jun, 50(6), 479 - 83 Dihydrophencomycin methyl ester, a new phenazine derivative from a marine Streptomycete; Pusecker K et al.; The novel 5,10-dihydrophencomycin methyl ester (4) and the known microbial metabolites (2-hydroxyphenyl)-acetamide (1), menaquinone MK9 (II, III, VIII, IX-H8) (2), and phencomycin (3a) were isolated from an unidentified marine Streptomyces sp . and the structures were elucidated by NMR methods . Compound 4 shows weak antibiotic activity against Escherichia coli and Bacillus subtilis. Planta Med, 1997 Jun, 63(3), 268 - 70 Phenolic and antibacterial constituents of Vahlia capensis; Majinda RR et al.; The n-butanol fraction of Vahlia capensis yielded kaempferol, quercetin, afzelin, astragalin, quercitrin, isoquercitrin, rutin, gallic acid, chiro-inositol, dulcitol, and a novel biflavonoid, VC-15B (vahlia biflavone) . The compounds were identified using 1D and 2D NMR techniques and FABMS . Vahlia biflavone and gallic acid were isolated, using bioassay-guided procedure and identified as the antibacterial components . Both compounds showed activity against Gram positive Staphylococcus aureus and Bacillus subtilis . Vahlia biflavone gave MIC values of 15.3 micrograms/ml and 30.6 micrograms/ml against S . aureus and B . subtilis, respectively while gallic acid gave a value of 71.3 micrograms/ml for both organisms. Mol Microbiol, 1997 Jun, 24(5), 905 - 15 The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division; Edwards DH et al.; The Bacillus subtilis divIVA gene, first defined by a mutation giving rise to anucleate minicells, has been cloned and characterized . Depletion of DivIVA leads to inhibition of the initiation of cell division . The residual divisions that do occur are abnormally placed and sometimes misorientated relative to the long axis of the cell . The DivIVA phenotype can be suppressed by disruption of the MinCD division inhibitor, suggesting that DivIVA controls the topological specificity of MinCD action and thus septum positioning . A DivIVA-GFP fusion targets to new and used sites of cell division, consistent with it having a direct role in topological specification. Biosci Biotechnol Biochem, 1997 Jun, 61(6), 1019 - 21 Effects of DNA topology on transformation efficiency of Bacillus subtilis ISW1214 by electroporation; Ohse M et al.; We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214 . Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form . Treatment of sc DNA with restriction endonculease linearized the DNA . The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6 +/- 0.6) x 10(5) transformants per microgram DNA higher than that (2.0 +/- 0.3) x 10(4) transformants per microgram DNA for the cr DNA, using the DNA concentration of 20 micrograms/ml at an electric field strength of 7 kV/cm and a capacitance of 10 microF with a single decayed pulse . The transformation efficiency (TE) for the ln DNA was zero . The variations of TE for different topological forms of DNA reflected their relative stability in the host cells . The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA. Bioessays, 1997 Jun, 19(6), 455 - 8 A regulatory switch involving a Clp ATPase; Lazazzera BA et al.; Clp ATPase chaperone proteins are found in procaryotes and eucaryotes . Recently, ClpC of Bacillus subtilis was found to be part of a regulatory switch(1) . ClpC, in combination with the MecA and ComS proteins, regulates the activity of a transcription factor, ComK, which is necessary for the development of genetic competence (the ability to bind and take up exogenous DNA) . The complex of ClpC:MecA:ComK renders ComK inactive . Interaction between ComS and the ternary complex releases active ComK . This regulatory switch controls ComK activity in response to cell density signals that affect production of ComS . Regulated interaction between Clp ATPase and target proteins might prove to be widespread. Microbiology, 1997 Jun, 143 ( Pt 6), 1933 - 40 The thioredoxin reductase system of mycoplasmas; Ben-Menachem G et al.; Representative species of the Mollicutes possess a thioredoxin reductase system (NTS) composed of a low-molecular-mass thioredoxin (TRX) and NADPH-binding thioredoxin reductase (NTR) . The TRXs of Mycoplasma pneumoniae and M . capricolum have molecular masses of 11-2 and 12 kDa, respectively, and are stable at 90 degree C for 10 min . Both TRXs reacted with monospecific polyclonal antibodies generated against the Bacillus subtilis TRX, but not with anti-Escherichia coli TRX antisera . The M . capricolum and M . pneumoniae NTRs were partially purified and were found to be active with the homologous TRX, but not with the TRX of B . subtilis or E . coli . The NTS activity had an optimal pH of 6.5-7.5 and was dependent on NADPH as an election donor, a requirement which could not be fulfilled by NADH . The genes encoding the TRX and NTR (trxA and trxB) or M . pneumoniae were cloned and sequenced . The comparative analysis of the predicted amino acid sequence of trxA showed that the 11.2 kDa protein (102 aa) shared 26-68% sequence similarity with products of other known trxA genes and contained the conserved active site Cys-Gly-Pro-Cys . The predicted amino acid sequence of trxB contained 315 residues with a conserved NADPH binding domain and FAD binding domains I and II . The cysteine dithiol redox active region had isoleucine rather than threonine at the active site, as compared with other NTRs . The high activity of the NTS in mycoplasmas suggests that mycoplasmas may have evolved the NTS to protect themselves from the consequences of their self-generated oxidative challenge. Microbiology, 1997 Jun, 143 ( Pt 6), 1855 - 9 The Bacillus subtilis 168 chromosome from sspE to katA; Cummings NJ et al.; We have cloned and sequenced a 24.5 kb region of the Bacillus subtilis 168 chromosome spanning the sspE and katA genes . The region contains a ribosomal RNA operon, rrnD, a tRNA gene set, trnD and 17 ORFs, 16 with putative ribosome-binding sites . Four of the ORFs (ORF2, ORF14, ORF16 and ORF17) match to known B . subtilis genes (sspE, thiA, senS and katA) . Eight of the remaining ORF products show similarities with proteins present in the databases, including an ATP-binding transport protein, a glutamate-1-semialdehyde aminotransferase, a thiol-specific antioxidant protein, a mitomycin radical oxidase and a ferric uptake regulation protein. J Bacteriol, 1997 Jun, 179(12), 3922 - 7 Contribution of partner switching and SpoIIAA cycling to regulation of sigmaF activity in sporulating Bacillus subtilis; Magnin T et al.; sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB . SpoIIAB has an alternative binding partner, SpoIIAA . To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigmaF depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigmaF-SpoIIAB-ADP, and sigmaF-SpoIIAB-ATP . The results suggested that SpoIIAB's choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell . The intracellular concentrations of sigmaF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3) . sigmaF and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5 . SpoIIAA-P increased steadily to a maximum at t3, but nonphosphorylated SpoIIAA was detectable only from t1.5, reached a maximum at t2.5, and then declined . Kinetic studies of the phosphorylation of SpoIIAA catalyzed by SpoIIAB suggested that the reaction was limited by a very slow release of one of the products (SpoIIAA-P or ADP) from SpoIIAB, with a turnover of about once per 20 min . This remarkable kinetic property provides an unexpected mechanism for the regulation of sigmaF . We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the same time as SpoIIAB, SpoIIAA cycles repeatedly between the phosphorylated and nonphosphorylated forms . This cycling sequesters SpoIIAB in a long-lived complex and prevents it from inhibiting sigmaF. J Bacteriol, 1997 Jun, 179(12), 3914 - 21 New classes of mutants in complementary chromatic adaptation provide evidence for a novel four-step phosphorelay system; Kehoe DM et al.; Complementary chromatic adaptation appears to be controlled by a complex regulatory system with similarity to four-step phosphorelays . Such pathways utilize two histidine and two aspartate residues for signal transduction . Previous studies of the signaling system controlling complementary chromatic adaptation have uncovered two elements of this pathway, a putative sensor, RcaE, and a response regulator, RcaC . In this work, we describe a second response regulator controlling complementary chromatic adaptation, RcaF, and identify putative DNA binding and histidine phosphoacceptor domains within RcaC . RcaF is a small response regulator with similarity to SpoOF of Bacillus subtilis; the latter functions in the four-step phosphorelay system controlling sporulation . We have also determined that within this phosphorelay pathway, RcaE precedes RcaF, and RcaC is probably downstream of RcaE and RcaF . This signal transduction pathway is novel because it appears to use at least five, instead of four, phosphoacceptor domains in the phosphorelay circuit. RNA, 1997 Jun, 3(6), 577 - 85 Ribonuclease P RNA: models of the 15/16 bulge from Escherichia coli and the P15 stem loop of Bacillus subtilis; Easterwood TR et al.; The Escherichia coli ribonuclease P RNA 15/16 internal bulge loop and the Bacillus subtilis P15 stem loop are important substrate binding sites for the CCA-3' terminus of pre-tRNA . Models of E . coli 15/16 bulge loop and the B . subtilis P15 stem loop have been constructed using MC-SYM, a constraint satisfaction program . The models use covariation analysis data for suggesting initial base pairings, chemical probing, and protection/modification results to determine particular pairing orientations, and mutational experimental analysis data for tRNA-RNase P RNA contacts . The structures from E . coli and B . subtilis, although different in secondary structure, have similar sequence and function . Using MC-SYM, we are able to illustrate how the 3' end of the pre-tRNA is able to interact with this segment of the catalytic RNase P RNA . In addition, we propose additional hydrogen bonding between A76 in the 3' terminus of the tRNA and the 15/16 region of E . coli and to the loop of B . subtilis. J Bacteriol, 1997 Jun, 179(11), 3509 - 18 Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist; Movahedzadeh F et al.; The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E . O . Davis, S . G . Sedgwick, and M . J . Colston, J . Bacteriol . 173:5653-5662, 1991) . In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase . A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression . However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon . Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites . The differences from the E . coli consensus patterns would explain the previously described lack of expression of the M . tuberculosis recA gene from its own promoter in E . coli . In addition, the M . tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes . The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence. Infect Immun, 1997 Jun, 65(6), 2321 - 8 High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets; Harth G et al.; Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is the world's leading cause of death from a single infectious agent, and new vaccines and drugs to combat it are urgently needed . The major extracellular proteins of M . tuberculosis, which are released into its phagosome in macrophages, its host cells in humans, are leading candidates for a vaccine and prime targets for new drugs . However, the development of these biologicals has been hampered by the unavailability of large quantities of recombinant extracellular proteins identical to their native counterparts . In this report, we describe the heterologous expression and secretion of four major M . tuberculosis extracellular proteins (the 30-, 32, 16-, and 23.5-kDa proteins--the first, second, third, and eighth most abundant, respectively) in rapidly growing, nonpathogenic mycobacterial species . Multiple attempts to obtain secretion of the proteins by using Escherichia coli- and Bacillus subtilis-based expression systems were unsuccessful, suggesting that high-level expression and secretion of these Mycobacterium-specific proteins require a mycobacterial host . All four recombinant proteins were stably expressed from the cloned genes' own promoters at yields that were 5- to 10-fold higher than those observed for the native proteins . The four proteins were purified to apparent homogeneity from culture filtrates by ammonium sulfate precipitation and ion-exchange and molecular sieve chromatography . The recombinant proteins were indistinguishable from their native counterparts by multiple criteria . First, N-terminal amino acid sequence determination demonstrated that processing of the leader peptides was highly accurate . Second, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed identical migration patterns . Third, mass spectrometry analysis confirmed that differences in mass were < or = 5 Da . A homolog of the M . tuberculosis 30-kDa protein was identified in M . smegmatis by means of DNA analyses and immunoscreening . This is the first time that secretion of recombinant M . tuberculosis extracellular proteins in their native form has been achieved . This study opens the door to mass production of correctly processed and secreted extracellular proteins of M . tuberculosis in a heterologous host and allows ready evaluation of their biologic and immunologic function. Biochemistry, 1997 May 27, 36(21), 6317 - 25 Recognition of the T stem-loop of a pre-tRNA substrate by the ribozyme from Bacillus subtilis ribonuclease P; Loria A et al.; The ribozyme from bacterial ribonuclease P (denoted P RNA) specifically recognizes the coaxially stacked T stem-loop and the acceptor stem of a tRNA substrate . This recognition is mediated primarily through tertiary interactions . At least four 2'-OH groups in the T stem-loop region have been implicated as direct contacts with Bacillus subtilis P RNA {Pan, T., et al . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 12510} . Effects of six single 2'-OH --> 2'-H substitutions and two base mutants of the G19-C56 tertiary interaction in tRNA on substrate binding (Kd) and the chemical step of the reaction (k2) have been determined using a tRNA(Phe) substrate containing a 2'-deoxy residue at the cleavage site . Our results show that at least five functional groups in the T stem-loop of tRNA directly participate in P RNA binding . They include the 2'-OH groups of residues 54, 56, 61, and 62 and possibly the 4-amino group of the conserved C56 . The 2'-OHs of residues 54, 61, and 62 are positioned within the same minor groove due to stacking of the reverse Hoogsteen U54-A58 pair on the G53-C61 Watson-Crick pair in the T stem . This groove is extended to the 4-amino group of C56 through the tertiary structure of tRNA . We use the term "tertiary groove" to describe alignment of functional groups through tertiary folding of an RNA . The binding also includes the 2'-OH of nucleotide C56 which is not located in this tertiary groove . Assuming additivity, these five interactions can contribute 7.4 kcal/mol or 10(5)-fold in binding but only -0.5 kcal/mol or approximately 2-fold in chemistry at 37 degrees C . The P RNA binding site for the T stem-loop includes at least the previously identified A230 as well as the A130 in B . subtilis P RNA . The Kd and k2 data from the A130G mutant of B . subtilis P RNA suggest that A130 may be proximal to residue 56 in tRNA . These results show how the highly structured T stem-loop region in a pre-tRNA substrate is bound by the B . subtilis P RNA . This is among the first examples of how a nonhelical RNA structure can be recognized by another RNA through tertiary interactions. Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5622 - 7 Diffusion control in an elementary protein folding reaction; Jacob M et al.; The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N &lrharr; U two-state reaction . Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose . The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together . It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding . Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy . We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein . This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior. J Biotechnol, 1997 May 23, 55(1), 43 - 53 Over-expression of the Saccharomyces cerevisiae exo-beta-1,3-glucanase gene together with the Bacillus subtilis endo-beta-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in yeast; van Rensburg P et al.; The EXG1 gene encoding the main Saccharomyces cerevisiae exo-beta-1,3-glucanase was cloned and over-expressed in yeast . The Bacillus subtilis endo-1,3-1,4-beta-glucanase gene (beg1) and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene (end1)