Bacillus subtilis

Nucleic Acids Res, 1996 Dec 1, 24(23), 4775 - 82
Structure and evolution of ribonuclease P RNA in Gram-positive bacteria; Haas ES et al.; The sequences and structures of RNase P RNAs of some Gram-positive bacteria, e.g . Bacillus subtilis, are very different than those of other bacteria . In order to expand our understanding of the structure and evolution of RNase P RNA in Gram-positive bacteria, gene sequences encoding RNase P RNAs from 10 additional species from this evolutionary group have been determined, doubling the number of sequences available for comparative analysis . The enlarged data set allows refinement of the secondary structure model of these unusual RNase P RNAs and the identification of potential tertiary interactions between P10.1 and L12, and between L5.1 and L15.1 . The newly-obtained sequences suggest that RNase P RNA underwent an abrupt, dramatic restructuring in the ancestry of the low-G+C Gram-positive bacteria after the divergence of the branches leading to the 'Clostridia and relatives' and the remaining low-G+C Gram-positive species . The unusual structures of the RNase P RNAs of Mycoplasma hyopneumoniae and M.floccularre are apparently derived from RNAs with Bacillus-like structure rather than from intermediate, partially restructured ancestral RNAs . The structure of the RNase P RNA from the photosynthetic Heliobacillus mobilis supports the relationship of this specie with Bacillus and Staphylococcus rather than the 'Clostridia and relatives' as suggested by the sequences of their small-subunit ribosomal RNAs.

J Bacteriol, 1996 Dec, 178(24), 7206 - 11
Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro; Lu Y et al.; Transcriptional attenuation of the pyrimidine biosynthetic (pyr) operon from Bacillus subtilis was reconstituted with an in vitro system that consisted of pyr DNA templates, B . subtilis RNA polymerase, four ribonucleoside triphosphates, and the purified B . subtilis PyrR regulatory protein . The templates used each specified one of the three known attenuation regions of the pyr operon . Runoff (read-though) and terminated transcripts of the predicted lengths were the only major products synthesized . Transcription of the template that specifies the 5' leader attenuation region of the operon was examined in detail . Termination of transcription at the attenuator was strongly promoted by the combination of PyrR plus UMP . The concentration of UMP required for half-maximal effect was 2.5 microM . UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UDP was only effective at 100 times the concentration of UMP . Other pyrimidine and purine metabolites tested did not affect termination . PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferase activity of PyrR, antagonized UMP-dependent transcriptional termination, but uracil did not . Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr attenuation regions . The results strongly support the model for transcriptional regulation of the B . subtilis pyr operon previously proposed by R . J . Turner, Y . Lu, and R . L . Switzer (J . Bacteriol . 176:3708-3722, 1994).

J Bacteriol, 1996 Dec, 178(24), 7112 - 9
The levanase operon of Bacillus subtilis expressed in Escherichia coli can substitute for the mannose permease in mannose uptake and bacteriophage lambda infection; Martin-Verstraete I et al.; Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, a maltose- and maltodextrin-specific porin of the outer membrane . LamB also serves as a receptor for several other bacteriophages . Lambda DNA requires, in addition to LamB, the presence of two bacterial cytoplasmic integral membrane proteins for penetration, namely, the IIC(Man) and IID(Man) proteins of the E . coli mannose transporter, a member of the sugar-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS) . The PTS transporters for mannose of E . coli, for fructose of Bacillus subtilis, and for sorbose of Klebsiella pneumoniae were shown to be highly similar to each other but significantly different from other PTS transporters . These three enzyme II complexes are the only ones to possess distinct IIC and IID transmembrane proteins . In the present work, we show that the fructose-specific permease encoded by the levanase operon of B . subtilis is inducible by mannose and allows mannose uptake in B . subtilis as well as in E . coli . Moreover, we show that the B . subtilis permease can substitute for the E . coli mannose permease cytoplasmic membrane components for phage lambda infection . In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not require the mannose transporter for infection.

J Bacteriol, 1996 Dec, 178(23), 7020 - 3
The yeast two-hybrid system detects interactions between Bacillus subtilis sigmaB regulators; Voelker U et al.; SigmaB, the general stress response sigma factor of Bacillus subtilis, is regulated by the products of seven genes (rsbR, S, T, U, V, W, and X) with which it is cotranscribed . Biochemical techniques previously revealed physical associations among RsbW, RsbV, and sigmaB but failed to detect interactions of RsbR, S, T, U, or X with each other or RsbV, RsbW, or sigmaB . Using the yeast two-hybrid system, we have now obtained evidence for such interactions . The yeast reporter system was activated when RsbS was paired with either RsbR or RsbT, RsbR was paired with RsbT, and RsbV was paired with either RsbU or RsbW . In addition, RsbW2 and RsbR2 dimer formation was detected . RsbX failed to show interactions with itself or any of the other sigB operon products.

J Bacteriol, 1996 Dec, 178(23), 7014 - 5
Significance of HPr in catabolite repression of alpha-amylase; Voskuil MI et al.; CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes . However, alpha-amylase synthesis by B . subtilis SA003 containing the ptsH1 mutation was repressed by glucose . Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B . subtilis.

J Bacteriol, 1996 Dec, 178(23), 6865 - 72
A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family; Braun EL et al.; The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms . In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability . We have described changes in the pattern of protein synthesis during the growth of S . cerevisiae cells to stationary phase (E . K . Fuge, E . L . Braun, and M . Werner-Washburne, J . Bacteriol . 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase . We report here the identification of the SNZ1 gene, which encodes this protein . We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes . SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization . Multiple SNZ1-related sequences were detected in some organisms, including S . cerevisiae . Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S . cerevisiae . The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S . cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest.

J Bacteriol, 1996 Dec, 178(23), 6730 - 5
Protein conformational change and nucleotide binding involved in regulation of sigmaF in Bacillus subtilis; Lord M et al.; We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions at the site of phosphorylation (serine 58), to interact with SpoIIAB . Native gel analysis revealed that SpoIIAAS58A could form a complex with SpoIIAB in the presence of ADP and more strongly in the presence of ATP . SpoIIAAS58N did not form a complex with SpoIIAB in the presence of ADP but displayed some interaction with SpoIIAB in the presence of ATP . SpoIIAAS58D was unable to form a complex with SpoIIAB in the presence of either ADP or ATP . Corresponding differences were found in the behavior of the three mutant proteins when studied by gel permeation with high-performance liquid chromatography and limited proteolysis . SpoIIAAS58A behaved like the wild-type SpoIIAA, SpoIIAAS58D like SpoIIAA-P, and SpoIIAAS58N in a way that was intermediate between the behaviors of SpoIIAA and SpoIIAA-P . Limited proteolysis was also used to show that on binding of ADP or ATP SpoIIAB undergoes a shift in conformation . The affinity of SpoIIAB for ADP and ATP was determined by limited proteolysis in the presence of a wide range of nucleotide concentrations . The results indicated that SpoIIAB has approximately equal affinity for ADP and for ATP.

Infect Immun, 1996 Dec, 64(12), 5178 - 86
Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin); Ishihara K et al.; A chymotrypsin-like protease from Treponema denticola ATCC 35405 was purified by chromatographic techniques . The purified enzyme consisted of three polypeptides (38, 43, and 72 kDa) . The protease exhibited specificity for peptide bonds containing phenylalanine and proline at the P1 and P2 positions, respectively, and was classified as a serine protease on the basis of inhibition studies . Naturally occurring protease inhibitors such as alpha1-antitrypsin and alpha1-antichymotrypsin had no effect on enzymatic activity . The enzyme degraded fibronectin, alpha1-antitrypsin, and gelatin while weakly degrading the immunoglobulin G heavy chain and type IV collagen . N-terminal amino acid sequences were determined for the 43- and 72-kDa proteins . On the basis of these sequences, the genes coding for the 43- and 72-kDa proteins were isolated and sequenced . The open reading frame which codes for the 72-kDa protein was designated prtP . This gene consists of 2,169 bp and codes for a protein with an Mr of 77,471 . The protein appeared to be composed of a signal peptide region followed by a prosequence and the mature protein domain . The deduced amino acid sequence exhibited similarity with that of the Bacillus subtilis serine protease subtilisin . The deduced properties of the sequence suggest that the 72-kDa protein is a chymotrypsin-like protease . However, the nature and function of the 43-kDa protein have not yet been determined.

J Biol Chem, 1996 Nov 29, 271(48), 31000 - 7
Activation of replication origins in phi29-related phages requires the recognition of initiation proteins to specific nucleoprotein complexes; Freire R et al.; Protein p6 of Bacillus subtilis phage phi29 activates the initiation of viral DNA replication by forming a multimeric nucleoprotein complex at the origins of replication, located at both ends of the linear genome . This activation requires a precise positioning of the protein p6 array with respect to the initiation site . To investigate this activation mechanism, we have purified the phi29 protein p6 counterparts from the related phages Nf and GA-1 and analyzed the formation of complexes with DNA . In the homologous protein p6-DNA complexes the phi29 and Nf protein arrays showed an identical positioning, different than that of the GA-1 protein array . In contrast, in the heterologous complexes the protein showed a different arrangement except in the case of the Nf protein-phi29 DNA complex . We have also purified the proteins involved in the initiation of replication (terminal protein and DNA polymerase) from phages Nf and GA-1 and measured the ability of the different p6 proteins to activate homologous and heterologous replication origins . The results obtained indicate that the activation requires not only the formation of a specific nucleoprotein complex but also its specific recognition by the proteins involved in the initiation of DNA replication.

Gene, 1996 Nov 28, 181(1-2), 147 - 51
The Bacillus subtilis chromosome region near 78 degrees contains the genes encoding a new two-component system, three ABC transporters and a lipase; Yamamoto H et al.; The nucleotide sequence of a 9444-bp segment around the 78 degrees region of the Bacillus subtilis (Bs) chromosome has been determined . Nine putative orfs were identified . The deduced amino acid sequences of the products of two of them (yfiJ and yfiK) exhibit high similarity to those of a sensor protein, DegS, and a transcriptional regulatory protein, DegU, of Bs, respectively . Three of them (yfiL, yfiM and yfiN) seem to be ABC transporter genes . One orf (designated as lipB), the closest to the sspE among the nine orfs, is the second lipase gene in Bs.

Gene, 1996 Nov 28, 181(1-2), 71 - 6
A xylose-inducible Bacillus subtilis integration vector and its application; Kim L et al.; The construction of a xylose-inducible expression vector is described . This vector allows the integration of any gene, coding for its authentic protein, at the amyE locus of Bacillus subtilis (Bs) . The controlable expression cassette consists of the repressor-encoding gene and the promoter of the Bacillus megaterium-derived operon for xylose utilization, sandwiched between the 5'- and 3'-ends of amyE . This thereby allows insertion of in vitro constructed transcriptional fusions at the amyE locus of the Bs chromosome . The versatility of this expression system was tested by fusing three different heat-shock genes to the xylose-inducible promoter and following their expression by Western immunoblot analysis . Whereas no increase in the amount of heat-shock protein could be detected under non-inducing conditions when compared to the isogenic wild-type strain, the three proteins were strongly induced after addition of xylose, depending on the gene . To determine the tightness and the induction factor of the system more accurately, the bgaB gene encoding a heat-stable beta-galactosidase (beta Gal) was analyzed . The background activity of beta Gal increased by a factor of at least 200 after addition of xylose . The system is not subject to catabolite, but rather to glucose repression.

J Biol Chem, 1996 Nov 22, 271(47), 30256 - 62
An alkaline D-stereospecific endopeptidase with beta-lactamase activity from Bacillus cereus; Asano Y et al.; We purified a novel extracellular D-stereospecific endopeptidase, alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cereus strain DF4-B . The Mr of the enzyme was 37,952, and it was composed of a single polypeptide chain . The optimal pH for activity was approximately 10.3 . The enzyme was strictly D-stereospecific toward oligopeptides composed of Dphenylalanine such as (D-Phe)3 and (D-Phe)4 . The enzyme also acted to a lesser extent on (D-Phe)6, Boc-(D-Phe)4 (where Boc is tert-butoxycarbonyl), Boc-(D-Phe)4 methyl ester, Boc-(D-Phe)3 methyl ester, Boc-(D-Phe)2, (D-Phe)2, and others, but not upon their corresponding peptides composed of L-Phe, (D-Ala)n (n = 2-5), (D-Val)3, and (D-Leu)2 . The mode of action of the enzyme was clarified with synthetic substrates ((D-Phe)2-D-Tyr and D-Tyr-(D-Phe)2) and eight stereoisomers of (Phe)3 . The enzyme had beta-lactamase activity toward ampicillin and penicillin G, although carboxypeptidase DD and D-aminopeptidase activities were undetectable . The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1164-base pair open reading frame consisting of 388 codons was identified as the adp gene . The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from Streptomyces lactamdurans and Bacillus subtilis, and class C beta-lactamases . Thus, the enzyme was categorized as a new "penicillin-recognizing enzyme."

Gene, 1996 Nov 21, 180(1-2), 57 - 61
Plasmids for ectopic integration in Bacillus subtilis; Guerout-Fleury AM et al.; Plasmids have been constructed that allow integration by a double recombination event at the thrC locus of the Bacillus subtilis (Bs) chromosome . These plasmids can be used either for construction of merodiploid strains and complementation analysis, or for construction of transcriptional fusions to the Escherichia coli lacZ gene . The plasmids contain an antibiotic (An) marker selectable in Bs, as well as an additional An marker outside of the region that can recombine into the chromosome . When used in conjunction with recipient strains containing a third An marker at their thrC locus, these plasmids allow easy identification of transformants issued from a marker exchange event without additional Campbell-type integration . The existing plasmids used for ectopic integration at the amyE locus have been modified similarly.

FEBS Lett, 1996 Nov 18, 397(2-3), 173 - 6
Bacillus subtilis DegU acts as a positive regulator for comK expression; Ogura M et al.; Bacillus subtilis ComK plays a critical role in competence development . We report that B . subtilis degR, a positive regulator for exoenzyme production, is negatively regulated by overproduced ComK caused by a mecA null mutation . To identify a positive regulator for comK expression in the mecA background, mutations that allowed the degR gene to be expressed were screened in Tn10 transposon insertion mutants . As a result, we identified degU insertion mutations as those having such a property . The degU mutation reduced comK-lacZ expression in a competence medium in both the wild-type and mecA cells in sporulation and competence media . These results indicate that the degU gene product acts as a positive regulator for comK expression even under the condition where the negative regulation of comK by MecA is released.

Nucleic Acids Res, 1996 Nov 15, 24(22), 4565 - 71
Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2); Cho EJ et al.; The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2) . It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis . The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B . subtilis alpha subunits, respectively . Using T7 expression system, we overexpressed the S . coelicolor alpha protein in E . coli . A small fraction of this protein was found to be assembled into E . coli RNA polymerase . Antibody against S . coelicolor alpha protein crossreacted with that of B . subtilis more than with the E . coli alpha subunit . The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity . Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly . Similar results were also obtained for natural alpha protein . Limited proteolysis with endoproteinase Glu-C revealed that S . coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E . coli alpha.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 63 - 9
Impaired oxidative stress resistance of Bacillus subtilis sigB mutants and the role of katA and katE; Engelmann S et al.; Two catalases of B . subtilis have been studied which are subject to two different regulatory mechanisms . Whereas KatA belongs to the group of proteins specifically induced by oxidative stress, KatE is a general delta B-dependent stress protein, not induced by oxidative stress . There are two mechanisms of oxidative stress resistance, the adaptive resistance induced by low H2O2 concentrations and an unspecific resistance acquired in glucose-starved cells . Mutants lacking KatA are defective in the adaptive resistance and both exponentially growing and glucose-starved cells are 100-fold more sensitive against lethal concentrations of H2O2 . Under both conditions, however, a katE mutant was just as resistant as the wild type . Therefore, the role of KatE in oxidative stress tolerance remains obscure . sigB mutants which are no longer able to induce delta B-dependent general stress proteins in glucose-starved cells are characterized by a strong impairment in the unspecific oxidative stress resistance but not in the H2O2-induced oxidative stress resistance . This is the first evidence that sigB mutants have an obvious phenotype compared to the wild type and indicates that delta B-dependent general stress proteins may function in providing starving cells with resistance against oxidative stress.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 41 - 8
Overproduction, purification and characterization of the HPB12-L24 ribosomal protein of Bacillus subtilis; Zouine M et al.; HPB12-L24 was previously described as a bifunctional histone-like and ribosomal protein in Bacillus subtilis . In order to confirm the identity of HPB12 and L24, and to study the properties of this protein, the rplX gene of B . subtilis encoding L24 has been overexpressed in Escherichia coli by an efficient protein overproduction system . A simple and rapid purification scheme using ammonium sulfate precipitation and cation-exchange chromatography is presented . 10 mg of pure L24 per g of Escherichia coli cells were obtained . The purified recombinant protein L24 is heat-stable, acid-soluble and binds preferentially supercoiled DNA like protein HPB12 . These results confirm the identity of HPB12 and L24 . Overexpression of rplX led to gross alterations of cell morphology and to an abnormal shape of nucleoids.

J Biol Chem, 1996 Nov 15, 271(46), 28898 - 902
Energetic implications for protein phosphorylation . Conformational stability of HPr variants that mimic phosphorylated forms; Huffine ME et al.; The HPr protein from Bacillus subtilis is a key protein in the phosphoenolpyruvate-sugar transport system . HPr has two biological phosphorylation sites . The active site histidine is transiently phosphorylated in the phosphotransferase reaction while phosphorylation of serine 46 diminishes the activity of HPr . Here, we use protein engineering and equilibrium protein folding experiments to determine if the two phosphorylation events are energetically coupled . Our approach is to use structural mimics of the two phosphorylated forms of HPr, where histidine 15 is replaced by a negatively charged glutamate and serine 46 is changed to an aspartate, both alone and in combination . The thermodynamic analysis of the differences in conformational stability between the single and double mutants shows that the two phosphorylation sites are not energetically coupled in the HPr protein . We also show that single mutants of the active site histidine residue can have dramatic effects on the conformational stability of HPr . Combined with structural information, the method employed here will be of general use in unraveling the biological effects of phosphorylation on protein activity.

Gene, 1996 Nov 14, 179(2), 287 - 9
A novel cloning system for direct screening using a suicidal strategy; Barros EV et al.; The pAC92 plasmid is a direct screening cloning vector which allows positive selection of recombinant clones (re-clones) . This new high-copy-number plasmid vector encodes ampicillin resistance and carries the Bacillus subtilis alpha-amylase (alpha-Amy)-encoding gene (amy) containing a multiple cloning site . The pAC92 plasmid confers to Escherichia coli transformants an amylolytic phenotype easily detected by iodine vapor staining . The re-clones are identified by insertional inactivation of alpha-Amy activity . During pAC92 construction, a bacterial growth defect was observed in host cells after some modifications of the promoter region that caused the increase in the amy expression . This suicide characteristic permitted the positive selection of re-clones . A second transformation step was performed to enhance the rate of re-clones per plate.

Biochim Biophys Acta, 1996 Nov 11, 1309(1-2), 42 - 6
Isolation and characterization of the Bacillus subtilis tryptophanyl-tRNA synthetase gene (trpS) conferring 5-fluorotryptophan resistance and temperature sensitivity; Chow KC et al.; The mutant trpS gene of Bacillus subtilis encoding a thermosensitive tryptophanyl-tRNA synthetase that also confers resistance to growth inhibition by 5-fluorotryptophan at permissive temperature has been isolated and characterized . A point mutation at codon 82 of the gene from a wild-type TCA codon for Ser to a TTA codon for Leu has been identified.

Genetika, 1996 Nov, 32(11), 1498 - 503
{Homology in natural cryptic plasmids of Bacillus subtilis}; Poluektova EU et al.; Peculiarities of DNA homology in a number of cryptic plasmids isolated from soil bacillary strains and related or identical to Bacillus subtilis were studied . Fragments generated after digestion of one of these plasmids, p1414, were employed as a probe for blot hybridization with identical fragments of other plasmids . The data obtained suggest that nearly all the studied plasmids possess a common homology site that occupies a significant plasmid portion . Regions of detectable and weak homology were located throughout this site . Moreover, plasmid p1414 was shown to carry a large site that lacks homology with plasmids belonging to two groups . Eventually, one of these plasmids demonstrates complete lack of homology with the remaining plasmids.

PDA J Pharm Sci Technol, 1996 Nov-Dec, 50(6), 391 - 8
Microbial barrier assessment of Tyvek stopper packaging for rubber closures; Moldenhauer JE et al.; Two types of Tyvek and high density polyethylene or polypropylene packaging used for sterilization of rubber closures were evaluated for Microbial Barrier properties . The packaging evaluated was "Ready to Sterilize" (1) stoppers and a second test package (Test 2) which was designated as appropriate for a clean room, filled with washed and siliconized stoppers and then heat sealed . Each type of packaging was subjected to three different sterilization temperatures (125 degrees C, 128 degrees C and 131 degrees C) in a production sterilizer (15-18 psi) . Following sterilization, a microbial barrier assessment was performed, using Bacillus subtilis niger (ATCC 9372), to determine whether the packaging could maintain a sterile barrier following sterilization . Results of the testing indicated that a microbial barrier was maintained for products in "Ready to Sterilize" packages at 125 degrees C and 128 degrees C . For products sterilized in the Test 2 container a microbial barrier could not be maintained at 128 degrees C, and no further testing was performed . Following sterilization at 131 degrees C physical defects were noted for the "Ready to Sterilize" bag and a microbial barrier could not be maintained.

Prikl Biokhim Mikrobiol, 1996 Nov-Dec, 32(6), 646 - 9
{The effect of electromagnetic field on the biosynthesis and various properties of extracellular proteinase and lectin from Bacillus subtilis 316 M}; Kudria VA et al.; The promoting effect of electromagnetic field (EMF) on biosynthesis and activity of extracellular proteinase and lectin in B . subtilis 316 M was observed . It caused 1.5- and 4-fold increase of the metabolites yield respectively . The EMF stimulated a 2-fold activation of lectin, rise of the enzyme activity and a shift of it pI from 11.4-11.5 to 9.2-9.3.

J Biotechnol, 1996 Nov 1, 51(2), 175 - 80
Dielectrophoretic separation of bacteria using a conductivity gradient; Markx GH et al.; Dielectrophoresis, the lateral motion induced on particles by non-uniform electric fields, is a sensitive function of the electrical conductivity of the particle suspending medium . This dependence is exploited in a new technique for separating bioparticles from suspended mixtures . The bioparticles are first immobilised by positive dielectrophoresis at electrodes in a separation chamber, and the conductivity of the liquid flowing through the chamber is then gradually and continuously increased so as to produce a conductivity gradient with time . The bioparticles are released from the electrodes according to their own dielectric properties and as a function of flow rate and medium conductivity . This is demonstrated for pure suspensions and mixtures of the bacteria Bacillus subtilis, Escherichia coli and Micrococcus luteus.

Lett Appl Microbiol, 1996 Nov, 23(5), 290 - 4
Alanine germination receptors of Bacillus subtilis; McCann KP et al.; The alanine-stimulated spore germination responses of Bacillus subtilis 168 have been dissected by combining physiological and genetical approaches . From the analyses the authors infer that there are three classes of alanine response . Two of the responses are mediated via the GerA proteins, with and without germinal adjuncts, the third is mediated via the GerB proteins and obligately requires adjuncts.

Microbiology, 1996 Nov, 142 ( Pt 11), 3163 - 70
Phosphate-starvation-inducible proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study; Eymann C et al.; A two-dimensional (2-D) gel electrophoresis study of Bacillus subtilis strain 168 identified 20 proteins that are strongly induced in response to phosphate starvation . The induction of nine of these phosphate-starvation-induced (Psi) proteins was dependent on a functional PhoR protein . PhoR is the histidine sensor-kinase component of a phosphate-concentration-sensing two-component regulatory system which, together with its partner response regulator PhoP, controls the expression of genes in the Pho regulon . Genes encoding PhoR-dependent Psi proteins are therefore likely to be members of the Pho regulon . Spo0A approximately P, the response regulator of the signal transduction pathway required for the induction of sporulation, has previously been shown to negatively affect the induction of the Pho regulon by repressing the phoP-phoR operon . The induction pattern of some PhoR-dependent Psi proteins was altered in a spo0A mutant such that their synthesis continued for longer than was found with the wild-type . The most abundant Psi protein, Psi1-3, was characterized by N-terminal sequencing of internal peptide fragments and shown to have a high similarity to an Escherichia coli protein which is involved in phosphate uptake during phosphate starvation.

Microbiology, 1996 Nov, 142 ( Pt 11), 3113 - 23
Sequencing of a 65 kb region of the Bacillus subtilis genome containing the lic and cel loci, and creation of a 177 kb contig covering the gnt-sacXY region; Yoshida K et al.; Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, this paper communicates the sequencing of a chromosome region containing the lic and cel loci (65 kb), which creates a 177 kb contig covering the region from gnt to sacXY . This 65 kb region contains 64 ORFs (62 complete and two partial genes) . The 14th, 15th and 17th genes correspond to licT, licS and katE, encoding the antiterminator for licS transcription, beta-glucanase (lichenase) and catalase 2, respectively . The 11th, 30th, 36th, 39th, 41st, 45th-48th, 51st and 58th genes are designated deaD, pepT, galE, aldY, msmX, cydABCD, sigY and katX because their products probably encode ATP-dependent RNA helicase, tripeptidase, UDP-glucose 4-epimerase, aldehyde dehydrogenase, multiple sugar-binding transport ATP-binding protein, the respective components of cytochrome d ubiquinol oxidase and ATP-binding cassette transporter, sigma-factor of RNA polymerase and catalase, respectively . The 60th-64th genes are celRABCD, which are probably involved in cellobiose utilization . Gene organization and gene features in the gnt-sacXY region are discussed.

Microbiology, 1996 Nov, 142 ( Pt 11), 3103 - 11
Systematic sequencing of the 283 kb 210 degrees-232 degrees region of the Bacillus subtilis genome containing the skin element and many sporulation genes; Mizuno M et al.; As part of the Bacillus subtilis genome sequencing project, we have determined a 283 kb contiguous sequence from 210 degrees to 232 degrees of the B . subtilis genome . This region contains the 48 kb skin element which is excised during sporulation by a site-specific recombinase . In this region, 310 complete ORFs and one tRNA gene were identified: 66 ORFs have been sequenced and characterized previously by other workers, e.g . acc, ans, bfm, blt, bmr, comE, comG, dnaK, rpoD and sin operons; cwiA, gpr and lysA genes; many sporulation genes and operons, spo0A, spoIIA, spoIIM, spoiiP, spoIIIA, spoIIIC, spoIVB, spoIVCA, spoIVCB and spoVA, etc . The products of 84 ORFs were found to display significant similarity to proteins with known function in data banks, e.g., proteins involved in nucleotide metabolism, lipid biosynthesis, amino acid transport (ABC transporter), phosphate-specific transport, the glycine cleavage system, the two-component regulatory system, cell wall autolysis, ferric uptake and sporulation . However, the functions of more than half of the ORFs (52%, 160 ORFs) are still unknown . In the skin element containing 60 ORFs, 32 ORFs (53%) encode proteins which have significant homology to gene products of the B . subtilis temperate phage phi 105 and/or the defective phage PBSX.

Microbiology, 1996 Nov, 142 ( Pt 11), 3097 - 101
New genes in the 170 degrees region of the Bacillus subtilis genome encode DNA gyrase subunits, a thioredoxin, a xylanase and an amino acid transporter; Rose M et al.; A DNA contig of 26.2 kb covering the 170 degrees region of the Bacillus subtilis strain 168 genome was isolated and sequenced . For DNA isolation, suitable restriction sites at the end of previously known genes were chosen to amplify adjacent unknown DNA regions by inverse PCR . On the basis of the DNA sequence, 26 ORFs were identified of which eglS and ccdA, as well as part of citB and tkt have been described previously . Here we report the complete sequences of the aconitase (citB) and transketolase (tkt) genes . Of the other proteins encoded on the 26.2 kb fragment, eight revealed similarities to previously described proteins . These included a pair of newly identified DNA gyrase subunits A (grlA) and B (grlB), a sodium/proton-dependent alanine carrier (alsT), a member of the thioredoxin family (TlpA), an endo-1,4-beta-xylanase (xynD) and a response regulator protein . Comparison of the physical and the genetic maps revealed several differences . According to its flanking sequences the lexA (dinR) gene which was previously mapped at 162 degrees was found to be adjacent to yneA localized at 170 degrees . Genes citB and eglS were located the opposite way round and closer together than expected from the genetic map (citB at 173 degrees and eglS at 170 degrees) . The prkA gene, which was mapped at 169 degrees, was not present on the respective fragment . Sequence comparison actually showed that prkA is located close to 70 degrees on the B . subtilis genome.

Microbiology, 1996 Nov, 142 ( Pt 11), 3089 - 96
Integrated mapping and sequencing of a 115 kb DNA fragment from Bacillus subtilis: sequence analysis of a 21 kb segment containing the sigL locus; Fabret C et al.; A sequence strategy which combines a low redundancy shotgun approach and directed sequencing has been elaborated . Essentially, the sequences, as well as the size of the fragments utilized for a low coverage shotgun approach, were exploited for the construction of a physical map of the region to be sequenced . The latter considerably simplified the subsequent directed sequencing steps . We report the physical mapping of a 115 kb segment which covers nearly 100 kb of the hisA-cysB region of the Bacillus subtilis chromosome and contains previously sequenced genes sigL and sacB . Sequencing and analysis of a 21305 bp segment, which includes the sigL locus, revealed 21 ORFs, apparently belonging to at least seven transcription units . This segment has a G + C content greater than 47%, compared to 43% characteristic of the flanking regions, and mainly consists of genes whose products seem to be involved in the synthesis of an exopolysaccharide . These observations leave open the possibility that the analysed fragment has been acquired through horizontal transfer.

Microbiology, 1996 Nov, 142 ( Pt 11), 3079 - 88
Sequence of the 305 degrees-307 degrees region of the Bacillus subtilis chromosome; Soldo B et al.; The nucleotide sequence of the Bacillus subtilis 168 chromosomal segment located between yvhJ (307 degrees) and secA (305 degrees) was determined . This 20.3 kb region encompasses 23 ORFs, 17 of which have been sequenced previously . Comparison of sequences obtained here with the previously obtained ones revealed seven discrepancies . The products of the sequenced genes are involved in the regulation of degradative enzymes, competence, flagellar motility and protein secretion . Putative functions of newly identified genes are based on sequence homologies.

Microbiology, 1996 Nov, 142 ( Pt 11), 3067 - 78
The dnaB-pheA (256 degrees-240 degrees) region of the Bacillus subtilis chromosome containing genes responsible for stress responses, the utilization of plant cell walls and primary metabolism; Wipat A et al.; Within the framework of the international programme to sequence the genome of Bacillus subtilis strain 168, we were allocated the region between dnaB (256 degrees) and pheA (240 degrees) . The sequencing of this region is now complete and we report our primary analysis of the 114 kb region containing 114 ORFs . In addition to previously characterized genes, we have identified genes involved in the utilization of plant cell wall polysaccharides, stress responses and the metabolism of amino acids, cell walls, DNA and fatty acids . We also discuss various structural and physical features, including the orientation of genes with respect to replication, putative start and stop codons, ribosome binding sites and rho-independent transcription terminators.

Microbiology, 1996 Nov, 142 ( Pt 11), 3057 - 65
Cloning and sequencing of a 40.6 kb segment in the 73 degrees-76 degrees region of the Bacillus subtilis chromosome containing genes for trehalose metabolism and acetoin utilization; Yamamoto H et al.; In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 40.6 kb chromosome segment, which contains the tre locus, has been cloned and sequenced . This region (40 601 bp; 73 degrees-76 degrees on the genetic map) contains 38 complete ORFs and one partial one . Three ORFs, the closest to the hsdC locus, correspond to the treP, treA and treR genes encoding enzyme IITre, trehalose-6-phosphate hydrolase and the repressor of the tre operon, respectively . A homology search for the products deduced from the 39 ORFs revealed that 23 exhibit significant similarity to known proteins, e.g . proteins involved in acetoin utilization, deoxyribonuclease, methyladenine glycosidase, hydroxyisobutyrate dehydrogenase, multidrug resistance proteins, protein phosphatase, cyclic-nucleotide phosphodiesterase, 5'-nucleotidase and NADP(H)-flavin oxidoreductase . Based on the gene organization and the results of the homology search, it is predicted that YfjG, YfjH, YfjI, YfjJ and YfjK form an acetoin dehydrogenase system (acetoin regulatory protein, and acetoin dehydrogenase components/subunits E3, E2, E1 beta and E1 alpha respectively) . yfkN, an extremely large ORF comprising 4386 nucleotides, seems to correspond to the fusion of the genes for 2',3'-cyclic-nucleotide 2'-phosphodiesterase and 5'-nucleotidase precursor.

Microbiology, 1996 Nov, 142 ( Pt 11), 3039 - 46
Sequence analysis of a 50 kb region between spo0H and rrnH on the Bacillus subtilis chromosome; Yasumoto K et al.; The 49630 bp spo0H-rrnH region of the Bacillus subtilis genome has been fully sequenced . The sequence contains one partial and 62 complete ORFs, one partial and three complete rRNA genes and a cluster of six tRNA genes . The direction of the transcription and translation of 61 ORFs is the same as that of the movement of the replication fork . A homology search of 40 ORFs in newly determined sequence revealed that 27 of them had significant similarity to known proteins such as elongation factor G, elongation factor Tu, pseudouridine synthase I and ribsosomal proteins . Two adjacent genes, ybaD and ybaE, appeared to encode proteins belonging to the ATP-binding cassette (ABC) family.

Microbiology, 1996 Nov, 142 ( Pt 11), 3033 - 7
The ampS-nprE (124 degrees-127 degrees) region of the Bacillus subtilis 168 chromosome: sequencing of a 27 kb segment and identification of several genes in the area; Winters P et al.; A stretch of DNA approximately 27 kb in length, adjacent to the nprE gene of Bacillus subtilis, has been sequenced . The sequenced fragment carries a total of 23 ORFs . Of these, 15 could be assigned probable functions based on homologies to characterized genes either in B . subtilis or in other organisms . The sequencing of this region has also allowed us to assign to this area adeC and strB, previously located on the other side of nprE, between nprE and the pyr operon.

Microbiology, 1996 Nov, 142 ( Pt 11), 3027 - 31
The 52 degrees-55 degrees segment of the Bacillus subtilis chromosome: a region devoted to purine uptake and metabolism, and containing the genes cotA, gabP and guaA and the pur gene cluster within a 34960 bp nucleotide sequence; Borriss R et al.; Within the framework of the international project for sequencing the entire Bacillus subtilis genome, we have determined the complete sequence of the segment flanking the purE-D gene cluster (55 degrees) as far as cotA (52 degrees) . This segment (34960 bp) contains, as well as 12 genes already identified as part of the pur operon, 17 putative ORFs and one partial one . Two of them (gabP and guaA) are known B . subtilis genes . The gene product of cotA (formerly pig) shows significant similarity to oxidoreductases (phenoxazine synthase and bilirubin oxidase) . The putative products of ORFs yeaB (Czd protein), yeaC (MoxR), yebA (CNG-channel and cGMP-channel proteins from eukaryotes), yebB (hypothetical 32.9 kDa protein of Escherichia coli), yecA (amino acid permease) and yecB (adenine deaminase) were similar to proteins in data banks.

Microbiology, 1996 Nov, 142 ( Pt 11), 3021 - 6
A 22 kb DNA sequence in the cspB-glpPFKD region at 75 degrees on the Bacillus subtilis chromosome; Noback MA et al.; A 21808 bp nucleotide sequence at 75 degrees on the genetic map of the Bacillus subtilis chromosome was determined . The sequence of this region is adjacent to the glpPFKD operon involved in glycerol utilization . Twenty-six ORFs were identified, one of which corresponds to the cspB gene, encoding a cold-shock protein . Seventeen of the deduced protein sequences of these ORFs displayed significant homology to known proteins in the data banks . One putative operon was identified, consisting of five ORFs, that is probably involved in the uptake and processing of copper . The location of cspB in this sequence does not confirm the genetic mapping data, indicating that the gene is closely linked to comK, which is located at 80 degrees on the B . subtilis chromosome.

Microbiology, 1996 Nov, 142 ( Pt 11), 3017 - 20
Mapping of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes; Bolotin A et al.; We constructed a PCR map of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome . It was established using known sequences of the spoIIIC, blt, aadK, sacC, spoVB and pheA loci and eight random sequence tags . The tags were generated using PFGE-purified DNA of yeast artificial chromosome (YAC) 11-17 from the yeast clone which carries the major part of this region . The ends of two other YACs were positioned on the map using total DNA extracted from yeast cells carrying them . The procedure allowed the placement of precisely known and new (putative) genes on the physical chromosome map and the generation of sufficient amounts of DNA for sequencing this region . Apart from allowing correction of the genetic map in this region, these results demonstrate how a collection of long segments of bacterial chromosome and Long Accurate PCR can be used for reliable high-resolution physical mapping of an extended chromosome area.

Microbiology, 1996 Nov, 142 ( Pt 11), 3005 - 15
Organization of the Bacillus subtilis 168 chromosome between kdg and the attachment site of the SP beta prophage: use of Long Accurate PCR and yeast artificial chromosomes for sequencing; Capuano V et al.; Within the Bacillus subtilis genome sequencing project, the region between lysA and ilvA was assigned to our laboratory . In this report we present the sequence of the last 36 kb of this region, between the kdg operon and the attachment site of the SP beta prophage . A two-step strategy was used for the sequencing . In the first step, total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection . Sequencing of the clones allowed us to establish a number of contigs . In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products . The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10,000, which does not affect significantly the final sequence quality . This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions . The 36 kb sequence contains 38 coding sequences (CDSs), 19 of which encode unknown proteins . Seven genetic loci already mapped in this region, xpt, metB, ilvA, ilvD, thyB, dfrA and degR were identified . Eleven CDSs were found to display significant similarities to known proteins from the data banks, suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA, the first bacterial homologue of chalcone synthase; exol, a 5' to 3' exonuclease, similar to that of DNA polymerase I of Escherichia coli; and bsaA, a stress-response-associated protein . The protein encoded by yplP has homology with the transcriptional NifA-like regulators . The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.

Mol Microbiol, 1996 Nov, 22(4), 693 - 701
Expression of the Bacillus subtilis gabP gene is regulated independently in response to nitrogen and amino acid availability; Ferson AE et al.; Expression from the Bacillus subtilis nrg-21 locus Increases 26-fold during nitrogen-limited growth . The DNA corresponding to this locus was cloned and sequenced . The nucleotide sequence revealed a gene that could encode a protein with sequence similarity to the Escherichia coll gamma-aminobutyric acid (GABA) permease . A transposon insertion in this locus eliminated the uptake of GABA and severely inhibited the utilization of GABA as a nitrogen source . Primer extension analysis revealed that the B . subtilis gabP gene was transcribed from two overlapping promoters . Transcription from the P1 promoter was repressed during growth in the presence of amino acids . The product of the codY gene proved to be required for this repression . Transcription from the P2 promoter increased during nitrogen-limited growth and was dependent upon the product of the tnrA gene . Deletion analysis revealed that activation of the P2 promoter during nitrogen-limited growth requires a nucleotide sequence located upstream of its -35 region . Regulation of gabP expression by the CodY and TnrA regulatory systems, which respond to different physiological signals, allows for a wide range of gabP expression during growth on various nitrogen sources.

Mol Microbiol, 1996 Nov, 22(4), 605 - 18
Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I; Bolhuis A et al.; Bacillus subtilis contains three chromosomally encoded type I signal peptidases (SipS, SipT and SipU), which remove signal peptides from secretory precursor proteins . In the present study the biological function of SipS and the regulation of its synthesis were analysed . Unlike the type I signal peptidase of Escherichia coli, SipS was essential neither for protein secretion nor viability of the cell . However, in the absence of SipS the rate of processing of several preproteins was reduced, and four of the seven major secreted proteins of B . subtilis were hardly detectable in the growth medium . Surprisingly, the processing of Bacillus amyloliquefaciens alpha-amylase and the secretion of at least two endogenous B . subtilis proteins was improved in the absence of SipS . These findings indicate that the substrate preference of SipS differs from that of SipT and SipU, and that SipS is an important factor determining the efficiency of protein secretion in B . subtilis . SipS is transcribed in a growth phase- and medium-dependent manner . In minimal medium, the growth phase-dependent transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that the expression of sipS is regulated by the same factors that control the expression of most genes for secreted degradative enzymes . Our observations suggest that B . subtilis can modulate its capacity and specificity for protein secretion through the controlled expression of sipS.

J Nat Prod, 1996 Nov, 59(11), 1056 - 60
Four new bioactive manzamine-type alkaloids from the Philippine marine sponge Xestospongia ashmorica; Edrada RA et al.; Analysis of the Philippine marine sponge Xestospongia ashmorica afforded four new manzamine congeners 1-4 and four known compounds 5 and 7-9 . Compound 1 is the 6-deoxy derivative of manzamine X, while 2-4 are the N-oxides of manzamine J (5), 3,4-dihydromanzamine A (6), and manzamine A (7), respectively . The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, 1H-detected direct, and long-range 13C-1H correlations) and mass spectrometric (EI, FAB-MS, and electrospray ionization) data . Alkaloid N-oxide structures were confirmed by conversion to the corresponding tertiary bases by reduction with Zn/HCl . This is the first report of the occurrence of bioactive manzamine N-oxides in marine sponges . Compound 7 exhibited insecticidal activity toward neonate larvae of the polyphagous pest insect Spodoptera littoralis (with an ED50 of 35 ppm) when incorporated in artificial diet and offered to larvae in a chronic feeding bioassay . Compound 7 was also active against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus . Cytotoxicity was studied in vitro using L1578y mouse lymphoma cells . From the alkaloids studied, the N-oxides 3 and 4 were the most active (ED50 = 1.6 micrograms/mL) followed by compound 7 (ED50 = 1.8 micrograms/mL).

Mol Microbiol, 1996 Nov, 22(3), 405 - 13
Re-examination of the role of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli; Leonardo MR et al.; In Escherichia coli, EnvZ senses changes in the osmotic conditions of the growth environment and controls the phosphorylated state of the regulatory protein, OmpR . OmpR-phosphate regulates the expression of the porin genes, ompF and ompC . To investigate the role of the periplasmic domain of EnvZ in sensing of osmolarity signals, portions of this domain were deleted . Cells containing the EnvZ mutant proteins were able to regulate normally the production of OmpF and OmpC in response to changes in osmolarity . The periplasmic domain of EnvZ was also replaced with the non-homologous periplasmic domain of the histidine kinase PhoR of Bacillus subtilis . Osmoregulation of OmpF and OmpC production in cells containing the PhoR-EnvZ hybrid protein was indistinguishable from that in cells containing wild-type EnvZ . Identical results were obtained with an envZ-pta/ack strain, which could not synthesize acetyl phosphate . Thus, acetyl phosphate was not involved in the regulation of ompF and ompC observed in this study . These results indicate that the periplasmic domain of EnvZ is not essential for sensing of osmolarity signals.

J Bacteriol, 1996 Nov, 178(22), 6640 - 3
flaD (sinR) mutations affect SigD-dependent functions at multiple points in Bacillus subtilis; Rashid MH et al.; A flaD (sinR) null mutation depressed sigD-lacZ expression only two- to fourfold, whereas a flaD1 point mutation depressed it almost completely . Introduction of pHYSigD, a sigmaD-overproducing plasmid, corrected the filamentous phenotype common to both sinR mutants; autolysin synthesis was restored partially and completely in the flaD1 and flaD (sinR) null strains, respectively . Flagellin synthesis and motility were not restored at all in either strain.

J Bacteriol, 1996 Nov, 178(22), 6632 - 4
The SpoIIAA protein of Bacillus subtilis has GTP-binding properties; Najafi SM et al.; SpoIIAA is the first protein of the spoIIA operon . Here we show that SpoIIAA can bind and hydrolyze GTP . The protein also accepts ATP, but with lower affinity . GDP competes poorly for binding of GTP . The GTPase activity of SpoIIAA is within the range found for other GTP-binding proteins.

J Bacteriol, 1996 Nov, 178(22), 6579 - 86
Mutation of the Bacillus subtilis alkyl hydroperoxide reductase (ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes; Bsat N et al.; In Bacillus subtilis, hydrogen peroxide induces the synthesis of catalase (KatA), alkyl hydroperoxide reductase (AhpCF), and a DNA-binding protein of the Dps family (MrgA) . KatA, AhpCF, heme biosynthesis enzymes, and MrgA are also induced upon entry into stationary phase under conditions of iron and manganese limitation . In an effort to define the peroxide regulon repressor, PerR, we used mini-Tn10 mutagenesis to identify loci affecting the regulation of mrgA . From this screen, we isolated two mini-Tn10 insertions in ahpC, the gene encoding the small subunit of AhpCF, that increase the transcription of mrgA-lacZ even in iron-supplemented minimal medium . Indeed, these ahpC::Tn10 insertions lead to elevated expression from all peroxide regulon promoters, including those for mrgA, katA, hemAXCDBL, and ahpCF . As a result, the ahpC::Tn10 mutants display an increased resistance to H2O2 . The ahpCF promoter region contains three sequences similar to the peroxide regulon consensus operator (per box) . We demonstrate that the ability of ahpC::Tn10 mutations to derepress mrgA requires aerobic growth . In contrast, a second distinct trans-acting regulatory mutation bypasses this requirement for aerobic growth . Since the peroxide regulon is activated in the absence of AhpCF, which degrades alkyl hydroperoxides, we propose that organic hydroperoxides may be physiologically relevant inducers in vivo.

J Bacteriol, 1996 Nov, 178(22), 6571 - 8
General and oxidative stress responses in Bacillus subtilis: cloning, expression, and mutation of the alkyl hydroperoxide reductase operon; Antelmann H et al.; The AhpC subunit of the Bacillus subtilis alkyl hydroperoxide reductase was identified as a general stress protein induced in response to heat or salt stress or after entry of the organism into the stationary phase . The ahp operon, encoding the two subunits AhpC and AhpF, was cloned and localized between the gntRKPZ operon and the bglA locus . Two-dimensional gel analyses revealed an especially strong induction of AhpC and AhpF in cells subjected to oxidative stress . Transcriptional studies showed a 3- to 4-fold induction of ahp mRNA after heat or salt stress or starvation for glucose and a 20-fold induction by oxidative stress, thus confirming the protein induction data for AhpC and AhpF . Stress induction occurred at a sigmaA-dependent promoter that overlaps with operator sites similar to the per box . Compared with the wild type, the ahpC mutant was resistant to hydrogen peroxide because of the derepression of the peroxide regulon (N . Bsat, L . Chen, and J . D . Helmann, J . Bacteriol . 178:6579-6586, 1996) but more sensitive to cumene hydroperoxide (CHP) during exponential growth . In contrast, stationary-phase wild-type and ahpC mutant cells displayed complete resistance to treatment with 1 mM CHP . Moreover, a sigmaB mutant was found to be extremely sensitive to CHP during vegetative growth and in stationary phase, which indicates that sigmaB-dependent general stress proteins are involved in the protection of cells against oxidative stress.

J Bacteriol, 1996 Nov, 178(22), 6518 - 24
A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis; Lee AI et al.; In Bacillus subtilis, the tryptophan-activated trp RNA-binding attenuation protein (TRAP) regulates expression of the seven tryptophan biosynthetic genes by binding to specific repeat sequences in the transcripts of the trp operon and of the folate operon, the operon containing trpG . Steinberg observed that strains containing a temperature-sensitive mutant form of tryptophanyl-tRNA synthetase, encoded by the trpS1 allele, produced elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess L-tryptophan (W . Steinberg, J . Bacteriol . 117:1023-1034, 1974) . We have confirmed this observation and have shown that expression of two reporter gene fusions, trpE'-'lacZ and trpG'-'lacZ, is also increased under these conditions . Deletion of the terminator or antiterminator RNA secondary structure involved in TRAP regulation of trp operon expression eliminated the trpS1 effect, suggesting that temperature-sensitive expression was mediated by the TRAP protein . Analysis of expression of mtrB, the gene encoding the TRAP subunit, both by examination of a lacZ translational fusion and by measuring the intracellular levels of TRAP by immunoblotting, indicated that the trpS1-induced increase in trp gene expression was not due to inhibition of mtrB expression or to alteration of the amount of TRAP present per cell . Increasing the cellular level of TRAP by overexpressing mtrB partially counteracted the trpS1 effect, demonstrating that active TRAP was limiting in the trpS1 mutant . We also showed that elevated trp operon expression was not due to increased transcription initiation at the upstream aroF promoter, a promoter that also contributes to trp operon expression . We postulate that the increase in trp gene expression observed in the trpS1 mutant is due to the reduced availability of functional TRAP . This could result from inhibition of TRAP function by uncharged tRNA(Trp) molecules or by increased synthesis of some other transcript capable of binding and sequestering the TRAP regulatory protein.

J Bacteriol, 1996 Nov, 178(22), 6451 - 8
Analysis of the peptidoglycan structure of Bacillus subtilis endospores; Popham DL et al.; Peptidoglycan was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains . Digestion with muramidase resulted in cleavage of the glycosidic bonds adjacent to muramic acid replaced by peptide or alanine side chains but not the bonds adjacent to muramic lactam . Reduction of the resulting muropeptides allowed their separation by reversed-phase high-pressure liquid chromatography . The structures of 20 muropeptides were determined by amino acid and amino sugar analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . In wild-type spores, 50% of the muramic acid had been converted to the lactam and 75% of these lactam residues were spaced regularly at every second muramic acid position in the glycan chains . Single L-alanine side chains were found on 25% of the muramic acid residues . The remaining 25% of the muramic acid had tetrapeptide or tripeptide side chains, and 11% of the diaminopimelic acid in these side chains was involved in peptide cross-links . Analysis of spore peptidoglycan produced by a number of mutants lacking proteins involved in cell wall metabolism revealed structural changes . The most significant changes were in the spores of a dacB mutant which lacks the sporulation-specific penicillin-binding protein 5* . In these spores, only 46% of the muramic acid was in the lactam form, 12% had L-alanine side chains, and 42% had peptide side chains containing diaminopimelic acid, 29% of which was involved in cross-links.

Arch Microbiol, 1996 Nov, 166(5), 293 - 300
Some like it cold: response of microorganisms to cold shock; Graumann P et al.; Bacteria respond to an abrupt decrease in temperature with a specific response, in which cold-induced proteins (CIPs) are transiently expressed at a higher level . Employing two-dimensional gel electrophoresis, several CIPs have been identified . In spite of this, the overall function of the cold shock response is unclear . Recently, the main attention has focused on a group of conserved cold shock proteins (CSPs) that have been shown to have the highest induction after cold shock and to play a major regulatory role in the physiology of adaptation to low temperatures . CSPs, of which Escherichia coli, Bacillus subtilis, and B . cereus possess a family comprising at least 3-7 proteins, are small acidic proteins that share over 45% of sequence identity . Recent evidence suggests that members of this wide-spread protein family can function both at the transcriptional and translational level in vitro . However, the exact mode of action has yet to be established . In addition, post-transcriptional regulation seems to play a major role in the induction of CSPs, a process in which the ribosome may be involved . This is in accordance with a model in which the ribosome has been proposed to be the sensor of temperature in bacteria.

Anal Chem, 1996 Nov 1, 68(21), 3705 - 12
Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry; Muddiman DC et al.; A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described . PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate . An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis . PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions . Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination . In the presence of guanidinium hydrochloride the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's primers, and salts) . Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with external countercurrent flow of 2.5 mM ammonium acetate . Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetronitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity . The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned . These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords . This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.

Genetics, 1996 Nov, 144(3), 871 - 81
Noncomplementing diploids from Bacillus subtilis protoplast fusion: relationship between maintenance of chromosomal inactivation and segregation capacity; Grandjean V et al.; Fusions of Bacillus subtilis protoplasts from two genetically marked strains produce noncomplementing heterodiploid bacteria . These noncomplementing diploids (Ncds) carry both parental chromosomes, but only one is expressed . Fusion products of strains polymorphic for NotI restriction sites provide new physical evidence to support the conclusion that Ncds are not an artifact of cross feeding or cell adhesion . We show that reversible chromosomal inactivation can only account for the biparental trait of unstable Ncds . Two types of cells were recovered from the late progeny of unstable Ncds: Ncds with irreversible chromosome silencing (stable Ncds) and secondary recombinants that displayed a genomic mosaic NotI profile . Segregants from an unstable Ncd population gave rise to two viable haploid cell types . By contrast, stable Ncds segregated into a population of viable and inviable haploid cells . We propose that the latter are derived from irreversible chromosome silencing . Our results indicate that clonal populations of stable Ncds are heterogenous and suggest that segregation and inactivation are independent parameters.

Appl Environ Microbiol, 1996 Nov, 62(11), 3948 - 53
Direct selection of cloned DNA in Bacillus subtilis based on sucrose-induced lethality; Bramucci MG et al.; Expression of the Bacillus subtilis or Bacillus amyloliquefaciens sacB gene in the presence of sucrose is lethal for a variety of bacteria . Sucrose-induced lethality can be used to select for inactivation of sacB by insertion of heterologous DNA in sensitive bacteria . This procedure has not been applicable to B . subtilis heretofore because expression of wild-type sacB is not detrimental to B . subtilis . The W29 mutation in the B . amyloliquefaciens sacB gene interferes with processing of the levansucrase signal peptide . The W29 mutation does not affect growth of B . subtilis in media lacking sucrose . However, this mutation inhibited growth of B . subtilis in media containing sucrose . Inactivation of the fructose polymerase activity encoded by sacB indicated that levan production was essential for sucrose-induced lethality . As a result, it was possible to select for cloned DNA in B . subtilis by insertional inactivation of the mutant sacB gene located on a multicopy plasmid vector in medium containing sucrose.

J Bacteriol, 1996 Nov, 178(21), 6361 - 5
Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis; Perkins JB et al.; Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon . Biotin and the B . subtilis birA gene product regulated synthesis of the transcripts . Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis . Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

J Bacteriol, 1996 Nov, 178(21), 6305 - 9
A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme; Ulijasz AT et al.; We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium . This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance . Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression . As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter . Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidoglycan {correction of peptidodoglycan} precursors are wrong.

J Bacteriol, 1996 Nov, 178(21), 6216 - 22
Secondary transporters for citrate and the Mg(2+)-citrate complex in Bacillus subtilis are homologous proteins; Boorsma A et al.; Citrate uptake in Bacillus subtilis is mediated by a secondary transporter that transports the complex of citrate and divalent metal ions . The gene coding for the transporter termed CitM was cloned, sequenced, and functionally expressed in Escherichia coli . Translation of the base sequence to the primary sequence revealed a transporter that is not homologous to any known secondary transporter . However, CitM shares 60% sequence identity with the gene product of open reading frame N15CR that is on the genome of B . subtilis and for which no function is known . The hydropathy profiles of the primary sequences of CitM and the unknown gene product are very similar, and secondary structure prediction algorithms predict 12 transmembrane-spanning segments for both proteins . Open reading frame N15CR was cloned and expressed in E . coli and was shown to be a citrate transporter as well . The transporter is termed CitH . A remarkable difference between the two transporters is that citrate uptake by CitM is stimulated by the presence of Mg2+ ions, while citrate uptake by CitH is inhibited by Mg2+ . It is concluded that the substrate of CitM is the Mg(2+)-citrate complex and that CitH transports the free citrate anion . Uptake experiments in right-side-out membrane vesicles derived from E . coli cells expressing either CitM or CitH showed that both transporters catalyze electrogenic proton/substrate symport.

J Bacteriol, 1996 Nov, 178(21), 6173 - 83
Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation; Atrih A et al.; The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined . Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan . The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry . This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid . The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases . Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively . Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex . The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall . The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively . This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis . The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues . Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate . The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.

Gene, 1996 Oct 31, 178(1-2), 119 - 23
Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes; Rowland BM et al.; Under iron-limiting conditions, Bacillus subtilis (Bs) produces the siderophore 2,3-dihydroxybenzoate (DHB) to acquire extracellular iron . In Escherichia coli (Ec), DHB is a precursor of the siderophore enterobactin, which suggested that Bs may possess similar biosynthetic enzymes . The sequences of two overlapping Bs clones capable of complementing Ec enterobactin mutants {Grossman, T.H., Tuckman, M., Ellestad, S . and Osburne, M.S . (1993) Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: Relationship between B . subtilis sfpo and Escherichia coli entD genes . J . Bacteriol . 175, 6203-6211} were analyzed and five open reading frames were identified . These genes are located near 291 degrees on the Bs chromosome and have been termed dhbA, dhbC, dhbE, dhbB and dhbF, based on similarities to Ec ent homologs . Amino-acid identities between gene product homologs are: EntA and DhbA, 41%; EntC and DhbC, 35%; EntE and DhbE, 48%; EntB and DhbB, 54%; and EntF and DhbF, 29% . DhbC is also 35% identical to the Bs menaquinone-specific isochorismate synthase, MenF, illustrating an example of gene duplication . Operon disruption studies suggested that the dhb genes comprise an operon of at least four genes.

J Mol Biol, 1996 Oct 25, 263(2), 259 - 68
Structure of the Bacillus subtilis phage SPO1-encoded type II DNA-binding protein TF1 in solution; Jia X et al.; The solution structure of a type II DNA-binding protein, the bacteriophage SPO1-encoded transcription factor 1 (TF1), was determined using NMR spectroscopy . Selective 2H-labeling, 13C-labeling and isotopic heterodimers were used to distinguish contacts between and within monomers of the dimeric protein . A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calculations using restrained molecular dynamics and simulated annealing protocols . The ensemble of 30 calculated structures has a root-mean-square deviation (r.m.s.d.) of 0.9 A, about the average structure for the backbone atoms, and 1.2 A for all heavy-atoms of the dimeric core (helices 1 and 2) and the beta-sheets . A severe helix distortion at residues 92-93 in the middle of helix 3 is associated with r.m.s.d . of approximately 1.5 A for the helix 3 backbone . Deviations of approximately 5 A or larger are noted for the very flexible beta-ribbon arms that constitute part of a proposed DNA-binding region . A structural model of TF1 has been calculated based on the previously reported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations . A comparison between the calculated average solution structure of TF1 and a solution structure of HU indicates a similarity in the dimeric core (excluding the nine amino acid residue tail) with pairwise deviations of 2 to 3 A . The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected . A 4 A deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substitution of Glu15 by Gly increases the thermostability of TF1 . The homology between TF1 and other proteins of this family leads us to anticipate similar tertiary structures.

Gene, 1996 Oct 24, 177(1-2), 275 - 6
Nucleotide sequence of the Bacillus coagulans homologue of the spoIIA operon of Bacillus subtilis; Park SG et al.; The spoIIA locus of Bacillus coagulans (Bc) was cloned into pTZ18R and the nucleotide sequence was determined . To clone the operon, one PCR primer corresponding to the C-terminal region of SpoIIAB, and a second corresponding to a region near the middle of SpoIIAC, were designed on the basis of the three previously published Bacillus spoIIA sequences . The Bc spoIIA sequence contains three open reading frames coding for putative proteins of 116, 147 and 252 aa.

Gene, 1996 Oct 24, 177(1-2), 129 - 32
Potassium permanganate susceptibility of sigma E-RNA polymerase-promoter complexes; Seyler RW Jr et al.; We used potassium permanganate (KMnO4) to identify unpaired thymidine (T) residues in promoter complexes formed by RNA polymerase (RNAP) associated with sigma E (sigma E-RNAP) from Bacillus subtilis . We found that a region of the spoIIID promoter from at least -10 to +1 becomes melted in the presence of this polymerase . In promoter complexes formed by RNAP associated with a mutant sigma E that melts promoter DNA inefficiently, we noted additional KMnO4 sensitivity at the -11 position of the spoIIID promoter . We suggest that the base pair at -11 is unpaired in both mutant and wild type (wt) complexes; however, close proximity of wt sigma E-RNAP with the T at -11 may protect it from KMnO4 attack . The absence of a close contact between the mutant sigma E-RNAP and the base at -11 may explain why this polymerase uses promoters less efficiently than wt sigma E-RNAP.

Gene, 1996 Oct 24, 177(1-2), 123 - 8
Isolation and characterization of csbB, a gene controlled by Bacillus subtilis general stress transcription factor sigma B; Akbar S et al.; In the bacterium Bacillus subtilis (Bs), the alternative transcription factor sigma B is activated by environmental stresses to control the expression of a large set of unlinked genes . However, the range of physiological functions mediated by these sigma B-controlled genes is presently unknown . We report here that the newly identified gene csbB is under the dual control of a sigma B-dependent and a sigma B-independent promoter . The predicted product of csbB is a 329 residue protein containing two potential membrane-spanning segments in its C-terminal region, leading us to speculate that one class of sigma B-controlled genes acts to modify the cell envelope as part of the general stress response.

Biochem Biophys Res Commun, 1996 Oct 23, 227(3), 762 - 7
Bacillus subtilis Ffh, a homologue of mammalian SRP54, can intrinsically bind to the precursors of secretory proteins; Bunai K et al.; We analyzed the binding activity of B . subtilis Ffh to the precursors of secretory proteins by purifying mature and precursor proteins of beta-lactamase derived from pUC18 and its derivatives, of which the signal peptide region was replaced with that of E . coli OmpA, B . subtilis AprE, PBP5* or an alkalophilic Bacillus sp . #1011 CGTase . Each of them was mixed with purified B . subtilis Ffh in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) . The tested precursor proteins, including those of E . coli, of which the signal sequences differ from those of B . subtilis in the number of charged amino acids and hydrophobicity, cross-linked with Ffh, whereas mature proteins did not . The addition of scRNA, the B . subtilis counterpart of mammalian SRP 7S RNA, into the mixture did not affect the complex formation . These findings suggest that B . subtilis Ffh intrinsically binds to several precursor proteins.

FEBS Lett, 1996 Oct 21, 395(2-3), 127 - 32
Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue; Takagi H et al.; Substitution of the conserved Gly127 for residues having a side chain markedly changed the substrate specificity of subtilisin E from Bacillus subtilis . The crystallographic findings suggested that Gly127 is responsible for accepting even the large P1 substrates, and the marked change of specificity was attributed to the introduction of a side chain in this position . To test this hypothesis, Gly127 was replaced with 3 non-charged amino acids, Ala, Ser and Val . When assayed with synthetic peptide substrates, all mutants purified from the periplasmic space in Escherichia coli showed a marked preference for small P1 substrate up to 150-fold relative to the wild-type . The kinetic data and molecular modeling analysis suggest that large hydrophobic P1 residues were unable to access the binding pocket due to steric hindrance.

J Biol Chem, 1996 Oct 18, 271(42), 26081 - 7
Circular ribozymes generated in Escherichia coli using group I self-splicing permuted intron-exon sequences; Puttaraju M et al.; A circularly permuted self-splicing group I intron from Anabaena was used to generate covalently closed circular trans-acting ribozymes in Escherichia coli . The RNA component of Bacillus subtilis RNaseP and an artificial trans-acting hepatitis delta virus ribozyme were expressed as the exon portion of the permuted intron . RNA isolated from these cells contained circular forms of the ribozymes, indicating that circles were generated from precursors expressed in these cells . Total RNA isolated from cells producing the circular RNA contained ribozyme activity . In contrast, a linear form of the delta virus ribozyme expressed as part of an unprocessed transcript yielded no detectable activity . These data extend previous in vitro and in vivo studies on splicing-mediated RNA circularization by demonstrating the intracellular production of circular ribozymes . These results have implications for the development of systems expressing therapeutic forms of small RNAs such as ribozymes and decoy-type competitors . Circular RNAs generated by splicing are devoid of flanking sequences that could potentially interfere with function . Also, because circular RNAs are not primary substrates for exonucleases, they may have increased in vivo half-lives relative to linear molecules with similar sequences.

Gene, 1996 Oct 17, 176(1-2), 81 - 4
Mapping of the replication origin of the Bacillus subtilis chromosome by the two-dimensional gel method; Moriya S et al.; Analysis of replication intermediates produced by in vitro replication of the Bacillus subtilis oriC plasmid revealed that replication initiated at an untranslatable DnaA box region downstream of the dnaA gene . In order to show that replication of the B . subtilis chromosome also starts at the same region in vivo, we have analyzed replication intermediates generated in vivo by the two-dimensional gel method . A bubble arc was detected when the downstream region was used as a probe . In contrast, only a simple Y arc was found when the upstream DnaA box region required for autonomous replication of the oriC plasmid was used as a probe . Furthermore, the bubble arc ranged from unit to almost double the size of a fragment in which the downstream region was located near the middle . These results indicate that replication of the B . subtilis chromosome initiates at the downstream DnaA box region of the dnaA gene and proceeds bidirectionally.

Gene, 1996 Oct 17, 176(1-2), 61 - 5
Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements and Escherichia coli mutant defective in the ATPase activity of SecA; Blanco J et al.; The secA gene of Streptomyces lividans was cloned using as probe a 57-mer oligonucleotide based on conserved sequences of the Escherichia coli secA and the Bacillus subtilis div genes . It encodes a protein of 946 amino acids (aa) with a deduced M(r) of 106,079, with high similarity to all known SecA proteins . All the previously described conserved motifs of SecA proteins were conserved in the S . lividans protein . The secA gene of S . lividans restored sensitivity to sodium azide in E . coli SecA4 (AzR) a mutant with an azide-resistant (ATPase defective) SecA protein . However, it did not complement the temperature-sensitive mutation in E . coli MM52 (SecAts) (a conditional lethal mutant defective in protein translocation) allowing only poor growth at the nonpermissive temperature . secA homologous sequences were present in 11 different species of Streptomyces and Nocardia.

J Biochem Biophys Methods, 1996 Oct 15, 33(1), 31 - 41
Preparation of general proteinase substrates using 3,5-dinitrosalicylaldehyde; Gallegos NG et al.; To search for new proteinases in Bacillus subtilis we have developed a general method for synthesizing chromogenic proteinase substrates using 3,5-dinitrosalicylaldehyde (DNSA) . Hammersten casein and soluble protein from extracts from B . subtilis cells were labeled with DNSA in the presence of NaBH4 . After dialysis (pH 7.8), the resultant 3,5-dinitro-2-hydroxybenzyl-casein (DNHB-casein) and DNHB-bacterial cell protein solutions were a light orange color . A model compound, N-benzyl-3,5-dinitro-2-hydroxybenzylamine was synthesized and estimated to have a molar absorption coefficient of 14,100 M-1 cm-1 at 366 nm at pH 8, which was used to calculate dye loading on casein . Chromogenic substrates prepared in this way should retain positive charges on lysine residues . DNHB-casein and DNHB-bacterial cell protein were incubated with varying concentrations of subtilisin BPN' for varying times, precipitated with trichloroacetic acid and centrifuged . The acid-soluble supernatant fractions were made basic with NaOH and absorbances were measured at 366 nm, the absorption maximum . Color production was proportional to subtilisin concentration and times of incubation; under the assay conditions used, the limit of detection of subtilisin was about 100 ng . Five proteinase activities were detected in soluble extracts of B . subtilis using DNHB-labeled proteins as substrates.

J Mol Biol, 1996 Oct 11, 262(5), 595 - 9
Evidence for intramolecular processing of prosubtilisin sequestered on a solid support; Volkov A et al.; Subtilisin E is synthesized in Bacillus subtilis as a preprosubtilisin . The prepeptide is removed by a signal peptidase, and the propeptide is cleaved from the mature protein by the catalytic domain of subtilisin itself in an autocatalytic fashion . A six residue histidine-tag was attached to the C terminus of prosubtilisin and mature subtilisin to enable immobilization on a metal chelating resin . Guanidine-HC1 denatured histidine-tagged subtilisin and prosubtilisin were immobilized on Co2+ charged Talon resin, then renatured by dialysis of the resin against renaturation buffer . Refolding of the immobilized prosubtilisin resulted in its quantitative autoprocessing and the formation of active enzyme . Mature subtilisin on the other hand refolded into an active conformation with very low efficiency, and at the same concentration the steady-state rate attained was at least a 1000 times lower than that from prosubtilisin . The results give very strong support for an intramolecular autoprocessing pathway for prosubtilisin, in addition to an intermolecular one demonstrated before . The results also demonstrate rather convincingly the very much higher yield of active enzyme refolded from prosubtilisin than from mature protein under sequestered unimolecular conditions.

Gene, 1996 Oct 10, 175(1-2), 59 - 63
Analysis of DNA flanking the treA gene of Bacillus subtilis reveals genes encoding a putative specific enzyme IITre and a potential regulator of the trehalose operon; Schock F et al.; Nucleotide sequencing revealed the genes treP encoding a putative specific enzyme IITre upstream from treA and treR encoding a potential regulator downstream from treA of Bacillus subtilis 168 . The treP gene encodes a 470-amino acid (aa) protein (50 kDa) showing high similarities to several different specific enzymes II of phosphoenolpyruvate-dependent phosphotransferase systems . treR encodes a 238-aa protein (28 kDa) with high homologies in its N-terminal part to DNA-binding proteins including a helixturn-helix motif . Homologies in its C-terminal part place it in the family of FadR-GntR transcriptional regulators . The three genes, treP-treA-treR, are probably organized in one operon expressed by a sigma A-dependent promoter 53 bp upstream from treP and a rho-independent terminator 28 bp downstream from treR.

FEBS Lett, 1996 Oct 7, 394(3), 340 - 4
1H NMR studies of the paramagnetic CuA center of cytochrome oxidase; Dennison C et al.; The dinuclear paramagnetic center of the soluble CuA domain of the cytochrome c oxidase from Bacillus subtilis has been studied using 1H NMR . The spectrum possesses remarkably sharp shifted resonances . Comparison with the spectrum of the CuA amicyanin variant provides the spin density distribution in the CuA site of cytochrome c oxidase . This represents the first paramagnetic NMR study of the dinuclear CuA center from the soluble domain of subunit II of cytochrome c oxidase.

Genes Cells, 1996 Oct, 1(10), 881 - 94
Compartmentalized distribution of the proteins controlling the prespore-specific transcription factor sigmaF of Bacillus subtilis; Lewis PJ et al.; BACKGROUND: Differential gene expression during sporulation in the prespore and mother cell of Bacillus subtilis is dependent on the correct timing and localization of the activity of specific transcription (sigma) factors . The first sigma factor activated is sigmaF, which directs gene expression specifically in the prespore compartment . Release of sigmaF activity is tightly controlled through a series of complex interactions involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor SpoIIAA and a phosphoprotein phosphatase SpoIIE . In vitro studies have shown that SpoIIAB binds to sigmaF, preventing transcription of the sigmaF regulon, and that it can also phosphorylate SpoIIAA, thereby inactivating it . However, non-phosphorylated SpoIIAA can displace sigmaF from SpoIIAB . The SpoIIE phosphatase provides a means of reactivating SpoIIAA-P . RESULTS: We have directly determined the cellular distributions of sigmaF, SpoIIAB, SpoIIAA-P and SpoIIAA during sporulation, using recently developed immunofluorescence methods . While sigmaF activity is restricted to the prespore, the protein is present in both compartments . As development proceeds the sigmaF signal disappears . The anti-sigma factor SpoIIAB is also distributed throughout both cells and rapidly disappears from both cellular compartments soon after sigmaF becomes active . Disappearance of SpoIIAB seems to be closely associated with the activation of the second prespore-specific sigma factor sigmaF . The distribution of phosphorylated SpoIIAA closely mimics that of SpoIIAB, being non-compartmentalized and disappearing soon after sigmaF activation occurs . Significantly, the active, non-phosphorylated form of the anti-anti-sigma factor, SpoIIAA, accumulates in the prespore just before sigmaF becomes active . CONCLUSION: These results support the hypothesis that the accumulation of SpoIIAA within the prespore is the single most important requirement for activation of sigmaF.

Curr Opin Genet Dev, 1996 Oct, 6(5), 531 - 7
Spore development in Bacillus subtilis; Piggot PJ; Cell-cell and starvation signals are funneled through the phosphorelay to initiate sporulation by activating the transcription regulator SpoOA . Activation of SpoOA leads to synthesis of the transcription factors sigmaF and sigmaE . Substantial advances have been made in our understanding of the signal circuitry of the phosphorelay and of the cell-type-specific activation of the sigma factors.

Infect Immun, 1996 Oct, 64(10), 4324 - 9
Clusterin, a putative complement regulator, binds to the cell surface of Staphylococcus aureus clinical isolates; Partridge SR et al.; The ability of Staphylococcus aureus Cowan I strain and a number of S . aureus clinical isolates to bind to the human blood glycoprotein clusterin was investigated . Binding of clusterin to these strains was tested by both enzyme-linked immunosorbent assay and flow cytometry . All of the S . aureus strains examined appeared to bind clusterin to some extent, while nonpathogenic control strains Bacillus subtilis BR151 and Escherichia coli JM109 did not . Three S . aureus isolates were selected for more detailed study; binding of labeled clusterin was saturable, inhibited in the presence of excess unlabeled clusterin, and prevented by pretreatment of bacteria with proteases . From the saturation binding studies, estimates of the affinity constants for the binding of clusterin to the bacteria ranged from 31 to 57 nM . Addition of clusterin to S . aureus cultures was also found to result in aggregation of the bacterial cells; aggregation was not detected when clusterin was added to B . subtilis BR151 or E . coli JM109 cultures . These results suggest that at least some S . aureus strains possess specific proteinaceous receptors for clusterin . Such receptors may be an important new bacterial virulence determinant for S . aureus, as clusterin has been proposed to have a role in the regulation of complement activity.

Intensive Care Med, 1996 Oct, 22(10), 1066 - 9
Retention of the antibiotic teicoplanin on a hydromer-coated central venous catheter to prevent bacterial colonization in postoperative surgical patients; Bach A et al.; OBJECTIVE: Antibiotic-coated intravascular catheters may be an effective means of decreasing bacterial colonization and subsequent catheter-related infection . The present study was designed to investigate the retention of the antibiotic teicoplanin on a hydromer-coated intravenous catheter and the effect of this antibiotic coating on catheter bacterial colonization . DESIGN: A prospective, randomized pilot study . SETTING: Operating rooms (ORs) and an intensive care unit (ICU) at a university hospital . PATIENTS: A consecutive group of 20 male patients undergoing major abdominal surgery . INTERVENTIONS: Control (C; n = 10) or teicoplanin-coated (T; n = 10) single-lumen central venous catheters were inserted before surgery in the OR . Catheters were withdrawn at the discretion of the physicians in the ICU after various periods . MEASUREMENTS: The teicoplanin content of the catheter material was assessed using a bioassay with Bacillus subtilis after complete elution of the antibiotic from the catheter . Bacterial colonization was measured using a quantitative culture technique after the catheter lumen had been flushed and the catheter segments sonicated . MAIN RESULTS: Nearly three-quarters of the initial teicoplanin coating (374 +/- 103 micrograms; mean +/- SD) were released during the first day of catheterization, and after 36 h of intravenous catheterization, no antibiotic was retained on the catheter . No significant difference could be found either in the incidence of bacterial colonization between test (n = 3) and control (n = 4) catheters or in the number of colony-forming units (CFU) on the catheter segments (T, 263 +/- 104 CFU/cm; C, 372 +/- 294 CFU/cm; mean +/- SEM) . CONCLUSION: The retention of teicoplanin antibiotic coating on hydromer catheters is only short term if catheters are inserted intravenously . This may limit clinical antibacterial efficacy.

Int J Pept Protein Res, 1996 Oct, 48(4), 357 - 63
Structure-biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin; Kang JH et al.; The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity . To investigate the structure-antimicrobial activity relationships, the basic amino acid-rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected . Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E . coli, and the peptide's toxicities were assayed by hemolysis . The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2) . The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine . However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine . The circular dichroism (CD) spectra of the short peptides in 30 mM SDS were correlated with their antimicrobial activities . These results suggested that the 11-residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity.

Proteins, 1996 Oct, 26(2), 236 - 8
Crystallization of NAD+ synthetase from Bacillus subtilis; Rizzi M et al.; NAD(+)-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD+ . Mutants of NAD+ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism . Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions . Investigation on the structural organization of NAD+ synthetase, an enzyme fundamental for NAD+ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD+-->NAD+ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp . and related bacteria.

Biochem Mol Biol Int, 1996 Oct, 40(3), 603 - 10
Evidence for the shikimate-3-phosphate interacting site in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis; Selvapandiyan A et al.; The role of basic amino acid residues that are highly conserved in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPs) in the binding of the substrate, shikimate-3-phosphate, has been assessed . Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substituted by glutamic acid and aspartic acid residues respectively by site-directed mutagenesis . Native and the mutant proteins were expressed using a two-vector system and the expressed proteins were purified to near homogeniety . The replacement of either Lys 19 or Arg 24 with a negatively charged residue nearly completely abolished the enzyme activity . The kinetic characterization of the purified wild type and the mutant proteins revealed that the substitution of positively charged residues in the N-terminal domain (K19 and R24) results in reduced affinity for shikimate-3-phosphate (S3P) . The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.

Am J Infect Control, 1996 Oct, 24(5), 364 - 71
Comparative sporicidal effect of liquid chemical germicides on three medical devices contaminated with spores of Bacillus subtilis; Sagripanti JL et al.; BACKGROUND: The relatively limited variety of surfaces and geometries challenged in current sporicidal testing reduces the predictive value of these analyses when extrapolated to the wide variety of medical devices . The unknown spore load being challenged and the qualitative nature (growth/no growth) of those tests further prevent precise comparison among liquid chemical disinfectants . Hence, the relative activity of different chemical substances has not been clearly established, hindering selection of the best agent for each clinical situation . METHODS: A micromethod was developed to assess sporicidal activity against Bacillus subtilis spores deposited on three different medical devices; carbon steel dental burs, silicone-rubber medical catheters, and titanium-alloy dental abutment screws . The spore load on each device and the recovery after three analytical steps were quantitatively assessed with spores radiolabeled with carbon 14 methionine . RESULTS: The killing of 2 to 7 x 10(6) spores loaded on three different devices and exposed to glutaraldehyde, formaldehyde, copper ascorbate, hydrogen peroxide, peracetic acid, sodium hypochlorite, or phenol for 30 minutes at 20 degrees C ranged from a 10(3)-fold decrease for 10% hydrogen peroxide to zero decrease for 5% phenol . Our results suggest that the nature of the surface being challenged may affect the sporicidal activity of some chemical agents . CONCLUSION: The quantitative data presented allow comparison of the sporicidal effect of different liquid chemical agents . These findings may help prevent an overestimation of sporicidal activity and possible transmission of pathogens from the surface of improperly decontaminated medical devices.

Mol Microbiol, 1996 Oct, 22(1), 75 - 85
Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis; Haijema BJ et al.; It has been hypothesized that the dinR gene product of Bacillus subtilis acts as a repressor of the SOS regulon by binding to DNA sequences located upstream of SOS genes, including dinR and recA . Following activation as a result of DNA damage, RecA is believed to catalyse DinR-autocleavage, thus derepressing the SOS regulon . The present results support this hypothesis: a dinR insertion mutation caused a high, constitutive expression of both dinR and recA, which could not be further elevated by SOS-induction . In addition, gel-retardation assays demonstrated a direct interaction between the dinR gene product and the recA and dinR promoter regions . Epistatic interactions and gel-retardation assays demonstrated that the previously reported competence-specific expression of recA directly depended upon the gene product of comK, the competence transcription factor . These data demonstrate the existence of a direct regulatory link between the competence signal-transduction pathway and the SOS reguion.

Mol Microbiol, 1996 Oct, 22(1), 43 - 51
An elongation factor Tu (EF-Tu) resistant to the EF-Tu inhibitor GE2270 in the producing organism Planobispora rosea; Soslo M et al.; Using a cell-free protein-synthesis system, we have established that the elongation factor (EF) Tu (EF-Tu) of the actinomycete Planobispora rosea, the producer of the thiazolyl peptide GE2270, a specific EF-Tu inhibitor, is highly resistant to its own antibiotic, while it is completely inhibited by kirromycin, which is another inhibitor of this factor . P . rosea was found to possess a single tuf gene, located between fus and rpsJ, encoding other components of the protein-synthesis machinery . The P . rosea tuf gene was expressed as a translational fusion to malE in Escherichia coli, and the resulting EF-Tu with an N-terminal Gly-Met extension was able to promote poly(U)-directed poly(Phe) synthesis in cell-free systems . This activity was not affected by GE2270, and the recombinant protein was incapable of binding the antibiotic, indicating that the P . rosea EF-Tu is intrinsically resistant to this inhibitor . Inspection of the translated tuf sequence revealed a number of amino acid substitutions in highly conserved positions . These residues, which are likely to be involved in conferring GE2270 resistance, map in EF-Tu domain II, as do the only two known mutations conferring resistance to this class of thiazolyl peptides in Bacillus subtilis.

Clin Orthop, 1996 Oct, (331), 159 - 63
Safety and efficacy of ethylene oxide sterilized polyethylene in total knee arthroplasty; Ries MD et al.; Eleven unassembled metal backed patellar interfaces were inoculated with 0.1 ml of Sportol (Bacillus subtilis variety niger spore) and then assembled . Ten of the 11 implants were exposed to 1/2 of a standard ethylene oxide sterilization cycle . The remaining implant was left unsterilized as a control . All the implants were separately incubated in soybean casein digest broth for 7 days at 30 degrees to 35 degrees C and tested for positive growth of Bacillus subtilis . To measure residual ethylene oxide content, 4 ultrahigh molecular weight polyethylene tibial inserts were exposed to a full ethylene oxide sterilization cycle . The implants were removed from the sterilization chamber and tested for residual ethylene oxide at 3, 5, 8, and 9 days after sterilization using an exhaustive extraction headspace technique . Residual ethylene oxide was measured in 3 additional implants 26 days after sterilization . No growth of Bacillus subtilis occurred on any of the 10 inoculated and ethylene oxide sterilized metal backed patellar components, whereas positive growth occurred on the inoculated, unsterilized control implant . Residual ethylene oxide measured in the tibial inserts at 3, 5, 8, and 9 days after sterilization was 23, 15, 12, and 9 ppm, respectively . Twenty-six days after sterilization, residual ethylene oxide was below the minimum detectable level of the measurement technique (5 ppm).

EMBO J, 1996 Oct 1, 15(19), 5125 - 34
Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis; Rizzi M et al.; NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide . The three-dimensional structure of NH3-dependent NAD+ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X-ray crystallography (at 2.6 and 2.0 angstroms resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques . The enzyme consists of a tight homodimer with alpha/beta subunit topology . The catalytic site is located at the parallel beta-sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg2+ ion are observed . Residue Ser46, part of the neighboring 'P-loop', is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido-NAD+ during the first step of the catalyzed reaction . The deamido-NAD+ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center . A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine-dependent GMP synthetase, supporting the hypothesis that NAD+ synthetase belongs to the newly discovered family of 'N-type' ATP pyrophosphatases.

Microbiology, 1996 Oct, 142 ( Pt 10), 2943 - 50
Chorismate synthase from Staphylococcus aureus; Horsburgh MJ et al.; The aroC gene encoding chorismate synthase and the ndk gene encoding nucleoside diphosphate kinase (Ndk) were cloned from Staphylococcus aureus . DNA sequencing suggests that aroC is located in an operon with aroB and aroA and encodes a protein of 388 amino acids with 61% identity to the aroF gene product of Bacillus subtilis . The ndk gene of S . aureus encodes a protein of 149 amino acids which exhibits a high degree of identity to other bacterial Ndk proteins . The 3' end of the S . aureus gerCC gene was also identified by sequencing and was located immediately upstream of ndk . The gerCA and gerCB genes were found to be located upstream of gerCC by Southern hybridization analysis . This observed linkage of the gerC genes with the ndk, aroC and aroB genes has been similarly observed in B . subtilis . The S . aureus chorismate synthase was overexpressed to a high level in Escherichia coli using a T7 promoter plasmid construct, the enzyme was purified to near homogeneity in two steps and found to be a homotetramer with a subunit molecular mass, estimated by electrospray mass spectrometry, of 43024 Da . The properties of S . aureus chorismate synthase are compared with those of the B . subtilis and E . coli enzymes.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10647 - 52
Structure of the replication terminus-terminator protein complex as probed by affinity cleavage; Pai KS et al.; The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin . The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry . The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP . Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization . We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease . From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts . Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex . The biological implications of the model have been discussed.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 267 - 71
Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms; Koulis A et al.; Hyperthermophiles exist in conditions which present an increased threat to the informational integrity of their DNA, particularly by hydrolytic damage . As in mesophilic organisms, specific activities must exist to restore and protect this template function of DNA . In this study we have demonstrated the presence of thermally stable uracil-DNA glycosylase activities in seven hyperthermophiles; one bacterial: Thermotoga maritima, and six archaeal: Sulfolobus solfataricus, Sulfolobus shibatae, Sulfolobus acidocaldarius, Thermococcus litoralis, Pyrococcus furiosus and Pyrobaculum islandicum . Uracil-DNA glycosylase inhibitor protein of the Bacillus subtilis bacteriophage PBS1 shows activity against all of these, suggesting a highly conserved tertiary structure between hyperthermophilic and mesophilic uracil-DNA glycosylases.

Appl Environ Microbiol, 1996 Oct, 62(10), 3687 - 96
Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis; Sauer U et al.; Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain . Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively . However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains . A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions . Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1 . For molecular characterization of B . subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived . In particular, the murein sacculus was found to constitute approximately 9% of B . subtilis biomass, three- to fivefold more than in Escherichia coli . Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway . Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation . Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.

J Bacteriol, 1996 Oct, 178(20), 6059 - 63
A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination; Moriyama R et al.; The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined . Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues . Enzyme activity was not found in germination exudate of B . subtilis spores, which differs from the case of B . cereus enzyme . A B . subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation . However, the sleB mutant was unable to complete germination mediated by L-alanine.

J Bacteriol, 1996 Oct, 178(20), 6036 - 42
Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing; Wu S et al.; We have identified a gene cluster located on the chromosomal SmaI I fragment of a highly methicillin resistant strain of Staphylococcus aureus, consisting of four open reading frames (ORFs), named after the number of deduced amino acid residues, in the sequential order orf333-orf108-orf159-orf256 . The gene cluster showed close similarities to the Bacillus subtilis sigB operon both in overall organization and in primary sequences of the gene products . The complete gene cluster (provisionally named sigma-B or sigB) was preceded by an sigmaA-like promoter (PA) and had an internal sigmaB-like promoter sequence (PB) between orf333 and orf108, suggesting a complex regulatory mechanism . The polypeptides encoded by orf333, -108, -159, and -256 showed 62, 67, 71, and 77% homologies, respectively, with the RsbU, RsbV, RsbW, and SigB polypeptides encoded by the B . subtilis sigB operon . A Tn551 insertional mutant, RUSA168 (insert in orf256 of the staphylococcal sigma-B operon), showed drastic reduction in methicillin resistance (decrease in MIC from 1,600 microg ml-1 to 12 to 25 microg ml-1off

J Bacteriol, 1996 Oct, 178(20), 6001 - 5
Identification and characterization of pbpC, the gene encoding Bacillus subtilis penicillin-binding protein 3; Murray T et al.; Penicillin-binding proteins (PBPs) are enzymes involved in the synthesis of peptidoglycan structures in Bacillus subtilis such as the vegetative cell wall and the spore cortex . The B . subtilis sequencing project has identified a gene (orf16, EMBL accession number D38161) which exhibits significant sequence similarity to genes encoding class B high-molecular-weight PBPs . We have found that orf16 encodes PBP3 and have renamed this locus pbpC . Transcriptional fusions to lacZ were used to demonstrate that pbpC is transcribed primarily during log-phase growth, with lower amounts expressed during sporulation . During spore germination and outgrowth, pbpC expression resumes coincident with an increase in the optical density of the culture . The major promoter for pbpC is located just upstream of the gene; a low level of expression during sporulation appears to originate from much further upstream . Loss of PBP3 does not produce any detectable change in phenotype with respect to cell morphology, growth, sporulation, spore heat resistance, or spore germination and outgrowth . This was also true when the pbpC mutation was combined with mutations affecting other PBP-encoding genes to produce double mutants . These findings are consistent with previous evidence that many PBPs of B . subtilis have redundant functions within the cell.

J Bacteriol, 1996 Oct, 178(20), 5910 - 5
CodY is required for nutritional repression of Bacillus subtilis genetic competence; Serror P et al.; The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids . This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and was relieved in strains carrying a null mutation in the codY gene . DNase I footprinting experiments showed that purified CodY binds directly to the srfA and comK promoter regions.

J Bacteriol, 1996 Oct, 178(19), 5817 - 21
Markerless deletions of pil genes in Myxococcus xanthus generated by counterselection with the Bacillus subtilis sacB gene; Wu SS et al.; In-frame deletions of pilA and pilS were constructed in Myxococcus xanthus with a plasmid integration-excision strategy facilitated by sacB . sacB conferred sucrose sensitivity upon its M . xanthus host only when it lay in the same orientation as adjacent M . xanthus genes . Gene orientation also affected the efficiency of sucrose counterselection in the sucrose-sensitive strains . The deltapilA mutant lacked pili and social motility, while the deltapilS mutant showed no defect in either phenotype.

J Bacteriol, 1996 Oct, 178(19), 5806 - 9
Evidence that the Bacillus subtilis pyrimidine regulatory protein PyrR acts by binding to pyr mRNA at three sites in vivo; Lu Y et al.; The Bacillus subtilis pyr operon is regulated by a transcriptional attenuation mechanism that requires the PyrR regulatory protein . Multicopy plasmids that could be transcribed to yield segments of RNA from the attenuation regions of the pyr operon induced derepression of chromosomal pyr genes, whereas plasmids that could not yield pyr RNA did not . We conclude that pyr RNA acts by titrating the PyrR protein and preventing it from regulating pyr attenuation.

Curr Microbiol, 1996 Oct, 33(4), 228 - 36
The aggregation-mediated conjugation system of Bacillus thuringiensis subsp . israelensis: host range and kinetics of transfer; Jensen GB et al.; The aggregation-mediated conjugation system in Bacillus thuringiensis subsp . israelensis encoded on the plasmid pXO16 is characterized by the formation of aggregates when Agr+ and Agr- cells are socialized in exponential growth . Using the aggregation phenotypes, we have identified potential recipients of the aggregation-plasmid pXO16 among Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus sphaericus, and 24 subspecies of B . thuringiensis . We found 14 Agr- strains, i.e., potential recipients of the aggregation system encoded by plasmid pXO16 . Five strains contained a conjugative apparatus of their own and were excluded from further examinations . To monitor the transfer of plasmid pXO16, we constructed a transposon insertion of the plasmid with Tn5401 . The study of the plasmid transfer of pXO16::Tn5401 indicated the secretion of bacteriocins from both donor strain and recipient strains . Only one out of the nine strains examined was unable to receive the aggregation-plasmid pXO16 and express the aggregation phenotype and the conjugative abilities . It was found that the transfer of plasmid pXO16 to Bacillus thuringiensis subsp . israelensis Agr- strains was 100% . All recipients had acquired the aggregation-plasmid pXO16 and converted to the Agr+ phenotype.

Gene, 1996 Sep 26, 174(1), 121 - 8
A gyrB-like gene from the hyperthermophilic bacterion Thermotoga maritima; Guipaud O et al.; We have cloned and sequenced two overlapping DNA fragments (3236 bp) containing a gene encoding the ATPase subunit of a type II DNA topoisomerase from the hyperthermophilic bacterion Thermotoga maritima (Tm Top2B) . The deduced protein is composed of 636 aa with a calculated molecular mass of 72415 Da . It shares significant similarities with the ATPase subunits of mesophilic bacterial DNA topoisomerases II, either DNA gyrase (GyrB) or DNA topoisomerase IV (ParE) . Although the highest similarity scores are obtained with GyrB proteins (55% identity with Bacillus subtilis DNA gyrase), a detailed phylogenetic analysis of all known DNA topoisomerases II does not allow us to determine if Tm Top2B corresponds to a DNA gyrase or a DNA topoisomerase IV . This hyperthermophilic Top2B protein exhibits a larger amount of charged amino acids than its mesophilic homologues, a feature which could be important for its thermostability . No gyrA-like gene has been found near top2B . A gene coding for a transaminase B-like protein was found in the upstream region of top2B.

Genes Dev, 1996 Sep 15, 10(18), 2348 - 58
Three sites of contact between the Bacillus subtilis transcription factor sigmaF and its antisigma factor SpoIIAB; Decatur AL et al.; The developmental regulatory protein sigmaF of Bacillus subtilis, a member of the sigma70-family of RNA polymerase sigma factors, is regulated negatively by the antisigma factor SpoIIAB, which binds to sigmaF to form an inactive complex . Complex formation between SpoIIAB, which contains an inferred adenosine nucleotide binding pocket, and sigmaF is stimulated strongly by the presence of ATP . Here we report that SpoIIAB contacts sigmaF at three widely spaced binding surfaces corresponding to conserved regions 2.1, 3.1, and 4.1 of sigma70-like sigma factors . This conclusion is based on binding studies between SpoIIAB and truncated portions of sigmaF, the isolation of mutants of sigmaF that were partially resistant to inhibition by SpoIIAB in vivo and were defective in binding to the antisigma factor in vitro, and the creation of alanine substitution mutants of regions 2.1, 3.1, or 4.1 of sigmaF that were impaired in complex formation . Because the interaction of SpoIIAB with all three binding surfaces was stimulated by ATP, we infer that ATP induces a conformational change in SpoIIAB that is needed for tight binding to sigmaF . Finally, we discuss the possibility that another antisigma factor, unrelated to SpoIIAB, may interact with its respective sigma factor in a similar topological pattern of widely spaced binding surfaces located in or near conserved regions 2.1, 3.1, and 4.1.

Genes Dev, 1996 Sep 15, 10(18), 2265 - 75
Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor; Yang X et al.; The general stress response of the bacterium Bacillus subtilis is governed by a signal transduction network that regulates activity of the sigma(B) transcription factor . We show that this network comprises two partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, which contribute to regulating sigma(B) . Each module consists of a phosphatase (X or U), an antagonist protein (S or V), and a switch protein/kinase (T or W) . In the downstream module, the W anti-sigma factor is the primary regulator of sigma(B) activity . If the V antagonist is phosphorylated, the W switch protein binds and inhibits sigma(B) . If V is unphosphorylated, it complexes W, freeing sigma(B) to interact with RNA polymerase and promote transcription . The phosphorylation state of V is controlled by opposing kinase (W) and phosphatase (U) activities . The U phosphatase is regulated by the upstream module . The T switch protein directly binds U, stimulating phosphatase activity . The T-U interaction is governed by the phosphorylation state of the S antagonist, controlled by opposing kinase (T) and phosphatase (X) activities . This partner-switching mechanism provides a general regulatory strategy in which linked modules sense and integrate multiple signals by protein-protein interaction.

J Biol Chem, 1996 Sep 6, 271(36), 21828 - 34
Characterization of the primary sigma factor of Staphylococcus aureus; Deora R et al.; RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays . Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S . aureus, it was hypothesized that plaC could encode the vegetative sigma factor . We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE . The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography . The purified protein, designated sigmaSA, cross-reacted with the B . subtilis anti-sigmaA antibody . E . coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S . aureus promoters hla, sea, and sec and from the E . coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E . coli sigma70 . sigmaSA, when added to the purified RNAP from S . aureus, stimulated transcriptional activity of the RNAP up to 72-fold . As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs . Disruption of the plaC gene on the S . aureus chromosome was lethal . We conclude that plaC encodes the primary sigma factor in S . aureus.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1507 - 9
Ansamycin antibiotics as free radical scavengers isolated from Streptomyces by using the bactericidal action of the hydroxyl radical; Morimitsu Y et al.; Ansamycin antibiotics (1-4) were isolated from the cultured broth of Streptomyces sp . USF-319 strain as a result of our screening for free radical scavengers . They inhibited the bactericidal effect of the Fenton reagent toward Bacillus subtilis by their radical scavenging activity . Some of them also showed inhibitory activity against lipid peroxidation and lipoxygenases.

Anaesthesist, 1996 Sep, 45(9), 814 - 8
{Comparative study of the efficiency of bacterial filters in long-term mechanical ventilation}; Nielsen HJ et al.; Two commercially available bacterial filters to be used as part of the mechanical ventilation unit during anaesthesia were tested for hygienic criteria . Manufacturers claim that bacterial breathing filters have a filtration capacity of about 99.995%, so that there would be no need for thermal disinfection of tubing and ventilation circuits after each use . One filter is designed for a single use only, the other can be used up to 24 times after sterilisation . Both filters consist of hydrophobic glass fibres . METHODS: During simulated mechanical ventilation for 24 h, an alcoholic suspension of Bacillus subtilis was atomized in front of the filters tested . A gelatin membrane filter was integrated in the ventilation circuit and captured the filtered gas behind the test filter . RESULTS: During simulated mechanical ventilation for 24 h, the filtration capacity of both the disposable and reusable filters (Table 2) did not confirm the manufacturers' short-term technical findings over 8 s (DIN-EN 143) . CONCLUSIONS: The use of bacterial filters during mechanical ventilation reduces the probability of bacterial contamination, but does not make sterilisation of the tubes and ventilation circuit unnecessary.

Plasmid, 1996 Sep, 36(2), 75 - 85
Mobilization of "nonmobilizable" plasmids by the aggregation-mediated conjugation system of Bacillus thuringiensis; Andrup L et al.; The aggregation-mediated conjugation system of Bacillus thuringiensis subsp, israelensis (Bti), encoded by the 200-kb plasmid pXO16, is highly potent in transferring itself and efficient in mobilizing other nonconjugative plasmids . In the present study we have analyzed the native Bacillus cereus plasmid pBC16 . This plasmid has previously been shown to harbor a mob gene (ORF beta) and a locus functioning as an oriT site in plasmid pLS20-mediated conjugation in Bacillus subtilis . However, in the conjugation system of Bti we found that a derivative of pBC16 deleted for both these loci was mobilizable, although at a reduced frequency . Another derivative of pBC16, containing a deletion spanning the first half of the coding region of the mob gene, was found to be nearly as mobilizable as the intact pBC16, suggesting its dispensability in the transfer process . Other plasmids based on the theta-replicating origins, pAM beta 1, pLS20, ori43, ori44, and ori60, were also consistently mobilized in the conjugation system encoded by Bti plasmid pXO16 . Analyzing the conjugation process by the use of scanning electron microscopy revealed the presence of connections between cells in the mating mixtures . These connections did not appear in monocultures of the donor strain or the recipient strain and may be conjugational junctions.

Mol Microbiol, 1996 Sep, 21(5), 989 - 99
Replacement of the lysine residue in the consensus ATP-binding sequence of the AddA subunit of AddAB drastically affects chromosomal recombination in transformation and transduction of Bacillus subtilis; Haijema BJ et al.; The ATP-dependent deoxyribonuclease enzyme complex (AddAB) of Bacillus subtilis possesses two consensus ATP-binding sequences, located in the N-terminal region of both subunits . The highly conserved lysine residues in both consensus ATP-binding sequences were replaced by glycine, resulting in the mutant enzyme complexes AddAB-A-K36G (AddA*B) and AddAB-B-K14G (AddAB*) . The mutation in subunit AddA reduced DNA repair and chromosomal transformation, and abolished bacteriophage PBS1-mediated transduction . This mutation also resulted in a complete loss of the ATP-dependent exonuclease and helicase activity . In contrast, the mutation in subunit AddB had only marginal effects . The recF and addAB genes are not required for transformation with plasmid DNA, but have overlapping activities in transformation with chromosomal DNA . By contrast to RecF, the AddAB enzyme is essential for PBS1-mediated transduction . However, recF has a more important function with respect to DNA repair than addAB.

Mol Microbiol, 1996 Sep, 21(5), 977 - 87
Identification of a ClpC ATPase required for stress tolerance and in vivo survival of Listeria monocytogenes; Rouquette C et al.; We identified a new chromosomal locus involved in the virulence of the facultative intracellular pathogen Listeria monocytogenes . This locus displays the same genetic organization as that of the clpC/mecB locus of Bacillus subtilis . It contains a thermoregulated operon of four genes, whose transcription is upregulated at 42 degrees C . The last gene of this operon is clpC, which encodes a protein of 826 amino acid residues, identified as a ClpC ATPase, sharing a strong peptide sequence identity (78%) with ClpC/MecB of B . subtilis . Tn917 insertions inactivating the entire operon, or only clpC, gave mutants highly susceptible to stress, including iron limitation, elevated temperatures and high osmolarity . The virulence of these mutants was severely impaired in the mouse . A clpC insertional mutant was also restricted in its capacity to grow in bone-marrow-derived macrophages . These results demonstrate that the ClpC ATPase of L . monocytogenes is a general stress protein involved in intracellular growth and in vivo survival of this pathogen in host tissues.

Mol Microbiol, 1996 Sep, 21(5), 913 - 24
Two-stage regulation of an anti-sigma factor determines developmental fate during bacterial endospore formation; Kellner EM et al.; During endospore formation in Bacillus subtilis an asymmetric division produces two cells, forespore and mother cell, which follow different developmental paths . Commitment to the forespore-specific developmental path is controlled in part by the activation of the forespore-specific RNA polymerase sigma factor, sigma F . Activity of sigma F is inhibited in the mother cell by the anti-sigma factor SpoIIAB . In the forespore, sigma F directs transcription of the structural gene for sigma G . However, sigma G does not become active until after engulfment of the forespore is complete . This sigma G activity is dependent upon the products of the spoIIIA operon . We showed that sigma G is present but mostly inactive in a spoIIIA mutant . We also demonstrated that the anti-sigma factor SpoIIAB can bind to sigma G in vitro . Moreover, a mutant form of sigma G that binds SpoIIAB inefficiently in vitro was shown to function independently of SpoIIIA during sporulation . These and previously reported results support a model in which SpoIIAB functions as an inhibitor of sigma G activity during sporulation . Therefore, we propose that the anti-sigma factor SpoIIAB antagonizes both sigma F and sigma G activities, and that this antagonism is relieved in the forespore in two stages . In the first stage, which follows septation, a SpoIIAA-dependent mechanism partially relieves SpoIIAB inhibition of sigma F activity in the forespore . In the second stage, which follows forespore engulfment, a SpoIIIA-dependent process inactivates SpoIIAB in the forespore, resulting in the activation of sigma G.

Can J Microbiol, 1996 Sep, 42(9), 919 - 26
Cloning and characterization of transcriptional promoters from Bacillus subtilis phage 2C; Daxhelet G et al.; Phage 2C is a Bacillus subtilis lytic phage, whose genome contains hydroxymethyluracil in place of thymine . To isolate promoters of early phage genes involved in the take-over of cellular metabolism, 2C DNA libraries were constructed in promoter-probe plasmids replicating in Escherichia coli and B . subtilis . Four different 2C DNA fragments strongly expressed reporter genes in E . coli but not in B . subtilis . All fragments originated from unique sequences of the genome and not from its terminal redundancies . One fragment was sequenced . Despite the presence of an sigma-A-RNA polymerase binding site upstream of the transcriptional initiation site of a 2C early gene, this fragment did not promote transcription in B . subtilis.

Nucleic Acids Res, 1996 Sep 1, 24(17), 3431 - 6
In vivo analysis of the plasmid pAM beta 1 resolution system; Janniere L et al.; The promiscuous plasmid pAM beta 1 from Gram-positive bacteria encodes a resolution system which differs from that of Tn3 in that (i) it requires a histone-like protein and an unusual resolvase-DNA interaction to promote recombination and (ii) it mediates in vivo DNA inversion in plasmid substrates . In this in vivo analysis, the pAM beta 1 resolution site is narrowed down to a 99 bp segment, the strand exchange is mapped within 10 bp and the serine residue at position 10 of the resolvase is shown to be essential for enzyme activity . In addition, data showing that the resolution system does not promote DNA inversion in the Bacillus subtilis chromosome are presented . Implications of this observation are discussed.

J Bacteriol, 1996 Sep, 178(18), 5480 - 6
Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr; Reizer J et al.; Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system . ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression . We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon . A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala {S46A HPr} and one in which His-15 is replaced by Ala {H15A HPr}) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo . The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar . By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect . In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA . These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.

J Bacteriol, 1996 Sep, 178(18), 5456 - 63
Reactivation of the Bacillus subtilis anti-sigma B antagonist, RsbV, by stress- or starvation-induced phosphatase activities; Voelker U et al.; sigma B is a secondary sigma factor that controls the general stress regulon in Bacillus subtilis . The regulon is activated when sigma B is released from a complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase . Two separate mechanisms cause sigma B release: an ATP-responsive mechanism that correlates with nutritional stress and an ATP-independent mechanism that responds to environmental insult (e.g., heat shock and ethanol treatment) . ATP levels are thought to directly affect RsbW's binding preference . Low levels of ATP cause RsbW to release sigma B and bind to an alternative protein (RsbV), while high levels of ATP favor RsbW-sigma B complex formation and inactivation of RsbV by an RsbW-dependent phosphorylation . During growth, most of the RsbV is phosphorylated (RsbV-P) and inactive . Environmental stress induces the release of sigma B and the formation of the RsbW-RsbV complex, regardless of ATP levels . This pathway requires the products of additional genes encoded within the eight-gene operon (sigB) that includes the genes for sigma B, RsbW, and RsbV . By using isoelectric focusing techniques to distinguish RsbV from RsbV-P and chloramphenicol treatment or pulse-chase labeling to identify preexisting RsbV-P, we have now determined that stress induces the dephosphorylation of RsbV-P to reactivate RsbV . RsbV-P was also found to be dephosphorylated upon a drop in intracellular ATP levels . The stress-dependent and ATP-responsive dephosphorylations of RsbV-P differed in their requirements for the products of the first four genes (rsbR, -S, -T, and -U) of the sigB operon . Both dephosphorylation reactions required at least one of the genes included in a deletion that removed rsbR, -S, and -T; however, only an environmental insult required RsbU to reactivate RsbV.

Photochem Photobiol, 1996 Sep, 64(3), 542 - 6
Damage to UV-sensitive cells by short UV in photographic flashes; Menezes S et al.; Light emitted by electronic photographic flash units is shown to damage bacteria and human skin fibroblasts deficient in repair systems, with survival curves very similar to those produced by 254 nm short UV . The lesions induced by these flashes are as photorepairable by the photolyase enzyme as those induced by 254 nm UV and result in equivalent survival rates . Biological dosimetry performed with microorganisms highly sensitive to UV (Escherichia coli K12 AB2480, deficient in excision and recombinational-dependent repair systems and Bacillus subtilis UVSSP spores, deficient in excision and in a specific spore repair process) revealed that each 1 ms flash of light from the photographic unit used in this work contained the equivalent of 0.25 J m-2 of 254 nm UV, when measured at a distance of 7.0 cm . This dose of UV was found to be lethal to both repair-deficient E . coli bacteria and repair-deficient human skin fibroblasts obtained from xeroderma pigmentosum donors, as well as mutagenic in B/r wild-type and HCR-mutant bacteria.

J Bacteriol, 1996 Sep, 178(17), 5235 - 42
The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis; Cordes C et al.; The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis . pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the Escherichia coli beta-galactosidase (lacZ) gene . Two processes affected the accumulation of pGP1 variants with deletions in the penP-lacZ region . First, divergent transcription from genes upstream of penP-lacZ increased pGP1 deletion frequencies up to about 10-fold . Second, the removal of the PenP signal peptide resulted in completely stable plasmids, indicating that the entry of the PenP fragment into the protein export pathway is an important factor in the instability of pGP1 . On the basis of these results, we propose a model in which the temporary anchoring of the plasmid to the membrane through the cotranscriptional and cotranslational entry of PenP into the protein export pathway creates domains of local hypersupercoiling, which we assume to be targets for deletion formation.

J Bacteriol, 1996 Sep, 178(17), 5159 - 63
Interaction of the trp RNA-Binding attenuation protein (TRAP) of Bacillus subtilis with RNA: effects of the number of GAG repeats, the nucleotides separating adjacent repeats, and RNA secondary structure; Babitzke P et al.; The 11-subunit trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates transcription and translation by binding to several (G/U)AG repeats present in the trp leader and trpG transcripts . Filter binding assays were used to study interactions between L-tryptophan-activated TRAP and synthetic RNAs . RNAs that contained GAG and/or UAG repeats were tested while the length and sequence of the nucleotides separating adjacent trinucleotide repeats were altered . TRAP-RNA complexes formed with transcripts containing GAG repeats were more stable than those with transcripts containing UAG repeats or alternating GAG and UAG repeats . The stability of TRAP-RNA complexes also increased substantially when the number of GAG repeats was increased from five to six and from six to seven . A gradual increase in complex stability was observed when the number of GAG repeats was increased from 7 to 11 . The optimal spacer between adjacent trinucleotide repeats was found to be 2 nucleotides, with A and U residues preferred over G and C residues . TRAP binding was specific for single-stranded RNA; TRAP could not bind to RNA containing GAG repeats base paired in a stable RNA duplex . Overall, our findings suggest that each L-tryptophan-activated TRAP subunit can bind one (G/U)AG repeat and that multiple TRAP subunit-RNA binding site interactions are required for stable TRAP-RNA association.

J Bacteriol, 1996 Sep, 178(17), 5144 - 52
Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis; Liu L et al.; The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes . Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development . In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken . Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells . Multicopy degU failed to suppress comS or sinR mutations . No suppression of comS or degU by multicopy sinR was observed . The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR . The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR . Cells bearing plasmid-amplified sinR were poorly competent . These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.

J Bacteriol, 1996 Sep, 178(17), 5130 - 7
Effects of lysine-to-glycine mutations in the ATP-binding consensus sequences in the AddA and AddB subunits on the Bacillus subtilis AddAB enzyme activities; Haijema BJ et al.; The N-terminal regions of both subunits AddA and AddB of the Bacillus subtilis AddAB enzyme contain amino acid sequences, designated motif I, which are commonly found in ATP-binding enzymes . The functional significance of the motif I regions was studied by replacing the highly conserved lysine residues of the regions in both subunits by glycines and by examination of the resulting mutant enzymes with respect to their enzymatic properties . This study shows that the mutation in subunit AddB hardly affected the ATPase, helicase, and exonuclease activities of the AddAB enzyme . However, the mutation in subunit AddA drastically reduced these activities, as well as the kcat for ATP hydrolysis . The apparent Km for ATP in ATP hydrolysis did not significantly deviate from that of the wild-type enzyme . These results suggest that the lysine residue in motif I of subunit AddA of the AddAB enzyme is not essential for the binding of the nucleotide but has a role in ATP hydrolysis, which is required for the exonuclease and helicase activities of the enzyme.

J Bacteriol, 1996 Sep, 178(17), 5121 - 9
Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes; Boch J et al.; Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media . Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B . subtilis . The DNA sequence of a 4.1-kb segment from the cloned chromosomal B . subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified . The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon . The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases . This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought . The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases . B . subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed . Deletion of the gbsAB genes destroyed the choline-glycine betaine synthesis pathway and abolished the ability of B . subtilis to deal effectively with high-osmolarity stress in choline- or glycine betaine aldehyde-containing medium . Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain . The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B . subtilis, even in medium of moderate osmolarity . A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis . gbsAB transcription was initiated from a promoter with close homology to sigma A-dependent promoters and was stimulated by the presence of choline in the growth medium.

Infect Immun, 1996 Sep, 64(9), 3758 - 64
Staphylocidal action of thrombin-induced platelet microbicidal protein is influenced by microenvironment and target cell growth phase; Koo SP et al.; Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic peptide released from rabbit platelets following exposure to thrombin in vitro . This peptide exerts potent in vitro microbicidal activity against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus . It is known that the microbicidal actions of other cationic antimicrobial peptides (e.g., neutrophil defensins) are influenced by environmental factors and target cell growth phase . However, whether these parameters affect tPMP microbicidal activity has not been studied . Thus, we assessed the in vitro bactericidal activity of tPMP against two tPMP-susceptible strains, Bacillus subtilis ATCC 6633 and S . aureus 502A, in various target cell growth phases or under various microenvironmental conditions . The conditions studied included differing bacterial growth phase (logarithmic versus stationary), temperature (range, 4 to 42 degrees C), pH (range, 4.5 to 8.5), cationicity (range, 0.1 mM to 2 M), anionicity (range, 0.08 to 5 microM), and neutral carbohydrates ranging in molecular weight (MW) from 180 to 37,700 (range, 50 to 500 mM) as well as rabbit platelet-free plasma and serum . tPMP staphylocidal activity was greater against logarithmic- than stationary-phase cells . tPMP bactericidal activity against both B . subtilis and S . aureus was directly correlated with temperature and pH, with microbicidal activity exhibited near the physiological range (37 to 42 degrees C and pH 7.2 to 8.5, respectively) . The presence of cations (Na+, K+, Ca2+, and Mg2+) decreased tPMP bactericidal activity in a time- and concentration-dependent manner, with complete inhibition at monovalent or divalent cation concentrations of > or = 250 or > or = 10 mM, respectively . Staphylocidal activity of tPMP was also inhibited by the polyanions polyanetholsulfonic acid and polyaspartic acid, at 0.1 and 0.4 microM, respectively . Coincident exposure with low-MW carbohydrates (glucose, sucrose, and melezitose) did not affect tPMP staphylocidal activity . However, higher-MW carbohydrates (raffinose and dextrans) decreased tPMP activity in a manner directly proportional to their concentration and MW . Solute-mediated inhibition of tPMP bactericidal activity was independent of solute osmolality but directly related to the duration of tPMP-solute coexposure . tPMP enhanced the staphylocidal activities of platelet-free plasma and heat-inactivated serum, while the activity of normal serum was not affected . These collective observations suggest that tPMP retains antimicrobial activities under physiological conditions which are likely to be relevant to host defense in vivo.

DNA Res, 1996 Aug 31, 3(4), 257 - 62
Cloning and sequencing of a 27.8-kb nucleotide sequence of the 79 degrees-81 degrees region of the Bacillus subtilis genome containing the sspE locus; Yamamoto H et al.; The nucleotide sequence of a 27830-bp DNA segment in the 79 degrees-81 degrees region of the Bacillus subtilis genome has been determined . This region contains 29 complete ORFs including the sspE gene, which encodes a small acid-soluble spore protein gamma and locates on the one side terminal of our assigned region . A homology search for the products deduced from the 29 ORFs revealed that nine of them exhibit significant similarity to known proteins, e.g . proteins involved in an iron uptake system, a multidrug resistance protein, a chloramphenicol resistance protein, epoxide hydrolase, adenine glycosylase, and a glucose-1-dehydrogenase homolog.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 9287 - 91
Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase; Vothknecht UC et al.; delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway . The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde . The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E . coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST) . GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde . The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1 . The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E . coli tRNA(Glu) and its activity was inhibited by hemin . It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12 . After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa . Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 8913 - 8
Protein p4 represses phage phi 29 A2c promoter by interacting with the alpha subunit of Bacillus subtilis RNA polymerase; Monsalve M et al.; Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached . Protein p4 binds at PA2c immediately upstream from RNA polymerase; repression involves a contact between both proteins that holds the RNA polymerase at the promoter . This contact is held mainly through p4 residue Arg120, which is also required for activation of the phi 29 late A3 promoter . We have investigated which region of RNA polymerase contacts protein p4 at PA2c . Promoter repression was impaired when a reconstituted RNA polymerase lacking the 15 C-terminal residues of the alpha subunit C-terminal domain was used; this polymerase was otherwise competent for transcription . Binding cooperativity assays indicated that protein p4 cannot interact with this mutant RNA polymerase at PA2c . Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues . Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase . Therefore, this domain of the alpha subunit can receive regulatory signals not only from transcriptional activators, but from repressors also.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 8841 - 5
TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis; Wray LV Jr et al.; Expression of the Bacillus subtilis nrgAB operon is derepressed during nitrogen-limited growth . We have identified a gene, tnrA, that is required for the activation of nrgAB expression under these growth conditions . Analysis of the DNA sequence of the tnrA gene revealed that it encodes a protein with sequence similarity to GlnR, the repressor of the B . subtilis glutamine synthetase operon . The tnrA mutant has a pleiotropic phenotype . Compared with wild-type cells, the tnrA mutant is impaired in its ability to utilize allantoin, gamma-aminobutyrate, isoleucine, nitrate, urea, and valine as nitrogen sources . During nitrogen-limited growth, transcription of the nrgAB, nasB, gabP, and ure genes is significantly reduced in the tnrA mutant compared with the levels seen in wild-type cells . In contrast, the level of glnRA expression is 4-fold higher in the, tnrA mutant than in wild-type cells during nitrogen restriction . The phenotype of the tnrA mutant indicates that a global nitrogen regulatory system is present in B . subtilis and that this system is distinct from the Ntr regulatory system found in enteric bacteria.

Genes Dev, 1996 Aug 15, 10(16), 2014 - 24
Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis; Solomon JM et al.; We have purified and characterized an extracellular peptide factor that serves as a cell density signal for both competence development and sporulation in Bacillus subtilis . This competence and sporulation stimulating factor (CSF) was purified from conditioned medium (culture supernatant) based on its ability to stimulate expression of srfA (comS) in cells at low cell density . CSF is a 5-amino-acid peptide, glu-arg-gly-met-thr (ERGMT), that is, the carboxy-terminal 5 amino acids of the 40-amino-acid peptide encoded by phrC . No detectable CSF was produced in a phrC null mutant . The activity of chemically synthesized CSF (ERGMT) was virtually indistinguishable from that of CSF that was purified from culture supernatants . At relatively low concentrations (1-10 nM), CSF stimulated expression of srfA, whereas high concentrations of CSF stimulated the ability of cells at low cell density to sporulate . Stimulation of srfA expression by CSF requires the oligopeptide permease encoded by spo0K, a member of the ATP-binding-cassette family of transporters, and the putative phosphatase encoded by rapC, the gene immediately upstream of phrC . RapC was found to be a negative regulator of srfA expression, suggesting that the target of RapC is the transcription factor encoded by comA . We propose that CSF is transported into the cell by the Spo0K oligopeptide permease and stimulates competence gene expression by inhibiting (either directly or indirectly) the RapC phosphatase.

Biochemistry, 1996 Aug 13, 35(32), 10493 - 505
Magnesium ions are required by Bacillus subtilis ribonuclease P RNA for both binding and cleaving precursor tRNAAsp; Beebe JA et al.; The multiple roles Mg2+ plays in ribozyme-catalyzed reactions in stabilizing RNA structure, enhancing the affinity of bound substrates, and increasing catalysis are delineated for the RNA component of ribonuclease P (RNase P RNA) by a combination of steady-state kinetics, transient kinetics, and equilibrium binding measurements . Divalent metal ions cooperatively increase the affinity of Bacillus subtilis RNase P RNA for B . subtilis tRNA(Asp) more than 10(3)-fold, consistent with at least two additional magnesium ions binding to the RNase P RNA.tRNA complex . Monovalent cations also decrease KD(tRNA) and reduce, but do not eliminate, the dependence on magnesium ions, demonstrating that nonspecific electrostatic shielding is not sufficient to explain the requirement for high salt . Both di- and monovalent cations promote the high affinity of tRNA by forming contacts in the binary complex that reduce the dissociation rate constant for tRNA . Additionally, the hyperbolic dependence of the hydrolytic rate constant on the concentration of magnesium with a K1/2 approximately equal to 36 mM suggests that a third low-affinity divalent metal ion stabilizes the transition state for pre-tRNA cleavage . Furthermore, many (about 100) magnesium ions bind independently to RNase P RNA with higher affinity than the K1/2 of any of the functionally characterized magnesium binding sites . Therefore, the magnesium binding sites that have differential affinity in either the "folded" species or binary complex are a small subset of the total number of associated magnesium ions . In summary, the importance of magnesium bound to RNase P RNA can be separated functionally into three crucial roles: at least three sites stabilize the folded RNA tertiary structure {Pan . T . (1995) Biochemistry 34, 902-909}, at least two sites enhance the formation of complexes of RNase P RNA with pre-tRNA or tRNA, and at least one site stabilizes the transition state for pre-tRNA cleavage.

Biochim Biophys Acta, 1996 Aug 7, 1276(1), 57 - 63
Purification and characterization of the succinate dehydrogenase complex and CO-reactive b-type cytochromes from the facultative alkaliphile Bacillus firmus OF4; Gilmour R et al.; The presence of a cytochrome bo-type terminal oxidase in Bacillus firmus OF4 had been suggested from the effects of CO on the spectra of reduced membrane cytochromes (Hicks, D.B., Plass, R.J . and Quirk, P.G . (1991) J . Bacteriol . 173, 5010-5016) . In that study the CO-binding b-type cytochrome was partially purified by anion exchange chromatography . No further purification was attempted but later HPLC analysis indicated the absence of significant heme O in the B . firmus OF4 membranes . The current work shows that the partially purified cytochrome b is actually composed of three different b-type cytochromes which can be separated and purified by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies . Two of the cytochromes were CO-reactive but lacked the characteristic multisubunit composition of known terminal oxidases . Neither purified cytochrome catalyzed quinol or ferrocytochrome c oxidation . The more abundant CO-reactive b-type cytochrome (cytochrome b560) had an apparent molecular mass of 10 kDa, whereas the other, more minor component (cytochrome b558), was partially purified and showed two bands of 23 and 17 kDa on SDS-PAGE . The functions of the cytochromes b560 and b558 remain unknown but together they account for the spectrum originally attributed to cytochrome bo . The third, non-CO reactive, cytochrome b was associated with substantial succinate dehydrogenase activity and was purified as a three subunit succinate dehydrogenase complex with high specific activity (17.7 mumol/min/mg) . Limited N-terminal sequence of each subunit demonstrated marked similarity to the complex from Bacillus subtilis . The cytochrome b of the alkaliphile enzyme was reduced about 50% by succinate compared to the level of reduction achieved by dithionite . The enzyme reacted with both napthoquinones and benzoquinones . The results presented indicate that Bacillus firmus OF4 contains a succinate dehydrogenase complex with very similar properties to the enzyme from Bacillus subtilis, but does not contain a cytochrome o-type terminal oxidase under the growth conditions studied.

J Biol Chem, 1996 Aug 2, 271(31), 18966 - 72
In vitro reconstitution of transcriptional antitermination by the SacT and SacY proteins of Bacillus subtilis; Arnaud M et al.; Expression of the sacPA and sacB genes of Bacillus subtilis is positively modulated by transcriptional regulatory proteins encoded by the sacT and sacY genes, respectively . Previous genetic studies led to the suggestion that SacT and SacY function as nascent mRNA binding proteins preventing early termination of transcription at terminators located in the leader regions of the corresponding genes . Here we report the overproduction, purification to near homogeneity, and characterization of the two antiterminators, SacT and SacY . Using mRNA band migration retardation assays and a reconstituted transcriptional antitermination system, the mRNA binding functions and antitermination activities of purified SacT and SacY are demonstrated under in vitro conditions . The results establish for the first time that members of the BglG family of antiterminators function in antitermination in the absence of other proteins in vitro . Purified SacT is shown to be phosphorylated by phosphoenolpyruvate in a phosphotransferase-catalyzed reaction dependent on Enzyme I and HPr . Unexpectedly, the purified SacT is shown to be functional in mRNA binding and in transcriptional antitermination independently of its phosphorylation state.

Biosci Biotechnol Biochem, 1996 Aug, 60(8), 1255 - 9
Cloning, sequencing, and expression of a beta-amylase gene from Bacillus cereus var . mycoides and characterization of its products; Yamaguchi T et al.; The cloned gene was composed of 1638 bp for coding plus promoter like and SD-like sequences ahead of it . The deduced amino acid sequence had high similarity with known beta-amylases . The N-terminal sequence of the cloned beta-amylase seemed to be a signal peptide . The gene was introduced into Bacillus subtilis 1A289 using pHY300PLK as a vector and the expressed protein was recovered from the culture media . The enzyme fraction produced was divided into two components upon the DEAE column chromatography . The amino acid sequence of one fraction (FrI) was the same as the mature enzyme, and the other (FrII) lacked the N-terminal amino acid residue (Ala) of the mature enzyme . The kinetic parameters of the hydrolysis catalyzed by the enzyme component FrI were measured, and the subsite affinities of the enzyme were evaluated . In conclusion, it was shown that the recombinant enzyme was the same as the mature enzyme functionally and proteochemically.

Appl Microbiol Biotechnol, 1996 Aug, 46(1), 55 - 60
Pulse-field gel-electrophoretic analysis of the amplification and copy-number stability of an integrational plasmid in Bacillus subtilis; Vazquez-Cruz C et al.; The stability of integration and amplification of an integrational plasmid in Bacillus subtilis was analyzed . A cat-containing plasmid was constructed that could be integrated into the amy locus to facilitate measurement of excision events . Pulse-field gel electrophoresis was used to measure the copy number in strains that were resistant to different levels of chloramphenicol . The stability of the amplified unit in strains containing from 2 to 18 tandem copies of the amplicon in the presence and absence of chloramphenicol and through different generation times was then determined . Our results demonstrate that, for any given strain, the copy number of the amplicon remains stable . Furthermore, this stability is maintained when a clone containing an amplicon of defined size is cultured through as many as 100 generations in the absence of selective pressure.

Mol Microbiol, 1996 Aug, 21(4), 763 - 75
Regulatory inputs for the synthesis of ComK, the competence transcription factor of Bacillus subtilis; Hahn J et al.; Competence in Bacillus subtilis is expressed post-exponentially in response to signals which are interpreted by a complex network of regulatory proteins . This network culminates in the transcriptional activation of a set of late-competence proteins that mediate DNA binding and uptake during transformation . ComK, a protein that binds to competence promoters and appears to activate their transcription, is itself synthesized in response to the signal-transduction network . ComK is known to be required for the transcription of its own gene . We have placed comK under control of the xylose-inducible PxylA promoter and used this construct to show that ComK synthesis is sufficient as well as necessary to induce competence . We have also confirmed that the Mec proteins act post-transcriptionally to inactivate ComK, probably by protein-protein interaction . We have further demonstrated that ComS is required to generate an upstream signal that causes reversal of Mec-induced inactivation of ComK . In addition to ComK itself, DegU, AbrB, and SinR are required for comK transcription; mutations in their genes are bypassed by PxylA-comK induction, and therefore their products appear not to act via the Mec proteins . Overproduction of ComK, in a loss-of-function mec mutant, is also known to bypass the need for DegU, SinR and AbrB . We propose that these proteins enhance the activity of ComK as a positive autoregulatory transcription factor, acting as coactivator proteins when ComK is present at low concentrations . Finally, we demonstrate that when ComK is synthesized from the PxylA promoter and mecA is inactivated by mutation, no additional growth-stage-regulated control of competence can be detected.

Yeast, 1996 Aug, 12(10), 953 - 63
The targeting of Bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part; Scotti PA et al.; We compared the ability of signal sequences from various Bacillus or yeast secreted proteins to direct Bacillus subtilis levansucrase into the secretion pathway of the yeast Saccharomyces cerevisiae . The efficiency of these sequences correlated with the overall hydrophobicity of their h-domain and was independent of their origin . Furthermore, the net charge of the proximal protein sequence downstream from the signal sequence contributed to the competence of the heterologous proteins to be secreted by yeast . Modification of this net charge allowed the protein to be translocated under the control of the yeast invertase signal sequence . Moreover, glycosylation of levansucrase did not modify significantly the fructosyl polymerase activity.

Mol Microbiol, 1996 Aug, 21(3), 511 - 8
CheC and CheD interact to regulate methylation of Bacillus subtilis methyl-accepting chemotaxis proteins; Rosario MM et al.; In this study, we have demonstrated that two unique proteins in Bacillus subtilis chemotaxis, CheC and CheD, interact . We have shown this interaction both by using the yeast two-hybrid system and by precipitation of in vitro translated products using glutathione-S-transferase fusions and glutathione agarose beads . We have also shown that CheC inhibits B . subtilis CheR-mediated methylation of B . subtilis methyl-accepting chemotaxis proteins (MCPs) but not of Escherichia coli MCPs . It was previously reported that cheC mutants tend to swim smoothly and do not adapt to addition of attractant; cheD mutants have very poorly methylated MCPs and are very tumbly, similar to cheA mutants . We hypothesize that CheC exerts its effect on MCP methylation in B . subtilis by controlling the binding of CheD to the MCPs . In absence of CheD, the MCPs are poor substrates for CheR and appear to tie up, rather than activate, CheA . The regulation of CheD by CheC may be part of a unique adaptation system for chemotaxis in B . subtilis, whereby high levels of CheY-P brought about by attractant addition would allow CheC to interact with CheD and consequently leave the MCPs, reducing CheA activity and hence the levels of CheY-P.

Mol Microbiol, 1996 Aug, 21(3), 501 - 9
The Bacillus subtilis soj-spo0J locus is required for a centromere-like function involved in prespore chromosome partitioning; Sharpe ME et al.; During sporulation in Bacillus subtilis a small prespore cell is formed by an asymmetric cell division . Pre-spore chromosome partitioning occurs by a specialised mechanism in which septation precedes chromosome movement . We show that the spo0J gene is needed to specify the orientation of the chromosome at the time of polar division and to impose directionality on the subsequent transport of the remainder of the chromosome through the septum . Both phenotypes may arise by disruption of a centromere-like apparatus that anchors the or/C region of the prespore chromosome in the pole of the cell.

Eur J Biochem, 1996 Aug 1, 239(3), 773 - 81
Analysis of global responses by protein and peptide fingerprinting of proteins isolated by two-dimensional gel electrophoresis . Application to the sulfate-starvation response of Escherichia coli; Quadroni M et al.; A set of 8 proteins (SSI, sulfate-starvation-induced proteins) was observed by comparative two-dimensional electrophoresis to be induced when Escherichia coli were grown using compounds other than sulfate or cysteine as the sole sulfur source . These proteins were isolated after two-dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry . The list of peptide masses served as a protein fingerprint which was used to search the databases, allowing four of the SSI proteins (SSI2, 5, 7, 8) to be identified with a high degree of confidence . To identify the other SSI proteins, and to obtain sequence information for as many of the proteins as possible, automated on-line HPLC MS/MS (fragmentation analysis using coupled mass scanning devices) data collection was performed . The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases . Genes encoding two further proteins (SSI 1 and 3) were identified in the 8.5' region of the Escherichia coli genome . N-terminal sequencing of all of the proteins confirmed the results of protein and peptide fingerprinting and in addition showed that SSI 6 shows 50% similarity to the Bacillus subtilis orfM gene product . SSI 4 was not found in the databases by any of these methods . The methods described are of general use for the rapid analysis of complex cell responses . MS data accumulation takes about 5 min/protein for protein fingerprinting and 30 min for peptide fingerprinting and requires approximately 100 fmol of material . N-terminal sequencing however, takes about 5 h/protein and approximately 1 pmol to obtain a 10 amino acid sequence for a search.

FEMS Microbiol Lett, 1996 Aug 1, 141(2-3), 129 - 37
A novel amidohydrolase gene from Bacillus subtilis cloning: DNA-sequence analysis and map position of amhX; Kempf B et al.; The nucleotide sequence of a new Bacillus subtilis gene (amhX) was determined that encodes a protein (AmhX) with strong sequence identity to amidohydrolases from both plant and bacterial species and a carboxypeptidase from the archaeon Sulfolobus sulfataricus . The amhX gene encodes a hydrophilic polypeptide of 383 amino acids with a molecular mass of 41.5 kDa . The amhX gene was overexpressed in E . coli by using the T7 RNA polymerase/promoter system and the transcription initiation sites for the amhX mRNAs in B . subtilis were determined by primer extension analysis . Chromosomal amhX mutations were constructed by marker replacement and the amhX gene was positioned at 25 degrees on the genetic and physical map of the B . subtilis chromosome.

Microbiology, 1996 Aug, 142 ( Pt 8), 2049 - 55
Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis; Salamitou S et al.; The Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes . Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes . Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background . We used lacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo- genetic backgrounds (spoOA, sigE and sigK) . cryIIIA was activated from the end of vegetative growth to stage II of sporulation (t3) in the wild-type strain . Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III . In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase . cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigma E mutant strain . Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors . Furthermore, an event linked with the thE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III.

Microbiology, 1996 Aug, 142 ( Pt 8), 2041 - 7
A Bacillus subtilis secreted phosphodiesterase/alkaline phosphatase is the product of a Pho regulon gene, phoD; Eder S et al.; A secreted phosphodiesterase/alkaline phosphatase, APaseD, was purified from a culture of Bacillus subtilis JH646MS . Its phosphodiesterase activity was reminiscent of an APase isolated and characterized previously . Immunoassay and N-terminal sequencing showed the two proteins to be identical . Using the first 20 amino acids of the mature protein, a BLAST search of GenBank was used to find an homologous sequence . An exact match was found but in a putative non-coding region . It was hypothesized that there was a base pair deletion in the phoD gene . A DNA fragment internal to the coding region was generated by PCR using template DNA from a strain which produced APaseD . The PCR fragment was cloned and used to interrupt the gene . Western blot analysis of the parent and the mutated strains showed that APaseD was missing in the mutant . Resequencing of the gene revealed a larger ORF encoding a protein similar in size to the 49 kDa APaseD estimated by SDS-PAGE . The promoter was then cloned, sequenced and used in phoD-lacZ promoter fusions which showed that the gene was phosphate-starvation-induced and dependent on PhoP and PhoR for expression.

Microbiology, 1996 Aug, 142 ( Pt 8), 2031 - 40
The phage-like element PBSX and part of the skin element, which are resident at different locations on the Bacillus subtilis chromosome, are highly homologous; Krogh S et al.; PBSX and skin are two unusual genetic elements resident on the Bacillus subtilis chromosome . PBSX is a phage-like element located at approximately 100 degrees which is induced by the SOS response and results in cell lysis with the release of phage-like particles . The phage particles contain bacterial chromosomal DNA and kill sensitive bacteria without injecting DNA . The skin element is located at approximately 230 degrees on the chromosome and is positioned within the sigK open reading frame (ORF) . It is excised at a particular stage of sporulation, leading to reconstitution of the complete sigK gene . In this paper, we show that there are phage-like operons present in the skin element which are highly homologous to the region of PBSX comprising part of the control region and the late operon . These operons are similar in terms of their gene organization, the percentage identity of the products of homologous ORFs and the positioning and strengths of ribosome-binding sites for each ORF . Although this high degree of conservation suggests that the phange-like operons in skin can be expressed, expression of the late operon was not detected during exponential growth, during sporulation or after induction of the SOS response . However two non-phage-like operons in the skin element are expressed and have distinct expression profiles that are dependent on the growth and developmental status of the cell.

Microbiology, 1996 Aug, 142 ( Pt 8), 2017 - 20
A Bacillus subtilis gene cluster similar to the Escherichia coli phosphate-specific transport (pst) operon: evidence for a tandemly arranged pstB gene; Takemaru K et al.; We have determined the complete nucleotide sequence of the Bacillus subtilis homologues of the Escherichia coli phosphate-specific transport (pst) genes in the framework of the international B . subtilis genome sequencing project . The pst genes in E . coli form an operon arranged in the order pstS, pstC, pstA, pstB and phoU . In the case of B . subtilis, there are also five ORFs presumably forming an operon . The deduced amino acid sequences of the products of these ORFs show striking similarities to their E . coli counterparts . Comparison of the organization of the pst operon of B . subtilis with that of E . coli revealed that the gene corresponding to phoU is missing, while there are two genes homologous to pstB in B . subtilis . The pst operon is located at 222 degrees on the B . subtilis chromosome.

Microbiology, 1996 Aug, 142 ( Pt 8), 2005 - 16
Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome; Sorokin A et al.; The standard strategies of genome sequencing based on lambda-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli . One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis, established by using yeast artificial chromosomes (YACs) . This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it . The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs . The complete 104,109 bp insert sequence of this YAC clone was thus established . The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13,695 and 20,303 bp) of the YAC insert . We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning, as well as for larger inserts of eukaryotic origin cloned in YACs . Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg . The organization of the newly determined region is presented . Of the 138 ORFs identified in the spollA-kdg region, 57 have no clear putative function from their homology to proteins in the databases.

J Bacteriol, 1996 Aug, 178(16), 5039 - 41
Identification of additional genes under the control of the transcription factor sigma F of Bacillus subtilis; Decatur A et al.; We describe the identification of five transcriptional units under the control of the sporulation transcription factor sigma F in Bacillus subtilis . These are csfA, csfB, csfC, csfD, and csfF, located at approximately 230 degrees, 2 degrees, 316 degrees, 205 degrees, and approximately 290 degrees, respectively, on the genetic map . Null mutations in csfA, csfB, csfC, or csfD, either alone or together, do not cause a noticeable defect in sporulation or germination.

J Bacteriol, 1996 Aug, 178(16), 5013 - 6
Identification of a novel gene of pyrimidine nucleotide biosynthesis, pyrDII, that is required for dihydroorotate dehydrogenase activity in Bacillus subtilis; Kahler AE et al.; An in-frame deletion in the coding region of a gene of previously unidentified function (which is called orf2 and which we propose to rename pyrDII) in the Bacillus subtilis pyr operon led to pyrimidine bradytrophy, markedly reduced dihydroorotate dehydrogenase activity, and derepressed levels of other enzymes of pyrimidine biosynthesis . The deletion mutation was not corrected by a plasmid encoding pyrDI, the previously identified gene encoding dihydroorotate dehydrogenase, but was complemented by a plasmid encoding pyrDII . We propose that pyrDII encodes a protein subunit of dihydroorotate dehydrogenase that catalyzes electron transfer from the pyrDI-encoded subunit to components of the electron transport chain.

J Bacteriol, 1996 Aug, 178(16), 4984 - 9
SpoIIE mutants of Bacillus subtilis comprise two distinct phenotypic classes consistent with a dual functional role for the SpoIIE protein; Barak I et al.; Mutations in the spoIIE locus of Bacillus subtilis block sporulation at the stage of asymmetric septation and prevent compartment-specific activation of the transcription factor delta F . Recent ultrastructural studies of spoIIE mutants led to the conclusion that cells blocked at the stage of asymmetric septation form primarily thick septal structures similar to those formed at the mid-cell site during vegetative growth, although in an earlier study Piggot (J . Bacteriol . 114:1241-1253, 1973) clearly detected a more complex range of phenotypes . We have examined the phenotypes of six spoIIE mutants, including one example of the previously studied null type, spoIIE21 . We confirmed that the spoIIE21 mutant and two other null mutants exhibit the classic thick-septum phenotype . However, two of the missense mutants, the spoIIE64 and spoIIE71 mutants, were found to display a strikingly different phenotype characterized by the presence of only thin asymmetric septa, frequently at both polar positions, as noted by Piggot . This phenotype is essentially identical to those of spoIIA (delta F) and spoIIG (delta E) null mutants, which also form sporulation septa that appear structurally normal at the level of electron microscopy . Despite the formation of apparently normal asymmetric septa, spoIIE64 and spoIIE71 mutants are fully defective in activation of delta F-dependent gene expression . These results indicate that the functional roles performed by SpoIIE in septum assembly and sigma factor regulation are distinct and separable.

J Bacteriol, 1996 Aug, 178(16), 4935 - 41
ADP-ribosylation of proteins in Bacillus subtilis and its possible importance in sporulation; Huh JW et al.; Endogenous ADP-ribosylation was detected in Bacillus subtilis, as determined in vitro with crude cellular extracts . The ADP-ribosylated protein profile changed during growth in sporulation medium, displaying a temporary appearance of two ADP-ribosylated proteins (36 and 58 kDa) shortly after the end of exponential growth . Mutants resistant to 3-methoxybenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained, and a significant proportion (15%) were found to be defective in both sporulation and antibiotic production . These mutants failed to ADP-ribosylate the 36- and 58-kDa proteins . The parent strain also lost the ability to ADP-ribosylate these proteins when grown in the presence of 3-methoxybenzamide at a concentration at which sporulation but not cell growth was severely inhibited . Results from genetic transformations showed that the mutation conferring resistance to 3-methoxybenzamide, named brgA, was cotransformed with the altered phenotypes, i.e., defects in ADP-ribosylation and sporulation . spoOA and spoOF mutants displayed an ADP-ribosylation profile similar to that of the parent strain, but a spoOH mutant failed to ADP-ribosylate any proteins, including the 36- and 58-kDa proteins . The significance of protein ADP-ribosylation in sporulation was further indicated by the observation that ADP-ribosylation of the 36-kDa protein could be induced by treatment with decoyinine, an inhibitor of GMP-synthetase, and by amino acid limitation, both of which resulted in an immediate decrease in GTP pool size eventually leading to massive sporulation . We propose that a new sporulation gene, which presumably controls sporulation via ADP-ribosylation of certain functional proteins, exists.

J Bacteriol, 1996 Aug, 178(16), 4861 - 9
Effects of mecA and mecB (clpC) mutations on expression of sigD, which encodes an alternative sigma factor, and autolysin operons and on flagellin synthesis in Bacillus subtilis; Rashid MH et al.; The expression of the major vegetative phase-specific autolysin genes (cwlB {lytC} and cwlG {lytD}) was greatly reduced by mecA and mecB null mutations . In contrast to the negative effects on late competence genes (such as comG) and levansucrase gene (sacB) expression, this positive effect of mec genes on autolysin gene expression was not mediated through the ComK protein but apparently through the level of the SigD protein . The pleiotropic effects of the mec mutations, i.e., the reduction of sigD expression and the overexpression of the ComK protein, seem not to be interwoven since the SigD- and ComK-dependent functions are clearly separable in the mec mutants . We also show that the synthesis of the flagellin protein, which is encoded by the SigD-dependent hag gene, was similarly affected by the mec mutations . Complementation analysis with a SigD-overproducing plasmid, pHYSigD, in mec mutants revealed the reversion of almost all of the SigD-dependent phenotypes except motility . This finding suggested that Mec proteins act on motility genes at two levels, one of which is apparently SigD independent . Finally, we discuss the transcriptional regulation of the sigD gene by multiple regulators, i.e., MecA, MecB, SinR (FlaD), and DegS-DegU, and its implications for cells in a global context.

J Bacteriol, 1996 Aug, 178(16), 4794 - 800
Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes; Morbidoni HR et al.; A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase . The B . subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP) . Also present is a sequence that encodes a homolog of E . coli plsX, a gene that plays a poorly understood role in phospholipid synthesis . The B . subtilis plsX gene weakly complemented an E . coli plsX mutant . The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E . coli, except that in E . coli the fabH gene lies between plsX and fabD . The absence of fabH in the B . subtilis cluster is consistent with the different fatty acid compositions of the two organisms . The amino acid sequence of B . subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E . coli acyl carrier protein, except that most of the protein retained the initiating methionine residue . The B . subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome.

J Bacteriol, 1996 Aug, 178(16), 4778 - 86
A sigma E dependent operon subject to catabolite repression during sporulation in Bacillus subtilis; Bryan EM et al.; To identify genes expressed at intermediate stages of Bacillus subtilis sporulation, we screened for sigma E-dependent promoters . One promoter that we found drives expression of an operon consisting of at least five open reading frames (ORFs) . The predicted products of the first three ORFs are very homologous to enzymes involved in fatty acid metabolism, including acetyl coenzyme A (acetyl-CoA) acetyltransferase (thiolase), 3-hydroxybutyryl-CoA dehydrogenase, and acyl-CoA dehydrogenase, respectively . We showed that the fourth ORF encoded a third isozyme of citrate synthase in B . subtilis . Genetic evidence and primer extension results showed that transcription of this operon is directed by the mother cell compartment-specific sigma factor, sigma E, and so the operon was named mmg (for mother cell metabolic genes) . Furthermore, we found that a sequence (mmgO) with homology to a catabolite-responsive element mediates glucose repression of mmg promoter activity during sporulation and that this repression was lost in a ccpA mutant.

J Bacteriol, 1996 Aug, 178(15), 4611 - 9
Cold shock stress-induced proteins in Bacillus subtilis; Graumann P et al.; Bacteria respond to a decrease in temperature with the induction of proteins that are classified as cold-induced proteins (CIPs) . Using two-dimensional gel electrophoresis, we analyzed the cold shock response in Bacillus subtilis . After a shift from 37 to 15 degrees C the synthesis of a majority of proteins was repressed; in contrast, 37 proteins were synthesized at rates higher than preshift rates . One hour after cold shock, the induction of CIPs decreased, and after 2 h, general protein synthesis resumed . The identified main CIPs were excised from two-dimensional gels and were subjected to microsequencing . Three small acidic proteins that showed the highest relative induction after cold shock were highly homologous and belonged to a protein family of which one member, the major cold shock protein, CspB, has previously been characterized . Two-dimensional gel analyses of a cspB null mutant revealed that CspB affects the level of induction of several CIPs . Other identified CIPs function at various levels of cellular physiology, such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, Ilvc, Gap, and triosephosphate isomerase).

J Bacteriol, 1996 Aug, 178(15), 4604 - 10
Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis; Yocum RR et al.; The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR . The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases . The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1 . Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine . Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B . subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B . subtilis, as others have already shown for Escherichia coli.

J Bacteriol, 1996 Aug, 178(15), 4576 - 81
Expression of the tre operon of Bacillus subtilis 168 is regulated by the repressor TreR; Schock F et al.; The tre locus from Bacillus subtilis containing the genes treP, treA, and treR has been analyzed for its regulation . We demonstrate that at least treP and treA form an operon whose expression is regulated at the transcriptional level . TreR activity has been investigated in in vivo and in vitro studies . An insertional inactivation of treR led to a constitutive expression of treP and treA . Upstream of treP we identified a 248-bp DNA fragment containing a potential sigmaA-dependent promoter and two palindromes reflecting potential tre operators which led to complex formation with TreR-containing protein extracts in DNA retardation experiments . This complex formation is abolished in the presence of trehalose-6-phosphate, which probably acts as an inducer . Therefore, we assume that treR encodes the specific Tre repressor involved in regulation of the expression of the tre operon.

J Bacteriol, 1996 Aug, 178(15), 4500 - 7
A compartmentalized regulator of developmental gene expression in Bacillus subtilis; Bagyan I et al.; We have identified a new Bacillus subtilis gene, spoVT, whose gene product is homologous to the transcriptional regulator AbrB and serves as a regulator of E sigmaG-controlled gene expression . SpoVT acts both positively and negatively in controlling sigmaG-dependent gene expression, providing an additional level of refinement to forespore gene regulation and feedback control of spoIIIG expression.

J Bacteriol, 1996 Aug, 178(15), 4375 - 80
Bacillus subtilis spore coat assembly requires cotH gene expression; Naclerio G et al.; Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer . We report the identification and characterization of a gene, herein called cotH, located at 300 degrees on the B . subtilis genetic map between two divergent cot genes, cotB and cotG . The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa . Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination . The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes . On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.

J Mol Biol, 1996 Jul 19, 260(3), 446 - 66
A concerted tryptophanyl-adenylate-dependent conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase revealed by the fluorescence of Trp92; Hogue CW et al.; A semi-conserved tryptophan residue of Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNATrp binding and recognition . The crystal structure of the Bacillus stearothermophilus TrpRS tryptophanyl-5'-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation . Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy . B . subtilis TrpRS has a single tryptophan residue, Trp92 . 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5'-adenylate (4FW-AMP) TrpRS complex . Complexation causes the Trp92 fluorescence to become quenched by 70% . Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled . This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes . Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form . From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNATrp is unlikely . Time-resolved fluorescence indicates conformational heterogeneity of B . subtilis Trp92 with the fluorescence decay being best described by three discrete exponential decay times . The decay-associated spectra (DAS) of the apo- and complexed-TrpRS show large variations of the concentration of individual fluorescence decay components . Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure . The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an alpha-helix . The amino acid residue cysteine is observed clearly within the quenching radius (3.6 angstroms) of the conserved tryptophan residue . These tryptophan and cysteine residues are neighbors, one helical turn apart . If this local alpha-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues . Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92 . A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small alpha-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit . This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding . We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.

J Mol Biol, 1996 Jul 19, 260(3), 369 - 77
In vitro protein-primed initiation of pneumococcal phage Cp-1 DNA replication occurs at the third 3' nucleotide of the linear template: a stepwise sliding-back mechanism; Martin AC et al.; Phage Cp-1 from Streptoccocus pneumoniae makes use of a protein-priming mechanism to start replication of its linear DNA: the first reaction consists of the addition of 5' dAMP to a molecule of the primer protein, an initiation event occurring at both DNA ends . After elongation of the initiation complex, the primer protein remains linked to the 5' end of the nascent DNA chain, and is subsequently referred to as terminal protein (TP) . In this paper, using DNA-free extracts from Cp-1-infected S . pneumoniae, we provide evidence that the formation of the covalent complex TP-dAMP is a template-instructed reaction and that ssDNA molecules can serve as templates for TP-primed replication . A mutational analysis of the 3' terminal nucleotides of Cp-1 DNA reveals that a precise DNA sequence is required for efficient template recognition, and that in vitro initiation of Cp-1 DNA replication is directed by the third nucleotide of the template . However, the two terminal nucleotides are recovered during the first steps of elongation . A new variant of the sliding-back mechanism for protein-primed initiation, firstly described for Bacillus subtilis phage phi29, is proposed to account for the maintenance of Cp-1 DNA ends . The results presented here reinforce the hypothesis that sliding-back must be a common feature in all genomes that use protein-priming to initiate replication.

J Mol Biol, 1996 Jul 12, 260(2), 196 - 206
On the connection between inherent DNA flexure and preferred binding of hydroxymethyluracil-containing DNA by the type II DNA-binding protein TF1; Grove A et al.; TF1 is a member of the family of type II DNA-binding proteins, which also includes the bacterial HU proteins and the Escherichia coli integration host factor (IHF) . Distinctive to TF1, which is encoded by the Bacillus subtilis bacteriophage SPO1, is its preferential binding to DNA in which thymine is replaced by 5-hydroxymethyluracil (hmU), as it is in the phage genome . TF1 binds to preferred sites within the phage genome and generates pronounced DNA bending . The extent to which DNA flexibility contributes to the sequence-specific binding of TF1, and the connection between hmU preference and DNA flexibility has been examined . Model flexible sites, consisting of consecutive mismatches, increase the affinity of thymine-containing DNA for TF1 . In particular, tandem mismatches separated by nine base-pairs generate an increase, by orders of magnitude, in the affinity of TF1 for T-containing DNA with the sequence of a preferred TF1 binding site, and fully match the affinity of TF1 for this cognate site in hmU-containing DNA (Kd approximately 3 nM) . Other placements of loops generate suboptimal binding . This is consistent with a significant contribution of site-specific DNA flexibility to complex formation . Analysis of complexes with hmU-DNA of decreasing length shows that a major part of the binding affinity is generated within a central 19 bp segment (delta G0 = 41.7 kJ mol-1) with more-distal DNA contributing modestly to the affinity (delta delta G = -0.42 kJ mol-1 bp-1 on increasing duplex length to 37 bp) . However, a previously characterised thermostable and more tightly binding mutant TF1, TF1(E15G/T32I), derives most of its extra affinity from interaction with flanking DNA . We propose that inherent but sequence-dependent deformability of hmU-containing DNA underlies the preferential binding of TF1 and that TF1-induced DNA bendings is a result of distortions at two distinct sites separated by 9 bp of duplex DNA.

J Mol Biol, 1996 Jul 12, 260(2), 165 - 77
Role of adenosine nucleotides in the regulation of a stress-response transcription factor in Bacillus subtilis; Alper S et al.; The RNA polymerase sigma factor sigma B is a stress-response regulatory protein in Bacillus subtilis . The activity of sigma B is controlled in part by RsbW, a protein that inhibits sigma B, and RsbV, a protein that counteracts this inhibition . We now demonstrate that purified RsbW is capable of forming alternative complexes with either sigma B or RsbV . Sigma B in the RsbW . sigma B complex was transcriptionally inactive . RsbV reversed this inhibition by sequestering RsbW in a RsbW-RsbV complex, thereby allowing sigma B to remain free and active . In contrast to interactions among the components of the homologous regulatory system for the sporulation transcription factor sigma F, the binding of RsbW to RsbV and sigma B did not require adenosine nucleotides . Experiments involving the exchange of proteins between the two regulatory systems demonstrated that RsbW and its homolog in the sigma F system, SpoIIAB, exhibit strong preference in binding to RsbV and sigma B, and SpoIIAA and sigma F, respectively, and that the difference in nucleotide-dependence of binding between these two systems is attributable to a difference between RsbW and SpoIIAB . In confirmation and extension of previous results, we show that RsbW is also a protein kinase that uses ATP to phosphorylate RsbV, thereby blocking the capacity of RsbV to bind to RsbW and activate transcription . A close correlation was observed between the concentration of ATP required for efficient RsbW-mediated phosphorylation of RsbV, inhibition of RsbW.RsbV comlex formation, and inhibition of sigma B-directed transcription . These results are consistent with the hypothesis that activation of sigma B under certain stress condition is due to a decrease in cellular ATP levels.

J Mol Biol, 1996 Jul 12, 260(2), 147 - 64
SpoIIAA governs the release of the cell-type specific transcription factor sigma F from its anti-sigma factor SpoIIAB; Duncan L et al.; The Bacillus subtilis transcription factor sigma F is a cell-type specific regulatory protein whose activity is governed by SpoIIAB and SpoIIAA and the nucleotides ATP and ADP . SpoIIAB is an anti-sigma factor that binds to sigma F in a manner that is stimulated by ATP, thereby trapping sigma F in an inactive complex . Alternatively, SpoIIAB binds to SpoIIAA in a manner that is stimulated by ADP to form a SpoIIAB.SpoIIAA complex in which SpoIIAB is sequestered from sigma F . SpoIIAB is also a protein kinase that uses ATP to phosphorylate, and thereby inactivate, SpoIIAA . Thus, ATP inhibits sigma F activity both by promoting formation of the SpoIIAB.sigma F complex and by phosphorylation of SpoIIAA . In extension of previous results, we use affinity chromatography to show that SpoIIAB is capable of forming long-lived complexes with sigma F and SpoIIAA and that the formation of these complexes is dependent on ATP and ADP, respectively . Using a DNA template lacking adenosine residues on the non-transcribed strand, we demonstrate that ATP is required for SpoIIAB-mediated inhibition of sigma F-directed RNA synthesis and that this inhibition is prevented by SpoIIAA in a manner that is stimulated by ADP . We show that ADP acts by protecting SpoIIAA from phosphorylation by SpoIIAB and that a mutant protein bearing an amino acid substitution at the site of phosphorylation in SpoIIAA is capable of preventing the inhibition of sigma F in a manner that does not depend on ADP . A principal finding from the investigation is that SpoIIAA restores activity to sigma F that had previously been inhibited by SpoIIAB . This is demonstrated both by the capacity of SpoIIAA to reverse SpoIIAB-mediated inhibition of sigma F-directed RNA synthesis and by its capacity to interact with and disrupt the SpoIIAB . sigma F complex . The results are consistent with a model in which sigma F is controlled by the cellular concentration of unphosphorylated SpoIIAA.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6992 - 7
Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis; Condon C et al.; The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA . Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region . The processed RNA is significantly more stable than the full-length transcript . Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript . However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form . This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions . The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination . Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.

FEBS Lett, 1996 Jul 8, 389(3), 281 - 4
The secG deletion mutation of Escherichia coli is suppressed by expression of a novel regulatory gene of Bacillus subtilis; Kontinen VP et al.; SecG, a membrane component of E . coli protein translocase, stimulates the translocation of proteins across the cell membrane through the cycle of topology inversion, which is coupled to the membrane-insertion and deinsertion cycle of SecA {Nishiyama et al . (1996) Cell 85, 71-81} . A gene of B . subtilis able to suppress the cold-sensitive phenotype of the secG deletion mutant of E . coli was cloned and found to encode a novel regulatory protein, ScgR . Similarity search revealed homology with known proteins such as GlnR of B . subtilis . Plasmid-encoded ScgR stimulated protein translocation in the deletion mutant . ScgR increased the proportion of cardiolipin at the expense of phosphatidylglycerol, but did not affect the composition of other lipid components of the cell, suggesting that the increased cardiolipin level compensates for the SecG function and thereby stimulates protein translocation.

J Mol Biol, 1996 Jul 5, 260(1), 54 - 69
The Bacillus subtilis DNA replication terminator; Smith MT et al.; The recent discovery of the Bacillus subtilis plasmid terminator TerLS20 with bidirectional fork arrest activity has provided the opportunity to probe further the structural and functional features of B . subtilis replication terminators in general . The minimal TerI and TerLS20 terminators each comprise two 13 nt segments flanking a central trinucleotide, which is almost completely conserved in all terminators . It corresponds to the region of overlap of the two RTP binding sites (A and B) on the DNA . It has been shown that, despite this conservation, considerable variation in this trinucleotide region still allows fork arrest activity . Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region, is not dependent upon stringently defined contacts with the bases in this region . A completely synthetic and highly symmetrical terminator was constructed by replacing the 13 nt segment of the A site of TerI with an opposed segment identical to that in the B site . The efficient bidirectional activity of this new terminator, TerSymB, established more firmly the need for two opposed RTP binding sites in a functional terminator . TerSymB was used to investigate the effect of sequence deviation in one of the 13 nt segments, from that in the B site, on bidirectionality of the terminator . It was found that the deviations introduced converted the terminator significantly towards polarity of action . The partial symmetry within each of the 13 nt segments of TerSymB, and the presumed recognition of this symmetry in the binding of a symmetrical dimer of RTP to each overlapping site, suggest that the bound dimers are centred over positions in the DNA sequence separated by 15 nt . This separation distance has been used in conjunction with the mode of binding of RTP to DNA proposed by Bussiere et al., based on their crystal structure for RTP, to model the interaction of the two dimers of RTP with unbent B-form DNA . Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucleotides centrally within the TerSymB sequence . The effects of these insertions on RTP binding and fork arrest activity were consistent with the proposed positioning of the RTP dimers within the terminator sequence, and interaction between the dimers bound to TerSymB . A model to account for the generation of RTP-terminator complexes with bidirectional or polar fork arrest activity utilising TerSymB or TerI-VI is presented.

IEEE Trans Biomed Eng, 1996 Jul, 43(7), 752 - 4
Effect of high-power microwave on indicator bacteria for sterilization; Wu Q; According to the superiority sterilization, a specially-designed microwave disinfector using high-power microwave energy was made . A series of sterilizing experiments have been made to determine the effect of microwave energy on several typical indicator bacteria such as Bacillus subtilis var . niger, Bacillus stearothermophilus, Bacillus pumilus E601, Staphylococcus aureus, Bacillus cereus . Under the conditions of different sterilization duration and unequal intensity of microwave power irradiation onto the bacteria, a useful result of killing bacteria has been observed, i.e., the Bacillus subtilis can be considered as an optimum indicator bacterium for microwave sterilization.

Microb Drug Resist, 1996 Summer, 2(2), 201 - 7
Glutamine synthetase and heteroresistance in methicillin-resistant Staphylococcus aureus; Stranden AM et al.; Inactivation of femC in methicillin-resistant Staphylococcus aureus (MRSA) results in lowered methicillin resistance and a reduction in the amidation of the iso-D-glutamate of the peptidoglycan stem peptide . The femC phenotype is due to insertional inactivation of the glutamine synthetase repressor gene glnR by Tn551, which has a polar effect on glutamine synthetase (glnA) transcription . The complete glutamine synthetase operon (glnRA) of S . aureus was cloned and sequenced, and its transcriptional start was determined . The deduced amino acid sequence of the staphylococcal glutamine synthetase showed 76% identity and 87% similarity to the Bacillus subtilis glutamine synthetase . The staphylococcal glnRA operon was shown to complement an Escherichia coli glutamine synthetase-negative mutant and to restore methicillin resistance in femC mutants . femC mutants revert to resistance in the presence of high concentrations of methicillin . These revertants, which still carried the femC lesion, were shown to retain the lowered amidation of the iso-D-glutamate peptidoglycan stem peptide . A new chromosomal locus hmrC was postulated to have mutated to allow expression of high methicillin resistance in these femC revertants . Although the highly resistant hmrC revertant resembled phenotypically the highly methicillin-resistant subclones occurring in heterogeneously resistant MRSA, we could show by transduction that the locus hmrC was distinct from chr*, a chromosomal site postulated to confer high methicillin resistance in heterogeneous MRSA . This suggests that S . aureus can adopt multiple ways to achieve high methicillin resistance.

Protein Eng, 1996 Jul, 9(7), 591 - 601
Novel features of serine protease active sites and specificity pockets: sequence analysis and modelling studies of glutamate-specific endopeptidases and epidermolytic toxins; Barbosa JA et al.; With a view to obtaining a better understanding of the structural determinants of P1 glutamate specificity in glutamate-specific endopeptidases (GSEs), the active sites and specificity pockets of such enzymes from Bacillus licheniformis (gse-bl), Bacillus subtilis (mpr) and Staphylococcus aureus (v8 protease) were modelled . This approach was extended to the epidermolytic toxins (ETs), responsible for the staphylococcal scalded skin syndrome . We identify a canonical structure for the S1 subsite, composed of H213 and T190, both of which we predict to interact directly with the P1 glutamate . The possible importance of R30 (for gse-bl and mpr) and of the N-terminus (for gse-bl, mpr and v8 protease) was also examined . In the case of mpr, a G193C substitution is predicted to participate in a novel disulphide bridge which stabilizes C193 in such a way as to maintain the oxyanion hole . In v8, the loss or substitution of several important structural components around D102 of the catalytic triad probably explains its reduced catalytic efficiency in comparison with other GSEs . In the case of the epidermolytic toxins K216 may be important for the previously reported phospholipase C-like activity, since the model predicts that it may stabilize the negative charge on the phosphonyl group.

Arzneimittelforschung, 1996 Jul, 46(7), 701 - 4
In vitro studies of the loss of antibacterial activity of oxytetracycline in presence of Ca(II) or Mg(II) ions; Naz S et al.; The results of a comparative study, which evaluated the in vitro effect on the antibacterial activity of oxytetracycline (OTC, CAS 79-57-2) in presence of Ca(II)/Mg(II) ions suggest that susceptibility of Staphylococcus aureus, Bacillus pumilis and Bacillus subtilis to OTC is reduced in presence of Ca(II)/Mg(II) ions . As the ratio of concentration of Ca(II)/Mg(II) to OTC was increased, antibacterial activity of OTC declined . In addition to the difference observed between the antibacterial effect of pure OTC and its Ca(II)/Mg(II) complexes, it was found that decline in antibacterial activity is greater for Mg(II)-OTC complex than Ca(II)-OTC complex for the same concentration of Ca(II)/Mg(II) ions.

Appl Environ Microbiol, 1996 Jul, 62(7), 2221 - 7
The two major spore DNA repair pathways, nucleotide excision repair and spore photoproduct lyase, are sufficient for the resistance of Bacillus subtilis spores to artificial UV-C and UV-B but not to solar radiation; Xue Y et al.; Bacterial endospores are 1 to 2 orders of magnitude more resistant to 254-nm UV (UV-C) radiation than are exponentially growing cells of the same strain . This high UV resistance is due to two related phenomena: (i) DNA of dormant spores irradiated with 254-nm UV accumulates mainly a unique thymine dimer called the spore photoproduct (SP), and (ii) SP is corrected during spore germination by two major DNA repair pathways, nucleotide excision repair (NER) and an SP-specific enzyme called SP lyase . To date, it has been assumed that these two factors also account for resistance of bacterial spores to solar UV in the environment, despite the fact that sunlight at the Earth's surface consists of UV-B, UV-A, visible, and infrared wavelengths of approximately 290 nm and longer . To test this assumption, isogenic strains of Bacillus subtilis lacking either the NER or SP lyase DNA repair pathway were assayed for their relative resistance to radiation at a number of UV wavelengths, including UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight, and sunlight from which the UV-B portion had been removed . For purposes of direct comparison, spore UV resistance levels were determined with respect to a calibrated biological dosimeter consisting of a mixture of wild-type spores and spores lacking both DNA repair systems . It was observed that the relative contributions of the two pathways to spore UV resistance change depending on the UV wavelengths used in a manner suggesting that spores irradiated with light at environmentally relevant UV wavelengths may accumulate significant amounts of one or more DNA photoproducts in addition to SP . Furthermore, it was noted that upon exposure to increasing wavelengths, wild-type spores decreased in their UV resistance from 33-fold (UV-C) to 12-fold (UV-B plus UV-A sunlight) to 6-fold (UV-A sunlight alone) more resistant than mutants lacking both DNA repair systems, suggesting that at increasing solar UV wavelengths, spores are inactivated either by DNA damage not reparable by the NER or SP lyase system, damage caused to photosensitive molecules other than DNA, or both.

J Bacteriol, 1996 Jul, 178(14), 4319 - 22
Identification, sequence, and expression of the gene encoding gamma-glutamyltranspeptidase in Bacillus subtilis; Xu K et al.; The Bacillus subtilis gene encoding gamma-glutamyltranspeptidase (GGT) activity encodes a protein of 587 amino acids having extensive homologies with other procaryotic GGTs . Inactivation of the gene abolished all measurable GGT activity, which in the wild type was found mainly to be excreted into the medium commencing at the end of vegetative growth.

J Bacteriol, 1996 Jul, 178(14), 4258 - 65
Replication fork arrest at relocated replication terminators on the Bacillus subtilis chromosome; Franks AH et al.; The replication terminus region of the Bacillus subtilis chromosome, comprising TerI and TerII plus the rtp gene (referred to as the terC region) was relocated to serC (257 degrees) and cym (10 degrees) on the anticlockwise- and clockwise-replicating segments of the chromosome, respectively . In both cases, it was found that only the orientation of the terC region that placed TerI in opposition to the approaching replication fork was functional in fork arrest . When TerII was opposed to the approaching fork, it was nonfunctional . These findings confirm and extend earlier work which involved relocations to only the clockwise-replicating segment, at metD (100 degrees) and pyr (139 degrees) . In the present work, it was further shown that in the strain in which TerII was opposed to an approaching fork at metD, overproduction of the replication terminator protein (RTP) enabled TerII to function as an arrest site . Thus, chromosomal TerII is nonfunctional in arrest in vivo because of a limiting level of RTP . Marker frequency analysis showed that TerI at both cym and metD caused only transient arrest of a replication fork . Arrest appeared to be more severe in the latter situation and caused the two forks to meet at approximately 145 degrees (just outside or on the edge of the replication fork trap) . The minimum pause time erected by TerI at metD was calculated to be approximately 40% of the time taken to complete a round of replication . This significant pause at metD caused the cells to become elongated, indicating that cell division was delayed . Further work is needed to establish the immediate cause of the delay in division.

J Bacteriol, 1996 Jul, 178(14), 4122 - 30
Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon; Bower S et al.; A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced . DNA sequence analysis indicated that B . subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes . These four genes and a homolog of the B . sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2 . bioI and orf2 show no similarity to any other known biotin biosynthetic genes . The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E . coli . Mutations in bioI caused B . subtilis to grow poorly in the absence of biotin . The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis . The B . subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B . sphaericus . Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B . subtilis birA gene.

Microbiology, 1996 Jul, 142 ( Pt 7), 1641 - 9
The genes of lepA and hemN form a bicistronic operon in Bacillus subtilis; Homuth G et al.; The IepA operon of Bacillus subtilis was found to be bicistronic and to consist of the two genes IepA and hemN, which encode a putative GTP-binding protein and an oxygen-independent coproporhyrinogen III oxidase, respectively . The IepA operon is located immediately upstream of the dnaK operon . Both operons are transcribed in the same direction and are not separated by an obvious transcription-terminator-like structure . The IepA operon is preceded by a potential vegetative promoter, and there is a putative strong intergenic terminator between IepA and hemN . Northern blot experiments revealed only a transcript corresponding to IepA, but expression of hemN was demonstrated in slot-blot and immunoblot experiments using antibodies raised against His-tagged HemN . The data suggest that most of the transcripts originating at the potential vegetative promoter are terminated at the intergenic terminator . Readthrough transcription into the downstream dnaK operon was not found.

Microbiology, 1996 Jul, 142 ( Pt 7), 1635 - 9
Lysine-induced premature transcription termination in the lysC operon of Bacillus subtilis; Kochhar S et al.; The expression of the Bacillus subtilis lysC operon, which encodes the first specific enzyme of lysine biosynthesis, is controlled by the availability of the end product, lysine . The question of whether lysine exerts its control by inducing premature termination of transcription was addressed using Northern blot analysis . Whereas lys-C-specific RNA from lysine-starved B . subtilis consisted primarily of the expected full-length mRNA (1.6 kb), that from bacteria grown with an excess of lysine consisted of a truncated 0.27 kb RNA in place of the full-length 1.6 kb transcript . On the other hand, a B . subtilis aecA mutant, in which the lysC operon was derepressed owing to a single nucleotide substitution in the region corresponding to the lysC leader transcript, produced full-length lysC mRNA, but no 0.27 kb RNA, even during growth with excess lysine . Mapping of the truncated 0.27 kb lysC RNA by hybridization with oligonucleotide probes showed that it corresponded to the upstream portion of the lysC leader transcript, extending from the transcription initiation site to a putative rho-independent terminator element . Quantitative transcript analysis by hybridization with specific oligonucleotides showed that lysine did not affect the number of lysC-specific RNA molecules but promoted the stoichiometric replacement of full-length mRNA with truncated 0.27 kb molecules . These results indicate that lysine regulates the expression of the lysC operon by effecting the premature termination of transcription at a rho-independent terminator site in the lysC leader region and that the site of the aecA mutation, far upstream of the putative terminator element, must play an essential role in premature transcription termination by a mechanism which is not yet understood.

J Bacteriol, 1996 Jul, 178(13), 3846 - 53
Homologous pairs of regulatory proteins control activity of Bacillus subtilis transcription factor sigma(b) in response to environmental stress; Kang CM et al.; In Bacillus subtilis, activity of the general stress transcription factor sigma B is controlled posttranslationally by a regulatory network that transmits signals of environmental and metabolic stress . These signals include heat, ethanol, or osmotic challenge, or a sharp decrease in cellular energy levels, and all ultimately control sigma B activity by influencing the binding decision of the RsbW anti-sigma factor . In the absence of stress, RsbW binds to sigma B and prevents its association with RNA polymerase core enzyme . However, following stress, RsbW binds instead to the RsbV anti-anti-sigma factor, thereby releasing sigma B to direct transcription of its target genes . These two principal regulators of sigmaB activity are encoded in the eight-gene sigB operon, which has the gene order rsbR-rsbS-rsbT-rsbU-rsbV-rsbW-sig B-rsbX (where rsb stands for regulator of sigma B) . Notably, the predicted rsbS product has significant amino acid identity to the RsbV anti-anti-sigma factor and the predicted rsbT product resembles the RsbW anti-sigma factor . To determine the roles of rsbS and rsbT, null or missense mutations were constructed in the chromosomal copies or each and tested for their effects on expression of a sigma B-dependent reporter fusion . On the basis of this genetic analysis, our principal conclusions are that (i) the rsbS product is a negative regulator of or" activity, (ii) the rsbT product is a positive regulator, (iii) RsbS requires RsbT for function, and (iv) the RsbS-RsbT and RsbV-RsbW pairs act hierarchically by a common mechanism in which key protein-protein interactions are controlled by phosphorylation events.

J Bacteriol, 1996 Jul, 178(13), 3796 - 802
Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis; Nakano MM et al.; Bacillus subtilis can grow anaerobically in the presence of nitrate as a terminal electron acceptor . The two component regulatory proteins, ResD and ResE, and an anaerobic gene regulator, FNR, were previously shown to be indispensable for nitrate respiration in B . subtilis . Unlike Escherichia coli fnr, B . subtilis fnr transcription was shown to be highly induced by oxygen limitation . fnr is transcribed from its own promoter as well as from a promoter located upstream of narK, the first gene in the narK-fnr dicistronic operon . DNA fragments containing the narK promoter, the fnr promoter, and both of the promoters were used to construct three lacZ fusions to examine the transcriptional regulation of the narK-fnr operon . ResDE was found to be required for transcriptional activation of fnr from the fnr-specific promoter, and FNR was required for activation of narK-fnr transcription from the FNR-dependent narK operon promoter under anaerobiosis . In order to determine if the requirement for ResDE in nitrate respiration is solely to activate fnr transcription, fnr was placed under control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter, Pspac . The observed defect in anaerobic growth of a Pspac-fnr delta resDE mutant in the presence of IPTG indicated that resDE has an additional role in B . subtilis anaerobic gene regulation.

J Bacteriol, 1996 Jul, 178(13), 3779 - 84
Role of CodY in regulation of the Bacillus subtilis hut operon; Fisher SH et al.; Bacillus subtilis mutants deficient in amino acid repression of the histidine utilization (hut) operon were isolated by transposon mutagenesis . Genetic characterization of these mutants indicated that they most likely contained transposon insertions within the codVWXY operon . The codY gene is required for nutritional regulation of the dipeptide permease (dpp) operon . An examination of hut expression in a delta codY mutant demonstrated that amino acid repression exerted at the hutOA operator, which lies immediately downstream of the hut promoter, was defective in a delta codY mutant . The codY gene product was not required for amino acid regulation of either hut induction or the expression of proline oxidase, the first enzyme in proline degradation . This indicates that more than one mechanism of amino acid repression is present in B . subtilis . An examination of dpp and hut expression in cells during exponential growth in various media revealed that the level of CodY-dependent regulation appeared to be related to the growth rate of the culture.

J Bacteriol, 1996 Jul, 178(13), 3701 - 9 sigma
Relative levels and fractionation properties of Bacillus subtilis sigma(B) and its regulators during balanced growth and stress; Dufour A et al.; sigma B is a secondary sigma factor that controls the general stress response in Bacillus subtilis . sigma B-dependent genes are activated when sigma B is released from an inhibitory complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase . Two separate pathways, responding either to a drop in intracellular ATP levels or to environmental stress (e.g., heat, ethanol, or salt), cause the release of sigma B from RsbW . rsbR, rsbS, rsbT, and rsbU are four genes now recognized as the upstream half of an operon that includes sigB (sigma B) and its principal regulators . Using reporter gene assays, we find that none of these four genes are essential for stationary-phase (i.e., ATP-dependent) activation of sigma B, but rsbU and one or more of the genes contained within an rsbR,S,T deletion are needed for stress induction of sigma B . In other experiments, Western blot (immunoblot) analyses showed that the levels of RsbR, RsbS, Rsb, and RsbU, unlike those of the sigB operon's four downstream gene products (RsbV, RsbW, RsbX and sigma B), are not elevated during sigma B activation . Gel filtration and immunoprecipitation studies did not reveal the formation of complexes between any of the four upstream sigB operon products and the products of the downstream half of the operon . Much of the detectable RsbR, RsbS, RsbT, and RsbU did, however, fractionate as a large-molecular-mass (approximately 600-kDa) aggregate which was excluded from our gel filtration matrix . The downstream sigB operon products were not present in this excluded material . The unaggregated RsbR, RsbS, and RsbU, which were retarded by the gel matrix, elated from the column earlier than expected from their molecular weights . The RsbR and RsbS fractionation profile was consistent with homodimers (60 and 30 kDa, respectively), while the RsbU appeared larger, suggesting a protein complex of approximately 90 to 100 kDa.

J Med Microbiol, 1996 Jul, 45(1), 57 - 63
A novel plasmid from Staphylococcus epidermidis specifying resistance to kanamycin, neomycin and tetracycline; Schwarz S et al.; The naturally occurring plasmid pSTS7 from Staphylococcus epidermidis mediated resistance to tetracycline via a tetL gene and to kanamycin and neomycin via an aadD gene . Plasmid pSTS7 showed partial restriction map and sequence homology to the previously described tetracycline resistance plasmid pNS1981 from Bacillus subtilis and to the kanamycin/neomycin/bleomycin resistance plasmid pUB110 from S . aureus . Sequence analysis of the regions flanking the two resistance genes in pSTS7 led to the identification of a novel site for interplasmid recombination which could explain the derivation of pSTS7 from the incompatible pNS1981- and pUB110-like parental plasmids under tetracycline-selective pressure.

Yeast, 1996 Jun 30, 12(8), 799 - 807
Lambda clone B22 contains a 7676 bp genomic fragment of Saccharomyces cerevisiae chromosome VII spanning the VAM7-SPM2 intergenic region and containing three novel transcribed open reading frames; Kail M et al.; A genomic clone of 7676 bp designated B22 from Saccharomyces cerevisiae has been sequenced . The 5' end matches the previously described gene, VAM7, and the 3' end matches the previously described gene, SPM2, both of which have been assigned to the left arm of chromosome VII . The intergenic region contains three transcribed open reading frames (ORFs) . The first is related to an uncharacterized ORF of Bacillus subtilis and more weakly to MesJ in Escherichia coli; this is found as a single transcript of 1.1 kb by Northern blotting . The second ORF encodes a small ras-like GTPase of 222 residues with strong homology to yeast Ypt8p and to mammalian Rab11; this is found as a single transcript of 1.1 kb by Northern blotting . The third ORF generates a transcript of 1.6 kb and encodes a protein of 382 residues including a perfect match to the consensus sequence of a C2H2 zinc finger domain; it shares a strong homology with yeast Mig1p and Cre-A from Aspergillus, Emericella and E . coli . This ORF also has a striking similarity to a putative 43 kDa zinc finger protein encoded by an ORF (YEL8) immediately downstream of YPT8, raising the possibility that a region between VAM7 and SPM2 on chromosome VII arose as a duplication of the YPT8-YEL8 region of chromosome V, followed by a translocation.

J Biotechnol, 1996 Jun 27, 47(2-3), 99 - 112
DNA repair in microgravity: studies on bacteria and mammalian cells in the experiments REPAIR and KINETICS; Horneck G et al.; The impact of microgravity on cellular repair processes was tested in the space experiments REPAIR and KINETICS, which were performed during the IML-2 mission in the Biorack of ESA: (a) survival of spores of Bacillus subtilis HA101 after UV-irradiation (up to 340 J m-2) in the experiment REPAIR; (b) in the experiment KINETICS the kinetics of DNA repair in three different test systems: rejoining of X-ray-induced DNA strand breaks (B1) in cells of Escherichia coli B/r (120 Gy) and (B2) in human fibroblasts (5 and 10 Gy) as well as (B3) induction of the SOS response after gamma-irradiation (300 Gy) of cells of Escherichia coli PQ37 . Cells were irradiated prior to the space mission and were kept in a non-metabolic state (metabolically inactive spores of B . subtilis on membrane filters, frozen cells of E . coli and human fibroblasts) until incubation in orbit . Germination and growth of B . subtilis were initiated by humidification, E . coli and fibroblasts were thawed up and incubated at 37 degrees C for defined repair periods (up to 4.5 h), thereafter they were frozen again for laboratory analysis . Relevant controls were performed in-flight (1 x g reference centrifuge) and on ground (1 x g and 1.4 x g) The results show no significant differences between the microgravity samples and the corresponding controls neither in the survival curves nor in the kinetics of DNA strand break rejoining and induction of the SOS response (proven by Student's t-test, 2 P = 0.05) . These observations provide evidence that in the microgravity environment cells are able to repair radiation-induced DNA damage close to normality . The results suggest that a disturbance of cellular repair processes in the microgravity environment might not be the explanation for the reported synergism of radiation and microgravity.

Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6616 - 20
Transcription activation by phage phi29 protein p4 is mediated by interaction with the alpha subunit of Bacillus subtilis RNA polymerase; Mencia M et al.; Regulatory protein p4 from Bacillus subtilis phage phi29 activates transcription from the viral late A3 promoter by stabilizing sigmaA-RNA polymerase at the promoter as a closed complex . Activation requires an interaction between protein p4 and RNA polymerase mediated by the protein p4 carboxyl-end, mainly through residue Arg-120 . We have obtained derivatives of B . subtilis RNA polymerase alpha subunit with serial deletions at the carboxyl-end and reconstituted RNA polymerase holoenzymes harboring the mutant alpha subunits . Protein p4 promoted the binding of purified B . subtilis RNA polymerase alpha subunit to the A3 promoter in a cooperative way . Binding was abolished by deletion of the last 15 amino acids of the alpha subunit . Reconstituted RNA polymerases with deletions of 15 to 59 residues at the alpha subunit carboxyl-end could recognize and transcribe viral promoters not activated by protein p4, but they had lost their ability to recognize the A3 promoter in the presence of protein p4 . In addition, these mutant reconstituted RNA polymerases could not interact with protein p4 . We conclude that protein p4 activation of the viral A3 promoter requires an interaction between the carboxyl-end of protein p4 and the carboxyl-end of the alpha subunit of B . subtilis RNA polymerase that stabilizes the RNA polymerase at the promoter.

FEBS Lett, 1996 Jun 24, 389(1), 84 - 7
Sequencing and functional analysis of the genome of Bacillus subtilis strain 168; Harwood CR et al.; The international programme to sequence the 4.2 Mb genome of Bacillus subtilis, a model Gram-positive bacterium, is a joint project involving European, Japanese and US research groups . To date ca . 3.0 Mb of the genome has been sequenced, with the remaining 1.2 Mb expected to be completed in 1997 . The amenability of B.subtilis to genetic manipulation, combined with the availability of extensive expertise on its biochemistry and physiology, makes this bacterium a valuable organism in which to investigate the properties of genes for which functions cannot be readily ascribed by standard methods.

EMBO J, 1996 Jun 17, 15(12), 3164 - 73
The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain; Pai KS et al.; The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini . The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers . The functional asymmetry is manifested in the polar impedance of replication fork movement . Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus . Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus . The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding . The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif.

Structure, 1996 Jun 15, 4(6), 679 - 90
Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis; Madhusudan et al.; BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis . It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue . This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases . Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay . RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution . The structure was solved by single isomorphous replacement and anomalous scattering techniques . The overall structural fold is (beta/alpha)5 and contains a central beta sheet . The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet . The active site accommodates a calcium ion . CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY . Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues . These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

Gene, 1996 Jun 12, 172(1), 17 - 24
The effect of Srb, a homologue of the mammalian SRP receptor alpha-subunit, on Bacillus subtilis growth and protein translocation; Oguro A et al.; To determine the signal recognition particle (SRP)-SRP receptor (Srb) system in Bacillus subtilis (Bs), we cloned the Bs srb gene, which encodes a homologue of the mammalian SRP receptor alpha-subunit {Oguro et al., DNA Res . 2 (1995) 95-100} . We sequenced a 6098-bp DNA containing srb and analyzed the gene organization . Primer extension experiment and Northern blot analysis revealed that srb constitutes an operon with two additional ORFs . A database search of known proteins revealed that one encodes a homologue of Escherichia coli RNase III {36.0% identical amino acids (aa)} and the other encodes a homologue of yeast Smc1 (26.6% identical aa) . We then constructed a Bs mutant in which srb expression was induced by IPTG . The depletion of Srb caused a defect in the cell growth and the cells became filamentous and twisted . Furthermore, pulse-chase experiments using this mutant revealed that the 17% of the beta-lactamase precursor accumulated in the cell after a 4-min chase in the absence of IPTG, although almost all of the precursors were converted into the mature from after a 1-min chase in the presence of IPTG.

Gene, 1996 Jun 12, 172(1), GC11 - 7
Parametric genome rearrangement; Blanchette M et al.; Algorithms inspired by comparative genomics calculate an edit distance between two linear orders based on elementary edit operations such as inversion, transposition and reciprocal translocation . All operations are generally assigned the same weight, simply by default, because no systematic empirical studies exist verifying whether algorithmic outputs involve realistic proportion of each . Nor do we have data on how weights should vary with the length of the inverted or transposed segment of the chromosome . In this paper, we present a rapid algorithm that allows each operation to take on a range of weights, producing an relatively tight upper bound on the distance between single-chromosome genomes, by means of a greedy search with look-ahead . The efficiency of this algorithm allows us to test random genomes for each parameter setting, to detect gene order similarity and to infer the parameter values most appropriate to the phylogenetic domain under study . We apply this method to genome segments in which the same gene order is conserved in Escherichia coli and Bacillus subtilis, as well as to the gene order in human versus Drosophila mitochondrial genomes . In both cases, we conclude that it is most appropriate to assign somewhat more than twice the weight to transpositions and inverted transpositions than to inversions . We also explore segment-length weighting for fungal mitochondrial gene orders.

Genes Cells, 1996 Jun, 1(6), 529 - 42
Subcellular localization of proteins governing the proteolytic activation of a developmental transcription factor in Bacillus subtilis; Resnekov O et al.; BACKGROUND: Spore formation in Bacillus subtilis takes place in a sporangium consisting of two compartments called the forespore and the mother cell . Late in development, when the forespore is wholly contained within the mother cell, gene transcription is coordinated between the compartments by an intercellular signal transduction pathway . This pathway operates at the level of proteolytic processing of the proprotein precursor (pro-sigma K to the mother-cell transcription factor sigma K . The conversion of pro-sigma K to sigma K is governed by the putative processing enzyme SpoIVFB and its negative regulator SpoIVFA, which are produced in the mother cell . RESULTS: We used fluorescence microscopy in conjunction with antibodies against SpoIVFA and SpoIVFB and a fusion of SpoIVFB to the Green Fluorescent Protein from Aquorea victoria to visualize these proteins in the sporangium . Both proteins were found to co-localize with the forespore region of the sporangium, a finding consistent with the idea that SpoIVFA and SpoIVFB, which are inferred to be integral membrane proteins, are located in the mother cell membrane that surrounds the forespore . CONCLUSIONS: We conclude that SpoIVFA and SpoIVFB are situated at the boundary between the forespore and the mother cell, at which location SpoIVFB could be activated by a signalling protein produced in the forespore.

J Ind Microbiol, 1996 Jun, 16(6), 377 - 82
Flagellar proteins of motile spores of Actinomycetes; Vesselinova NI et al.; Flagella of some of the actinoplanete genera were purified and the molecular sizes of their flagellin subunits compared by SDS-PAGE analysis to flagellins of cells of other bacteria . Several species of Actinoplanes have a major flagellar protein of subunit sizes of 42-43 kDa and a lesser amount of a second protein, possibly a minor flagellin subunit, of 60 kDa . The flagellar protein sizes of other actinoplanetes ranged from 32-43 kDa (major) and 48-58 kDa (minor) . Antibodies formed against the 42-kDa protein of A . rectilineatus showed cross-reactivity in Western blots against flagellar proteins of spores of other Actinoplanes species, two species of Dactylosporangium and an Ampullariella species . Cross-reactivity was also observed with motile cells of two other actinomycetes, Arthrobacter atrocyaneus and a Geodermatophilus species, and with Bacillus subtilis . No cross-reactivity was observed with Escherichia coli or Planomonospora parontospora flagellar proteins . The amino acid composition and partial N-terminal sequence of the 42-kDa flagellar protein of A . rectilineatus was compared to literature data for other bacterial flagellins and found to be most similar to B . subtilis 168.

Biol Trace Elem Res, 1996 Jun, 52(3), 209 - 25
Bioavailability of selenium accumulated by selenite-reducing bacteria; Combs GF Jr et al.; The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (SeO) . A laboratory strain of Bacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na2SeO3) . After harvest, the washed, lyophilized B . Subtilis and M . arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of SeO . These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet . Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite . A fourth experiment revealed that gray SeO had a bioavailability of 2% of selenite, but that the bioavailability of red SeO depended on the way it was prepared (by reduction of selenite) . When glutathione was the reductant, bioavailability resembled that of gray SeO and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3-4%) . These findings suggest that aerobic bacteria such as B . subtilis and M . arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.

Mol Microbiol, 1996 Jun, 20(5), 919 - 25
Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors; Pelicic V et al.; The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose . In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis . A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrF::K(m)) into the M . smegmatis genome . Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose . This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants . Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation . This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.

Appl Environ Microbiol, 1996 Jun, 62(6), 1977 - 83
Increased inactivation of Saccharomyces cerevisiae by protraction of UV irradiation; Sommer R et al.; The principle of equi-effectivity of the product of intensity and exposure time (principle of Bunsen-Roscoe) of UV irradiation has been assumed to be valid for the inactivation of microorganisms in general . Earlier studies claimed higher survival of Escherichia coli B/r with fractionated irradiation compared with single-exposure survival . However, data on the inactivation effect of protraction of UV irradiation are not available . By means of a specially designed UV irradiation apparatus which secured absolute UV dose measurements throughout the experiments, the effects of variation of UV irradiation intensities (253.7 nm) and exposure times were tested on the inactivation of a bacterial virus (Staphylococcus aureus phage A994), a vegetative bacterial strain (E . coli ATCC 25922), and bacterial spores (Bacillus subtilis ATCC 6633) as well as three haploid laboratory strains (RC43a, YNN281, and YNN282) and two diploid strains (commercial bakery yeast strain and laboratory strain YNN281 x YNN282) or yeast (Saccharomyces cerevisiae) and spores of the latter diploid yeast strain . Each test organism was exposed to three UV intensities (0.02, 0.2, and 2 W/m2), with corresponding exposure times resulting in three dose levels for each intensity . Differences in inactivation rates were tested by analyses of variance and Newman-Keuls tests . Virus and bacteria showed no differences in inactivation rates by variation of intensities and exposure times within selected UV doses; hence, the principle of Bunsen-Roscoe could not be rejected for these strains . However, in the eukaryotic test strains of S . cerevisiae longer exposure times with lower intensities led to enhanced inactivation in both haploid and diploid strains, with a more pronounced effect in the diploid yeast strains, whereas in yeast spores in this dose rate effect could not be observed.

RNA, 1996 Jun, 2(6), 564 - 73
Slow folding kinetics of RNase P RNA; Zarrinkar PP et al.; Understanding the folding mechanisms of large, highly structured RNAs is important for understanding how these molecules carry out their function . Although models for the three-dimensional architecture of several large RNAs have been constructed, the process by which these structures are formed is only now beginning to be explored . The kinetic folding pathway of the Tetrahymena ribozyme involves multiple intermediates and both Mg2+-dependent and Mg2+-independent steps . To determine whether this general mechanism is representative of folding of other large RNAs, a study of RNase P RNA folding was undertaken . We show, using a kinetic oligonucleotide hybridization assay, that there is at least one slow step on the folding pathway of RNase P RNA, resulting in conformational changes in the P7 helix region on the minute timescale . Although this folding event requires the presence of Mg2+, the slow step itself does not involve Mg2+ binding . The P7 and P2 helix regions exhibit distinctly different folding behavior and ion dependence, implying that RNase P folding is likely to be a complex process . Furthermore, there are distinct similarities in the folding of RNase P RNA from both Bacillus subtilis and Escherichia coli, indicating that the folding pathway may also be conserved along with the final structure . The slow folding kinetics, Mg2+-independence of the rate, and existence of intermediates are basic features of the folding mechanism of the Tetrahymena group I intron that are also found in RNase P RNA, suggesting these may be general features of the folding of large RNAs.

Microbiology, 1996 Jun, 142 ( Pt 6), 1417 - 21
Determination of a 12 kb nucleotide sequence around the 76 degrees region of the Bacillus subtilis chromosome; Yamamoto H et al.; The nucleotide sequence of a 12361 bp DNA segment in the 76 degrees region of the Bacillus subtilis chromosome has been determined . Ten putative ORFs were identified . The deduced amino acid sequences of the products of two of them (glv-1 and glv-2) exhibited high similarity to those of glvG (6-phospho-beta-glucosidase gene) and glvC {permease (the IIC domain) of the phosphotransferase system (PTS)}, respectively, in the glv operon of Escherichia coli . The C-terminal region of Glv-2 exhibited similarity to the entire region of GlvB (the IIB domain of PTS) of E . coli, suggesting fusion of the glvC and glvB genes in B . subtilis . glv-1, yfiA and glv-2 seem to form an operon of a phosphoenolpyruvate:sugar PTS, followed by a presumed four-membered operon of an ABC transport system . Moreover, a presumed sugar symporter and its regulatory genes were located in this region.

FEMS Microbiol Lett, 1996 Jun 1, 139(2-3), 235 - 8
Immunological detection of the GerA spore germination proteins in the spore integuments of Bacillus subtilis using scanning electron microscopy; Yasuda Y et al.; To clarify the molecular mechanisms that trigger spore germination of Bacillus subtilis, the location of GerA proteins (GerAA, GerAB and GerAC), which were reported to be putative gene products of a receptor for one of the germinants, L-alanine, was investigated by immunological techniques using anti-GerA peptide antibodies . Four antibodies were raised against the corresponding epitopes, two in GerAA, one in GerAB and the other in GerAC molecules . The binding of all four antibodies to the inner surface of the cortex-less spore coat fragments could be seen by scanning immunoelectron microscopy with colloidal gold particles . The result agreed with the fact, previously reported, that the colloidal gold particles were visualized just inside the spore coat layer by transmission immunoelectron microscopy using another anti-GerAB peptide antibody.

J Bacteriol, 1996 Jun, 178(12), 3668 - 70
General stress transcription factor sigmaB of Bacillus subtilis is a stable protein; Redfield AR et al.; The sigmaB subunit of Bacillus subtilis RNA polymerase governs the expression of a large general stress regulon . The results of pulse-chase and immunoprecipitation experiments showed that sigmaB is stable both in the presence and in the absence of the RsbW anti-sigma factor, the principal regulator of sigmaB in response to environmental signals.

J Bacteriol, 1996 Jun, 178(12), 3658 - 60
Characterization of the major citrate synthase of Bacillus subtilis; Jin S et al.; The major citrate synthase of Bacillus subtilis (CS-II) was purified to near homogeneity and shown to correspond to the product of the citZ gene . Accumulation of CS-II during exponential growth and stationary phases paralleled expression of the citZ gene . The physical and kinetic properties of CS-II were similar to those of citrate synthase enzymes from Bacillus megaterium and from eukaryotic cells but differed from those of citrate synthases from many gram-negative bacteria.

J Bacteriol, 1996 Jun, 178(12), 3486 - 95
Role of DNA repair in Bacillus subtilis spore resistance; Setlow B et al.; Wet-heat or hydrogen peroxide treatment of wild-type Bacillus subtilis spores did not result in induction of lacZ fusions to three DNA repair-related genes (dinR, recA, and uvrC) during spore outgrowth . However, these genes were induced during outgrowth of wild-type spores treated with dry heat or UV . Wet-heat, desiccation, dry-heat, or UV treatment of spores lacking major DNA-binding proteins (termed alpha-beta- spores) also resulted in induction of the three DNA repair genes during spore outgrowth . Hydrogen peroxide treatment of alpha-beta-spores did not result in induction of dinR- and rerA-lacZ but did cause induction of uvrC-lacZ during spore outgrowth . Spores of a recA mutant were approximately twofold more UV sensitive and approximately ninefold more sensitive to dry heat than were wild-type spores but were no more sensitive to wet heat and hydrogen peroxide . In contrast, alpha-beta- recA spores were significantly more sensitive than were alpha-beta- spores to all four treatments, as well as to desiccation . Surprisingly, RecA levels were quite low in dormant spores, but RecA was synthesized during spore outgrowth . Taken together, these data (i) are consistent with previous suggestions that some treatments (dry heat and UV with wild-type spores; desiccation, dry and wet heat, hydrogen peroxide, and UV with alpha-beta- spores) that kill spores do so in large part by causing DNA damage and (ii) indicate that repair of DNA damage during spore outgrowth is an important component of spore resistance to a number of treatments, as has been shown previously for UV.

J Bacteriol, 1996 Jun, 178(11), 3399 - 401
Phenotypic differentiation of "smart" versus "naive" bacteriophages of Bacillus subtilis; McVeigh RR et al.; The temperate bacteriophages of Bacillus subtilis differ dramatically in their response to the induction of the SOS system during the development of competence and following DNA damage . While all temperate bacteriophages are induced following DNA damage, the "naive" bacteriophages (i.e., phi105 and SPO2) are also induced during the development of competence . On the other hand, "smart" bacteriophages (i.e., phi3T and SPbeta) are not induced during the development of competence, and furthermore, once competence has developed, these prophages can no longer be induced by DNA damage.

J Bacteriol, 1996 Jun, 178(11), 3380 - 3
Analysis of suppressor mutations of spoIVCA mutations: occurrence of DNA rearrangement in the absence of site-specific DNA recombinase SpoIVCA in Bacillus subtilis; Sato T et al.; The spoIVCA gene of Bacillus subtilis encodes a site-specific recombinase, which excises a 48-kb skin element from the chromosomal DNA by DNA rearrangement and creates a new composite gene, sigK, on the chromosome . From spoIVCA mutants, we have isolated Spo+ revertants which have no skin element but have an intact sigK gene . This result suggests that the DNA rearrangement can occur in the absence of spoIVCA.

J Bacteriol, 1996 Jun, 178(11), 3260 - 9
Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism; Tong S et al.; Three genes involved in gluconate metabolism, gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, respectively, were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map . The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction . The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein . The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases . While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E . coli,gntV . The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis . Kinetic analysis of GntU indicates an apparent Km for gluconate of 212 microM, indicating that this is a low-affinity transporter . Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are separated by only 3 bp, form an operon . Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression . These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.

J Biol Chem, 1996 May 31, 271(22), 13140 - 6
Identification of a region of Bacillus subtilis Ffh, a homologue of mammalian SRP54 protein, that is essential for binding to small cytoplasmic RNA; Kurita K et al.; Bacillus subtilis Ffh and scRNA are homologues of mammalian SRP54 and SRP RNA, respectively, which are components of the eukaryotic signal recognition particle (SRP) . Ffh (446 amino acids) interacts with scRNA to form a stable complex in vivo . Here, we identified an RNA-binding domain of Ffh . The results obtained using a series of deletion mutants show that amino acid positions 364 to 432 in the C-terminal region of Ffh correlates with its ability to bind RNA . The amino acid sequence of this region is well conserved among members of the SRP54 protein family . This sequence contains two hydrophobic regions (h2, 364 to 391, and h3, 416 to 435), separated by the positively charged amino acid motif, 398RRKRIAKGSG407 . Among the basic amino acid residues in this region, Arg-401 was essential for binding to scRNA, but Arg-399 and Lys-400 were not . The co-existence of Arg-398 and Lys-404 was necessary for the same affinity as wild type Ffh . The two glycine residues of the 405GSG407 were also essential . MH23 peptide (91 amino acids) encompassing from 356 to 446, consisting of h2-RRKRIAKGSG-h3, bound scRNA with the same affinity as wild type Ffh, whereas a 24-amino acid synthetic peptide 392DIINASRRKRIAKGSGTSVQEVNR415 did not . The region containing two hydrophobic segments separated by the positively charged motif is the minimal requirement of Ffh for RNA binding.

J Biol Chem, 1996 May 31, 271(22), 13162 - 8
Identification of protein synthesis elongation factor G as a 4.5 S RNA-binding protein in Escherichia coli; Shibata T et al.; Escherichia coli 4.5 S RNA is metabolically stable and abundant . It consists of 114 nucleotides, and it is structurally homologous to domain IV of mammalian signal recognition particle (SRP) RNA . In this study, we found two 4.5 S RNA-binding proteins in cell extracts by means of a gel mobility shift assay . One protein was identified as Ffh, which has been characterized as 4.5 S RNA-binding protein . The other protein was separated from Ffh by two consecutive column chromatographic elutions and by monitoring the 4.5 S RNA binding activity . After the second chromatography, a dominant protein with an approximate molecular weight of 78,000 was associated with 4.5 S RNA binding activity . A sequence of the NH2-terminal 19 residues of the 78-kDa protein was completely identical to that of the protein elongation factor G (EF-G) of E . coli, and further it cross-reacted with antiserum against E . coli EF-G . The results obtained using a synthetic oligo RNA corresponding to the 23 S rRNA defining the EF-G binding site indicated that 4.5 S RNA and 23 S rRNA are competitive in 4.5 S RNA binding and that a decanucleotide sequence conserved between them serves as a binding site for EF-G . Conservation of the SRP RNA binding activity of EF-G from Bacillus subtilis suggests that the binding of EF-G to SRP RNA is essential for its function.

J Biol Chem, 1996 May 24, 271(21), 12269 - 74
Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA; Baumann C et al.; In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein) . TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring . Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied . Results from glycerol gradients and mobility shift gels indicate that two TRAP 11-mers bind to each trp leader RNA . A filter binding assay was used to determine an apparent binding constant of 8.0 +/- 1.3 x 10(9) m-1 (Kd = 0.12 +/- 0.02 nM) for TRAP and an RNA containing residues +36 to +92 of the trp leader RNA in 1 mM L-tryptophan at 37 degrees C . The temperature dependence of Kapp was somewhat unexpected demonstrating that the delta H of the interaction is highly unfavorable at + 15.9 kcal mol-1 . Therefore, the interaction is completely driven by a delta S of +97 cal mol-1 K-1 . The interaction between tryptophan-activated TRAP and trp leader RNA displayed broad salt and pH activity profiles . Finally, the rate of RNA dissociation from the RNA-TRAP.tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (> 40 microM) but increased in lower tryptophan concentrations . This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.

Mol Gen Genet, 1996 May 23, 251(2), 245 - 51
Implication of a repression system, homologous to those of other bacteria, in the control of arginine biosynthesis genes in Streptomyces coelicolor; Soutar A et al.; As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis in Streptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine . However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold . The response was not due to a general starvation effect . To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolated S . coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-type S . coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor . A subfragment carrying the 5' end of argC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism in Bacillus subtilis . It is therefore likely that in S . coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterised B . subtilis and Escherichia coli repressors.

Eur J Biochem, 1996 May 15, 238(1), 287 - 95
Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis; Svensson B et al.; Synthesis of heme A from heme B (protoheme IX) most likely occurs in two steps with heme O as an intermediate . Bacillus subtilis CtaB, an integral membrane protein, functions in farnesylation of heme B to form heme O . CtaA, also a membrane protein, is required for heme A synthesis from heme O and appears to be a monooxygenase and/or a dehydrogenase . Wild-type ctaA and ctaB expressed together from plasmids in B . subtilis resulted in CtaA containing equimolar amounts of low-spin heme B and heme A; this form of CtaA was named cyt ba-CTA . A mutant ctaB gene was identified and characterised . It encodes a truncated CtaB polypeptide . Wild-type ctaA and the mutant ctaB gene on plasmids resulted in CtaA containing mainly low-spin heme B; this variant was named cyt b-CTA . The heme B component in cyt ba-CTA and cyt b-CTA showed identical properties; a mid-point redox potential of +85 mV, an EPR g(max) signal at 3.7, and a split alpha-band light absorption peak . The heme A component in cyt ba-CTA showed a mid-point potential of +242 mV, an EPR g(max) signal at 3.5, and the alpha-band light absorption peak at 585 nm . It is suggested that the CtaA protein contains two heme binding sites, one for heme B and one for substrate heme . The heme B would play a role in electron transfer, i.e . function as a cytochrome, in the monooxygenase and/or dehydrogenase reaction catalysed by CtaA whereas heme O/heme A would be substrate/product.

Ann N Y Acad Sci, 1996 May 15, 782, 311 - 22
Stability of plasmid pHV1431 in free and immobilized cell cultures . Effect of temperature; Craynest M et al.; The segregational and structural stability of pHV1431 has been examined in Bacillus subtilis grown at 30 and 37 degrees C in continuous cultures without selection pressure . Immediately after appearance of plasmid-free cells in the reactor, a competition was observed between bacteria that favored plasmid-free cells because of the faster growth . A stronger instability was found at 30 degrees C compared to that at 37 degrees C . At 30 degrees C after 50 hours of culture, 2% of the cells carried the plasmid, whereas at 37 degrees C this percentage was reached after 130 hours . In both cases, no structural instability was observed . To improve the stability, the recombinant Bacillus subtilis (pHV1431) was immobilized in kappa-carrageenan gel beads . In comparison to free cell systems, a higher cell concentration was obtained . Moreover, the plasmid was maintained stable for longer periods; after 150 hours of culture 40% of cells in the reactor still carried the plasmid at both temperatures.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5043 - 8
Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis; Kast P et al.; Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate . Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond . To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway . The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected . The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential . Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement . The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

Biochemistry, 1996 May 14, 35(19), 6136 - 43
Stopped-flow, laser-flash photolysis studies on the reactions of CO and O2 with the cytochrome caa3 complex from Bacillus subtilis: conservation of electron transfer pathways from cytochrome c to O2; Hill BC; The reaction of CO and O2 with fully reduced cytochrome caa3 from Bacillus subtilis has been studied by rapid reaction spectrophotometry . The fully reduced caa3 complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a3-CO . This adduct is photosensitive, and its recombination rate is proportional to CO concentration with a bimolecular value of 1.2 x 10(5)M-1 s-1 . When the CO compound of the reduced complex is exposed to O2, the rate of oxidation proceeds at 0.1 s-1, which is assigned as the CO off rate . These kinetic constants give an equilibrium dissociation constant for the CO complex of 0.83 microM . Photolysis of the CO adduct in the presence of O2 reveals three reaction phases over the first 3 ms and an additional phase on the second time scale . A kinetic model is proposed in which fully reduced oxidase first combines with O2 and then electron transfer commences from both cytochrome a and a3, followed rapidly by electron input from CuA and the cytochrome c domain . An equivalent kinetic model has been used to account for the reactivity of mammalian cytochrome c oxidase in its electrostatic complex with soluble cytochrome c {Hill, B . C., (1994) J . Biol . Chem . 269, 2419-2425} . However, unlike the mitochondrial complex, the reactivity of cytochrome c in the B . subtilis caa3 complex is unaffected by ionic strength . Thus the cytochrome c moiety in the B . subtilis caa3 complex seems to be fixed in a reactive orientation by its covalent association with the rest of the oxidase complex . The pathway of electron transfer from cytochrome c to O2 appears very well conserved from B . subtilis to the mammalian respiratory chain, making the B . subtilis protein a good model to probe intersite electron transfer within the cytochrome c-cytochrome oxidase complex.

J Biol Chem, 1996 May 10, 271(19), 11455 - 61
The Spo0A protein of Bacillus subtilis inhibits transcription of the abrB gene without preventing binding of the polymerase to the promoter; Greene EA et al.; Repression of transcription of the abrB gene is essential to expression of many of the postexponential genes in Bacillus . The repression is due to the activity of the response regulator protein Spo0A . We have used in vitro transcription and DNase I and hydroxyl radical footprinting to explore the mechanism of transcription inhibition . Spo0A binds to specific DNA sequences (0A boxes), and two such boxes are found downstream of the tandem promoters for the abrB gene . The data indicate that both RNA polymerase and Spo0A bind simultaneously to a DNA fragment containing the promoters and the 0A boxes . The Spo0A prevents the polymerase from inducing DNA strand denaturation at the promoter for the abrB gene.

J Appl Bacteriol, 1996 May, 80(5), 529 - 34
An assessment of the Sartorius MD8 microbiological air sampler; Parks SR et al.; Tests described in this paper show that gelatine membrane filters used in the MD8 microbiological air sampling system collected monodispersed aerosols between 0.7 and 1.0 microns containing viable Bacillus subtilis var . niger spores, with an efficiency of 99.9995% . Gelatine membrane filters linked to the MD8 control pump system were as effective as the well established Casella slit-to-agar device for collecting some viable bacteria, nebulized under controlled experimental conditions and naturally occurring airborne micro-organisms in a pharmaceutical plant . By using a long flexible hose connection to the control pump, the head could be positioned where sampling was required in locations remote from the pump exhaust, making it suitable for microbiological monitoring in critical locations such as laminar flow stations and isolators.

FEMS Microbiol Lett, 1996 May 1, 138(2-3), 97 - 103
Cyclic AMP-independent catabolite repression in bacteria; Saier MH Jr; Until recently, only one mechanism of catabolite repression in bacteria, a mechanism dependent on the cyclic AMP receptor protein of Escherichia coli, was understood in molecular detail . Two cyclic AMP-independent catabolite repression mechanisms are currently under study . One such mechanism, found in E . coli, involves the catabolite repressor/activator (Cra) protein (formerly designated the fructose repressor FruR) which represses sugar catabolic systems and activates sugar anabolic systems . When catabolites bind to Cra, Cra dissociates from the DNA causing catabolite activation and catabolite repression, respectively . The second such mechanism, found in Bacillus subtilis, involves a catabolite-activated, ATP-dependent protein kinase that phosphorylates a specific seryl residue in the small phosphocarrier protein, HPr, of the phosphotransferase system . HPr(ser-P) binds to a transcription factor, CcpA, to promote DNA binding . DNA binding of the complex in turn promotes catabolite repression or catabolite activation, depending on the target operon . The characterization of these novel mechanisms establishes that cyclic AMP-independent catabolite control is operative in bacteria, and that multiple mechanisms of catabolite control evolved independently of each other.

Plasmid, 1996 May, 35(3), 164 - 73
Functional comparison of conjugative transposons Tn916 and Tn925; Showsh SA et al.; During interspecies matings between Bacillus subtilis and Bacillus thuringiensis subsp . israelensis, transfer of conjugative transposon Tn916 was detected at a frequency of 1.1 x 10(-4) transconjugants per donor . Tn916-dependent transfer of plasmids pC194 and pE194 was detected at frequencies of 1.4 x 10(-5) and 3.2 x 10(-7) transconjugants per donor, respectively . Similar frequencies were obtained during parallel matings with otherwise isogenic strains that contain Tn925 instead of Tn916 . Tn916- or Tn925-dependent transfer of plasmids pC194 or pUB110 from the recipient to the donor (retrotransfer) was not observed during inter- or intraspecies matings . Transposon-mediated plasmid transfer by Tn916 and Tn925 is a Rec independent event . Thus, the data from studies in which otherwise isogenic donor and recipient strains were used indicated that Tn916 and Tn925 are, from a functional point of view, much more similar than previously suggested.

Mol Microbiol, 1996 May, 20(4), 843 - 52
Interaction of CodY, a novel Bacillus subtilis DNA-binding protein, with the dpp promoter region; Serror P et al.; The product of the codY gene is required for nutritional repression of the Bacillus subtillis dipeptide permease operon (dpp), an operon expressed at early stationary phase in nutrient-rich medium . Though unrelated to any known DNA-binding protein, CodY was shown to bind specifically to the dpp promoter region . DNase I footprinting experiments revealed that the CodY-protected region encompasses the dpp transcription start site and overlaps with the region protected by another regulatory protein, AbrB . CodY and AbrB were found to compete, in vitro, for binding to the dpp promoter region . Binding of CodY was altered in mutants defective in nutritional regulation.

Mol Microbiol, 1996 May, 20(4), 713 - 23
Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon; Kruger E et al.; clpC of Bacillus subtilis is part of an operon containing six genes . Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins . Two promoters (PA and PB) were mapped upstream of the first gene . PA resembles promoters recognized by the vegetative RNA polymerase E sigma A . The other promoter (PB) was shown to be dependent on sigma B, the general stress sigma factor in B . subtilis, suggesting that clpC, a potential chaperone, is expressed in a sigma B-dependent manner . This is the first evidence that sigma B in B . subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by sigma 32 in Escherichia coli, indicating a new function of sigma B-dependent general stress proteins . PB deviated from the consensus sequence of sigma B promoters and was only slightly induced by starvation conditions . Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the sigma B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions . However, in a sigB mutant, the sigma A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency . Only the downstream sigma A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin . These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter . Involvement of additional elements in this mode of induction are discussed.

Genome Res, 1996 May, 6(5), 448 - 53
A new approach using multiplex long accurate PCR and yeast artificial chromosomes for bacterial chromosome mapping and sequencing; Sorokin A et al.; An efficient approach for structural studies on bacterial chromosomes is presented . It is based on high-resolution PCR map construction by using a multiplex long accurate PCR (MLA PCR) protocol and a YAC clone carrying the region to be mapped as indicator . The high-resolution PCR map of the bacillus subtilis rrnB-dnaB region is presented as an example . Data are also presented on the use of DNA generated by LA PCR for sequencing; they are relevant to LA PCR induced mutations and justify the application of such mapping for sequencing long stretches of bacterial chromosomes.

Biotech Histochem, 1996 May, 71(3), 130 - 6
Highly selective acridine and ethidium staining of bacterial DNA and RNA; Bruno JG et al.; The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels . Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents . EtBr stained both DNA and RNA in the gels . Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> or = 100 microM) of AO, lower concentrations (13.9 microM) of CPO, and did not stain with 0.5 microgram/ml EtBr in denaturing gels . The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining . The highest concentrations of AO (120 microM) and CPO (13.9 microM) were shown to detect purified DNA in gels with a sensitivity in the range of 25-50 ng per band . This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.

Biosci Biotechnol Biochem, 1996 May, 60(5), 773 - 8
A method to invert DNA segments of the Bacillus subtilis 168 genome by recombination between two homologous sequences; Toda T et al.; We developed a method that allows rapid isolation of mutant Bacillus subtilis 168 carrying an inversion of a specific DNA segment of the genome . Two incomplete neomycin resistance gene cassettes were integrated at both ends of the 1652-kb segment to be inverted . Reciprocal recombination within the 590-bp homologous region of these two cassettes created an intact neomycin resistance gene with concomitant inversion of the 1652-kb segment flanked by the two cassettes . Structure of the mutant genome was verified by analyzing the physical map for rare cutting endonucleases, SfiI, NotI, I-CeuI, and I-SceI . The inversion rate was estimated to be 6.9 +/- 1.4 x 10(-8)/cell/cell division at 37 degrees C . The method should be in principle applicable not only to other regions of the B, subtilis genome but also to other bacterial genomes.

Nat Genet, 1996 May, 13(1), 95 - 7
A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria; Meissner PN et al.; Variegate porphyria (VP), a low-penetrant autosomal dominant inherited disorder of haem metabolism, is characterised by photosensitivity (Fig . 1) and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia . Acute attacks, often precipitated by inappropriate drug therapy, are potentially fatal . While earlier workers thought the distal haem biosynthetic enzyme ferrochelatase may be involved in the genesis of VP, it was shown in the early 1980's, and is now accepted, that VP is associated with decreased protoporphyrinogen oxidase activity (PPO) (E.C.1.3.3.4) . VP prevalence is much higher in South Africa than elsewhere; probably due to a founder effect with patients descending from a 17th century Dutch immigrant . PPO cDNAs from Bacillus subtilis, Myxococcus xanthus, human placenta and mouse liver have been cloned, sequenced and expressed . Human and mouse cDNAs consist of open reading frames 1431 nucleotides long, encoding a 477 amino acid protein . The human PPO gene contains thirteen exons, spanning approximately 4.5 kb . We have identified a C to T transition in codon 59 (in exon 3) resulting in an arginine to tryptophan substitution (R59W) . A protein expressed from an in vitro-mutagenized PPO construct exhibits substantially less activity than the wild type . The R59W mutation was present in 43 of 45 patients with VP from 26 of 27 South African families investigated, but not in 34 unaffected relatives or 9 unrelated British patients with PPO deficiency . Since at least one of these families is descended from the founder of South African VP, this defect may represent the founder gene defect associated causally with VP in South Africa.

Biochem J, 1996 May 1, 315 ( Pt 3), 895 - 900
1H-NMR characterization of L-tryptophan binding to TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis; Ramesh V et al.; A 1H-NMR study of the binding of L-tryptophan to the trp RNA-binding attenuation protein of Bacillus subtilis (TRAP), an ondecamer (91.6 kDa), has been implemented . The assignment of the aromatic indole ring proton resonances of the bound tryptophan ligand has been successfully carried out by two-dimensional chemical exchange experiments . The observation of only a single set of chemical shifts of the bound ligand demonstrates that the tryptophan binding site is identical in all the 11 subunits of the protein . Further, the large change in ligand chemical shifts suggests that the conformation of tryptophan ligand undergoes a significant rearrangement after complex formation with TRAP . This is further substantiated by the extensive ligand-induced chemical shift changes observed to the protein resonances and identification of several strong ligand-protein intermolecular nuclear Overhauser effects . A correlation of these preliminary NMR data with the X-ray crystal structure of the TRAP-tryptophan complex also suggests, tentatively, that the observed changes to the NMR spectra of the protein might correspond to changes associated with residues surrounding the tryptophan binding pocket owing to complex formation.

EMBO J, 1996 May 1, 15(9), 2249 - 55
Multiple substrate binding sites in the ribozyme from Bacillus subtilis RNase P; Pan T et al.; The ribozyme from Bacillus subtilis RNase P (P RNA) recognizes an RNA structure consisting of the acceptor stem and the T stem-loop of tRNA substrates . An in vitro selection experiment was carried out to obtain potential RNA substrates that may interact with the P RNA differently from the tRNA substrate . Using a P RNA-derived ribozyme that contains most, if not all, of the structural elements thought to be involved in active site formation of P RNA, but lacks the putative binding site for the T stem-loop of tRNA, a single RNA substrate was isolated after nine rounds of selection . This RNA is a competent substrate for the ribozyme used in selection as well as for the full-length P RNA . Biochemical characterization shows that this selected substrate interacts at a different site compared with the tRNA substrate . The selection experiment also identified a self-cleaving RNA seemingly different from other known ribozymes . These results indicate that a biological ribozyme can contain different binding sites for different RNA substrates . This alternate binding site model suggests a simple mechanism for evolving existing ribozymes to recognize RNA substrates of diverse structures.

J Bacteriol, 1996 May, 178(10), 2853 - 60
Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain; Cheng J et al.; Deletion of the tetA(L) chromosomal region of Bacillus subtilis in a strain designated JC112 increased the strain's sensitivity to low tetracycline concentrations . It also resulted in phenotypic changes that correlate with the previously found role of TetA(L) in mediating electrogenic NA+/H+ antiport . Growth of JC112 was impaired relative to that of the wild type at both pH 7.0 and 8.3; Na(+)- and K(+)-dependent pH homeostases were impaired at alkaline pH . The phenotype of JC112 was complemented by plasmid-borne tetA(L) and related tet(K) genes; the antiport activity conferred by the tet(K) gene had an apparently higher preference for K+ over Na+ than that conferred by tetA(L) . The data were consistent with TetA(L) being the major Na+(K+)/H+ antiporter involved in pH homeostasis in B . subtilis as well as a significant Na+ extrusion system . The phenotype of JC112 was much more pronounced than that of an earlier transposition mutant, JC111, with a disruption in the putative tetA(L) promoter region . Northern (RNA) blot analysis of tetA(L) RNA from wild-type and JC111 strains revealed the same patterns . That JC111 nevertheless exhibited some Na+ and alkali sensitivity may be accounted for by disruption of regulatory features that, in the wild type, allow increased tetA(L) expression under specific conditions of pH and monovalent cation concentration . Evidence for several different regulatory effects emerged from studies of lacZ expression from the transposon of JC111 and from a tetA(L)-lacZ translational fusion introduced into the amyE locus of wild-type and JC112 strains.

J Bacteriol, 1996 May, 178(10), 2813 - 7
Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis; Zhang L et al.; A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric division . The development programs in these two cells involve the compartmentalized activities of sigma E in the larger mother cell and of sigma F in the smaller prespore . Activation of sigma E requires expression of the sigma F-directed gene spoIIR . By immunofluorescence microscopy of a strain containing a spoIIR-lacZ fusion, we have shown that spoIIR is transcribed exclusively in the prespore . By placing spoIIR under the control of PspoIIE, it was possible to express spoIIR before the spore septum was formed . Strains containing the PspoIIE-spoIIR construct activated sigma E only in the mother cell in organisms that underwent the asymmetric sporulation division . Thus, compartmentalization of sigma E activity did not require the compartmentalization of spoIIR expression . Nor did the compartmentalization of sigma E require SpoIIAA, SpoIIAB, sigma F, or sigma F-dependent transcription, all of which are required for prespore-specific gene expression . It is inferred that although sigma F and sigma E direct compartmentalized gene expression, neither of these sigma factors, nor the genes under their control, directs the process of compartmentalization.

J Bacteriol, 1996 May, 178(9), 2637 - 44
Transcriptional analysis of bglPH expression in Bacillus subtilis: evidence for two distinct pathways mediating carbon catabolite repression; Kruger S et al.; In Bacillus subtilis, aryl-beta-glucosides such as salicin and arbutin are catabolized by the gene products of bglP and bglH, encoding an enzyme II of the phosphoenolpyruvate sugar-phosphotransferase system and a phospho-beta-glucosidase, respectively . These two genes are transcribed from a single promoter . The presence of a transcript of about 4,000 nucleotides detected by Northern (RNA) blot analysis indicates that bglP and bglH are part of an operon . However, this transcript is only present when cells are grown in the presence of the inducing substrate, salicin . In the absence of the inducer, a transcript of about 110 nucleotides can be detected, suggesting that transcription terminates downstream of the promoter at a stable termination structure . Initiation of transcription is abolished in the presence of rapidly metabolized carbon sources . Catabolite repression of bglPH expression involves the trans-acting factors CcpA and HPr . In a ccpA mutant, transcription initiation is relieved from glucose repression . Furthermore, we report a catabolite responsive element-CcpA-independent form of catabolite repression requiring the ribonucleic antiterminator-terminator region, which is the target of antitermination, and the wild-type HPr protein of the phosphotransferase system . Evidence that the antitermination protein LicT is a crucial element for this type of regulation is provided.

Philos Trans R Soc Lond B Biol Sci, 1996 Apr 29, 351(1339), 537 - 42
Control of the cell-specificity of sigma F activity in Bacillus subtilis; Errington J et al.; Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division . Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity . Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control . It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA . SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro . In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F . In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free . Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio . Genetic experiments have implicated a fourth protein, called SpoIIE, in this system . It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F . First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA . Second it controls the formation of the septum that generates the prespore compartment . Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.

Mol Gen Genet, 1996 Apr 24, 251(1), 108 - 12
Degradation of the Bacillus subtilis xynA transcript is accelerated in response to stress; Allmansberger R; A popular method for the investigation of transcriptional regulation of gene expression is direct measurement of mRNA levels . As an internal control the level of a transcript from a constitutively expressed gene is often determined . To measure the induction rate of stress-responsive genes from Bacillus subtilis the transcript of the constitutively expressed xynA gene was used as a control . But the results presented in this communication prove that the degradation rate of the xynA transcript rises considerably in response to different kinds of stress . This response to stress is not dependent on protein biosynthesis.

FEBS Lett, 1996 Apr 22, 384(3), 235 - 9
Cloning of a potential cytochrome P450 from the archaeon Sulfolobus solfataricus; Wright RL et al.; Abstract A gene, CYP119, for a potential cytochrome P450 has been isolated and sequenced from the extreme acidothermophilic archaeon Sulfolobus solfataricus . The gene predicts a polypeptide of 368 amino acids containing the consensus heme-binding sequence Phe-Gly-Xaa-Gly-Xaa-His-Xaa-Cys-Xaa-Gly- Xaa3-Ala-Arg-Xaa-Glu . It most closely resembles the cytochrome P450s found in the bacterium Bacillus subtilis, with which it shares 129 identical amino acid residues (35%) . This first sequence of a potential archaeal cytochrome P450 represents an important step in tracing the complex evolutionary history of this biologically important enzyme family.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3253 - 8
The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication; Manna AC et al.; The replication terminator protein (RTP) of Bacillus subtilis causes polar fork arrest at replication termini by sequence-specific interaction of two dimeric proteins with the terminus sequence . The crystal structure of the RTP protein has been solved, and the structure has already provide valuable clues regarding the structural basis of its function . However, it provides little information as to the surface of the protein involved in dimer-dimer interaction . Using site-directed mutagenesis, we have identified three sites on the protein that appear to mediate the dimer-dimer interaction . Crystallographic analysis of one of the mutant proteins (Y88F) showed that its structure is unaltered when compared to the wild-type protein . The locations of the three sites suggested a model for the dimer-dimer interaction that involves an association between two beta-ribbon motifs . This model is supported by a fourth mutation that was predicted to disrupt the interaction and was shown to do so . Biochemical analyses of these mutants provide compelling evidence that cooperative protein-protein interaction between two dimers of RTP is essential to impose polar blocks to the elongation of both DNA and RNA chains.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3238 - 42
SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis; Arigoni F et al.; Cell-specific activation of the transcription factor sigma F during sporulation in Bacillus subtilis is controlled by a regulatory pathway involving the proteins SpoIIE, SpoIIAA, and SpoIIAB . SpoIIAB is an antagonist of sigma F, and SpoIIAA, which is capable of overcoming SpoIIAB-mediated inhibition of sigma F, is an antagonist of SpoIIAB . SpoIIAA is, in turn, negatively regulated by SpoIIAB, which phosphorylates SpoIIAA on serine 58 . SpoIIAA is also positively regulated by SpoIIE, which dephosphorylates SpoIIAA-P, the phosphorylated form of SpoIIAA . Here, isoelectric focusing and Western blot analysis were used to examine the phosphorylation state of SpoIIAA in vivo . SpoIIAA was found to be largely in the phosphorylated state during sporulation in wild-type cells but a significant portion of the protein that was unphosphorylated could also be detected . Consistent with the idea that SpoIIE governs dephosphorylation of SpoIIAA-P, SpoIIAA was entirely in the phosphorylated state in spoIIE mutant cells . Conversely, overexpression of spoIIE led to an increase in the ratio of unphosphorylated SpoIIAA to SpoIIAA-P and caused inappropriate activation of sigma F in the predivisional sporangium . We also show that a mutant form of SpoIIAA (SpoIIAA-S58T) in which serine 58 was replaced with threonine was present exclusively as SpoIIAA-P, a finding that confirms previous biochemical evidence that the mutant protein is an effective substrate for the SpoIIAB kinase but that SpoIIAA-S58T-P cannot be dephosphorylated by SpoIIE . We conclude that SpoIIE plays a crucial role in controlling the phosphorylation state of SpoIIAA during sporulation and thus in governing the cell-specific activation of sigma F.

J Biol Chem, 1996 Apr 12, 271(15), 9120 - 8
Cloning and characterization of the yeast HEM14 gene coding for protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides; Camadro JM et al.; Protoporphyrinogen oxidase, which catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX, is the molecular target of diphenyl ether type herbicides . The structural gene for the yeast protoporphyrinogen oxidase, HEM14, was isolated by functional complementation of a hem14-1 protoporphyrinogen oxidase-deficient yeast mutant, using a novel one-step colored screening procedure to identify heme-synthesizing cells . The hem14-1 mutation was genetically linked to URA3, a marker on chromosome V, and HEM14 was physically mapped on the right arm of this chromosome, between PRP22 and FAA2 . Disruption of the HEM14 gene leads to protoporphyrinogen oxidase deficiency in vivo (heme deficiency and accumulation of heme precursors), and in vitro (lack of immunodetectable protein or enzyme activity) . The HEM14 gene encodes a 539-amino acid protein (59,665 Da; pI 9.3) containing an ADP- beta alpha beta-binding fold similar to those of several other flavoproteins . Yeast protoporphyrinogen oxidase was somewhat similar to the HemY gene product of Bacillus subtilis and to the human and mouse protoporphyrinogen oxidases . Studies on protoporphyrinogen oxidase overexpressed in yeast and purified as wild-type enzyme showed that (i) the NH2-terminal mitochondrial targeting sequence of protoporphyrinogen oxidase is not cleaved during importation; (ii) the enzyme, as purified, had a typical flavin semiquinone absorption spectrum; and (iii) the enzyme was strongly inhibited by diphenyl ether-type herbicides and readily photolabeled by a diazoketone derivative of tritiated acifluorfen . The mutant allele hem14-1 contains two mutations, L422P and K424E, responsible for the inactive enzyme . Both mutations introduced independently in the wild-type HEM14 gene completely inactivated the protein when analyzed in an Escherichia coli expression system.

Mol Gen Genet, 1996 Apr 10, 250(6), 761 - 6
Suppression of the Bgl+ phenotype of a delta hns strain of Escherichia coli by a Bacillus subtilis antiterminator binding site; Beloin C et al.; Bacillus subtilis, like Escherichia coli, possesses several sets of genes involved in the utilization of beta-glucosides . In E . coli, all these genes are cryptic, including the genes forming the bgl operon, thus leading to a Bgl- phenotype . We screened for B . subtilis chromosomal DNA fragments capable of reverting the Bgl+ phenotype associated with an E . coli hns mutant to the Bgl- wild-type phenotype . One B . subtilis chromosomal fragment having this property was selected . It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from the bgl gene . Deletion studies as well as subcloning experiments allowed us to prove that the putative B . subtilis of the E . coli bgl operon . We propose that this repression results from the titration of the BglG antiterminator protein of E . coli bgl operon by our putative B . subtilis bglP RAT sequence . Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.

Mol Gen Genet, 1996 Apr 10, 250(6), 742 - 9
Dissection of the Bacillus subtilis spoOE binding site for the global regulator AbrB reveals smaller recognition elements; Strauch MA; AbrB is a global transcriptional regulator of many genes that are expressed as Bacillus subtilis exits from active growth into stationary phase and sporulation . Previous results have suggested that binding of abrB at some promoters involves multiple sites of recognition and is a cooperative process . It is shown here that the binding site at spoOE can be subdivided into 5' and 3' halves, each capable of directing AbrB binding . In addition, the central portion of the intact site can promote AbrB binding . Examination of various heterologous and homologous tandem combinations of the half-sites confirms that the native site is a complex array of overlapping suboptimal sites, the precise arrangement of which is required for optimal AbrB binding . Other data suggest that binding of multiple AbrB units is needed for stable complex formation . A binding mechanism involving numerous steps of intermediate affinity is envisioned.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2790 - 4
A stationary-phase stress-response sigma factor from Mycobacterium tuberculosis; DeMaio J et al.; Alternative RNA polymerase sigma factors are a common means of coordinating gene regulation in bacteria . Using PCR amplification with degenerate primers, we identified and cloned a sigma factor gene, sigF, from Mycobacterium tuberculosis . The deduced protein encoded by sigF shows significant similarity to SigF sporulation sigma factors from Streptomyces coelicolor and Bacillus subtilis and to SigB, a stress-response sigma factor, from B . subtilis . Southern blot surveys with a sigF-specific probe identified cross-hybridizing bands in other slow-growing mycobacteria, Mycobacterium bovis bacille Calmette-Guerin (BCG) and Mycobacterium avium, but not in the rapid-growers Mycobacterium smegmatis or Mycobacterium abscessus . RNase protection assays revealed that M . tuberculosis sigF mRNA is not present during exponential-phase growth in M . bovis BCG cultures but is strongly induced during stationary phase, nitrogen depletion, and cold shock . Weak expression of M . tuberculosis sigF was also detected during late-exponential phase, oxidative stress, anaerobiasis, and alcohol shock . The specific expression of M . tuberculosis sigF during stress or stationary phase suggests that it may play a role in the ability of tubercle bacilli to adapt to host defenses and persist during human infection.

Microb Drug Resist, 1996 Spring, 2(1), 123 - 9
D-alanine deprivation of Bacillus subtilis teichoic acids is without effect on cell growth and morphology but affects the autolytic activity; Wecke J et al.; Using insertional inactivation of the different genes of the dlt operon in Bacillus subtilis, we searched for metabolic and morphological changes caused by D-alanine ester deprivation of lipoteichoic acid and wall teichoic acid . There were no alterations of cell growth, basic metabolism, cellular content of phosphorus-containing compounds, ultrastructure, cell separation, and surface charge . The only alteration observed was an enhancement of endogenous and beta-lactam-induced cell lysis . Since this enhancement is doubtless correlated with the D-alanine ester deprivation of the teichoic acids, the present view based on in vitro experiments, that negatively charged LTA is inhibitory to autolysins, may be questioned . We propose that negatively charged lipoteichoic acid and/or wall teichoic acid serve in vivo to fix the cationic autolysins within the cell wall-membrane complex by electrostatic interaction . Positively charged D-alanine ester substituents decrease the binding capacity of the teichoic acids for autolysins by charge compensation.

Microb Drug Resist, 1996 Spring, 2(1), 119 - 21
Effect of the SinR protein on the expression of the Bacillus subtilis 168 lytABC operon; Margot P et al.; Transcription of the lytABC operon was determined by extension of primers on RNAs isolated from strains bearing a deficient sinR gene . A SinR null mutant, in which part of the sinR gene was deleted, exhibits a pattern identical to that characteristic of FlaB (SigD) deficient mutants, i.e., loss of the signal corresponding to the SigD-dependent promoter, but not of that recognized by the SigA form of the RNA polymerase . However, strains bearing either flaD1 or flaD2, two different point mutations of gene sinR, were characterized by a complete loss of signals corresponding to both promoters . Thus, modified FlaD1 and FlaD2 proteins behave like a repressor affecting the expression of lytABC more severely than does the absence of SinR, The most obvious interpretation of this observation is a direct interaction between the SinR protein and the promoters recognized by the SigD form of the RNA polymerase.

Microb Drug Resist, 1996 Spring, 2(1), 113 - 8
Peptidoglycan hydrolases of Bacillus subtilis 168; Smith TJ et al.; There are multiple peptidoglycan hydrolases associated with Bacillus subtilis 168 and these potentially lethal enzymes have been implicated in a number of important cellular processes . Several enzymes have been studied at the molecular level and their structural genes characterized . This information has begun to identify roles for individual enzymes in motility, cell separation, differentiation, and phage lysis . It has become apparent that in many cases important autolytic functions can be performed by more than one enzyme, so the complex web of mutually compensatory components can be unraveled only by making multiple mutants . One such multiple mutant has revealed the presence of several previously unknown minor autolysins, the functions of which are currently obscure.

Microb Drug Resist, 1996 Spring, 2(1), 91 - 3
Cellular signals regulating antibiotic sensitivities of bacteria; Matsuhashi M et al.; The effects of bacterial masses upon the drug resistance of neighboring bacteria were investigated . The experiments were performed with plastic Petri dishes divided into two identical compartments . A growing mass of Bacillus subtilis (signal emitter cell) in one compartment exerted enhancing effects upon the erythromycin and streptomycin resistance of Bacillus carboniphilus (signal recipient) cells, sparsely seeded in the other compartment, through the plastic wall and the air . These effects of the growing mass of cells are attributed to the emission of "sonic" signals.

Microb Drug Resist, 1996 Spring, 2(1), 9 - 15
Overall protein content and induced enzyme components of the periplasm of Bacillus subtilis; Pooley HM et al.; Estimates for the overall protein content of the periplasm of Escherichia coli range from 4 to 16% of cellular protein . A cursory examination of known sources of contamination inherent to the methods employed for measurement leads to the conclusion that even the lower value may represent an overestimate of the periplasmic protein in E . coli . The protoplast supernatant fraction (PSF) of Bacillus subtilis defines operationally a potential periplasm, which, after correction for cytoplasmic contamination, yielded, in B . subtilis strains 168 and W23, calculated values of 9 and 3%, respectively, of cell protein as being periplasmic . 26 Among enzymes typically periplasmic in E . coli, at least two, RNases and a 5'-nucleotidase, were located in the B . subtilis periplasm . Compared to other cell fractions, RNase activity in the periplasm was associated with several protein bands forming a unique profile . Samples from all growth phases of cells cultured under phosphate-limitation and phosphate-excess revealed that a major part of both investigated activities was induced by phosphate depletion and located outside the plasma membrane . The current belief that a periplasm containing soluble enzymes does not exist in gram-positive bacteria is examined in light of the absence of an outer membrane permeability barrier, and of a clearly defined electron-transparent zone located between the plasma membrane and the cell wall of B . subtilis . Previous results of studies of protein secretion, and cell wall permeability, are reinterpreted by assuming that the thick charged cell wall of gram-positive bacteria can act as the outer permeability barrier, and as such be the functional equivalent of the outer membrane of gram-negative organisms.

Microbiology, 1996 Apr, 142 ( Pt 4), 733 - 40
Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis; Lewis PJ et al.; Wild-type and mutant forms of the gene encoding green fluorescent protein (GFP) from Aequorea victoria have been introduced into Bacillus subtilis as translational fusions to the prespore-specific and mother-cell-specific genes dacF and spoIVA . In both cases, the protein was readily detected by fluorescence microscopy, and its synthesis was correctly localized . The S65T substitution, which improves the quantum yield and rate of development of fluorescence, also produced a spectral shift that allowed the protein to be colocalized with DNA, after staining with 4',6-diamidino-2-phenylindole . Three different translational fusions to the N-terminal region of GFP all produced active protein . Moreover, a full-length SpoIVA-GFP fusion showed proper targeting to the surface of the spore, albeit at low temperature and in the presence of wild-type SpoIVA protein . A mutation in the gfp gene which changes the light emitted by the protein from green to blue was found not to be useful because of the intrinsic autofluorescence of B . subtilis in the blue part of the spectrum.

Int J Syst Bacteriol, 1996 Apr, 46(2), 470 - 5
Bacillus vallismortis sp . nov., a close relative of Bacillus subtilis, isolated from soil in Death Valley, California; Roberts MS et al.; Five Bacillus strains isolated from Death Valley soil were shown to belong to a previously unidentified species, for which we propose the name Bacillus vallismortis . The type strain is strain DV1-F-3 (= NRRL B-14890) . On the basis of previously published restriction digestion data, B . vallismortis is most closely related to Bacillus subtilis . At this time B . vallismortis can be distinguished from B . subtilis only by differences in whole-cell fatty acid compositions, DNA sequences, and levels of reassociation of genomic DNA.

Mol Microbiol, 1996 Apr, 20(1), 201 - 12
Altering the level and regulation of the major sigma subunit of RNA polymerase affects gene expression and development in Bacillus subtilis; Hicks KA et al.; In Bacillus subtilis, the major sigma factor, sigma-A (rpoD), and the minor sigma factor, sigma-H (spo0H), are present during growth and are required for the initiation of sporulation . Our experiments indicate that sigma-A and sigma-H compete for binding to core RNA polymerase . We used a fusion of rpoD to the LacI-repressible IPTG-inducible promoter, Pspac, to vary the levels of sigma-A in the cell . Increasing the amount of sigma-A caused a decrease in expression of genes controlled by sigma-H, and a delay in the production of heat-resistant spores . Decreasing the amount of sigma-A, in a strain deleted for the chromosomal rpoD, caused an increase in expression of genes controlled by sigma-H . As rpoD itself is controlled by at least two promoters recognized by RNA polymerase that contains sigma-H, the effect of sigma-A levels on expression of sigma-H-controlled promoters represents a feedback mechanism that might contribute to maintaining appropriate levels of sigma-A . While the level of sigma-A was important for efficient sporulation, our results indicate that the normal transcriptional control of rpoD, in the context of the rpoD operon and the numerous promoters in that operon, is not required for efficient sporulation or germination, provided that the sigma-A level from a heterologous promoter is comparable to that in wild-type cells.

Mol Microbiol, 1996 Apr, 20(1), 1 - 7
Structure, function and controls in microbial division; Vicente M et al.; Several crucial genes required for bacterial division lie close together in a region called the dcw cluster . Within the cluster, gene expression is subject to complex transcriptional regulation, which serves to adjust the cell cycle in response to growth rate . The pivotally important FtsZ protein, which is needed to initiate division, is now known to interact with many other components of the division machinery in Escherichia coli . Some biochemical properties of FtsZ, and of another division protein called FtsA, suggest that they are similar to the eukaryotic proteins tubulin and actin respectively . Cell division needs to be closely co-ordinated with chromosome partitioning . The mechanism of partitioning is poorly understood, though several genes involved in this process, including several muk genes, have been identified . The min genes may participate in both septum positioning and chromosome partitioning . Coupled transcription and translation of membrane-associated proteins might also be important for partitioning . In the event of a failure in the normal partitioning process, Bacillus subtilis, at least, has a mechanism for removing a bisected nucleoid from the division septum.

Antimicrob Agents Chemother, 1996 Apr, 40(4), 852 - 7
Na+/H+ antiport activity conferred by Bacillus subtilis tetA(L), a 5' truncation product of tetA(L), and related plasmid genes upon Escherichia coli; Cheng J et al.; An Escherichia coli transformant expressing the Bacillus subtilis tetA(L) gene from a weak promoter was challenged by growth on medium with low, increasing tetracycline concentrations . Changes in the substrate preference ratios of the TetA(L)-mediated resistances and antiports were examined in view of recent findings suggesting that TetA(L) catalyzes efflux of Na+ in exchange for protons in addition to having the ability to catalyze metal-tetracycline/H+ antiport . After growth of the transformant on 1 microgram or more of tetracycline per ml for 12 to 15 h, the tetA(L) gene in the plasmid was found to be disrupted by an IS10 element 50 bp from the 5' end of the coding sequence . This disrupted recombinant plasmid, pKB1, conferred greater tetracycline resistance and higher levels of membrane metal-tetracycline/proton antiport than the original plasmid, pJTA1, but conferred lower NA+ resistance and Na+/H+ antiport levels than the original plasmid . The results indicate that the 5' end of the gene is necessary for optimal Na+/H+ antiport but that some such activity as well as robust tetracycline/H+ antiport persists in its absence . Two plasmid genes, tet(K) and qacA, were compared with tetA(L) vis-a-vis their abilities to enhance the Na+/H+ antiporter activity of everted vesicles from E . coli transformants . tet(K), which is more closely related to tetA(L), catalyzed 22Na+ uptake by energized vesicles, whereas the less closely related qacA gene did not.

Genes Dev, 1996 Apr 1, 10(7), 794 - 803
Bifunctional protein required for asymmetric cell division and cell-specific transcription in Bacillus subtilis; Feucht A et al.; During sporulation in Bacillus subtilis an asymmetric cell division gives rise to unequal progeny called the prepore and the mother cell . Gene expression in the prespore is initiated by cell-specific activation of the transcription factor sigma(F) . Three proteins participate in the regulation of sigma(F) activity . The first, SpoIIAB, is an inhibitor of sigma(F), that is, an anti-sigma factor . SpoIIAB is also a protein kinase that catalyzes phosphorylation of the second regulatory protein SpoIIAA (the anti-anti-sigma factor), and thus inactivates it . A third protein, SpoIIE, was shown recently to be able to dephosphorylate SpoIIAA-P in vitro . Here we show that SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate . First, we confirm by the use of in vivo experiments that it regulates the release of sigma(F) activity by dephosphorylating SpoIIAA-P . Second, we show that SpoIIE is needed for normal formation of the asymmetric septum that separates the prespore from the mother cell . Combination of these two functions in a single polypeptide may serve to couple the release of the cell-specific transcription factors with the formation of the differentiating cells.

Protein Sci, 1996 Apr, 5(4), 786 - 8
Crystallization and preliminary structural analysis of Bacillus subtilis adenylosuccinate lyase, an enzyme implicated in infantile autism; Redinbo MR et al.; Adenylosuccinate lyase (ASL) from Bacillus subtilis has been crystallized and structural analysis by X-ray diffraction is in progress . ASL is a 200-kDa homotetramer that catalyzes two distinct steps of de novo purine biosynthesis leading to the formation of AMP and IMP; both steps involve the beta-elimination of fumarate . A single point mutation in the human ASL gene has been linked to mental retardation with autistic features . In addition, ASL plays an important role in the bioprocessing of anti-HIV therapeutics . B subtilis ASL, which shares 30% sequence identity and 70% sequence similarity with human ASL, has been crystallized and data to 3.3 A have been collected at 100 K . The space group is P6(1)22 or P6(5)22 with a = b = 129.4 A; the length of the c-axis varies between 275 and 290 A, depending on the crystal . An analysis of solvent content indicates a dimer in the asymmetric unit, although a self-rotation function and an analysis of native Pattersons failed to identify unambiguously the location of any noncrystallographic symmetry axes . Structure determination by isomorphous replacement is in progress.

Biosci Biotechnol Biochem, 1996 Apr, 60(4), 630 - 3
Directed mutagenesis, Ser-56 to Pro, of Bacillus subtilis phosphatidylserine synthase drastically lowers enzymatic activity and relieves amplification toxicity in Escherichia coli; Saha SK et al.; Amino acid residue 56 of the phosphatidylserine synthase of Bacillus subtilis was changed from Ser to Pro by using modified primers in PCR amplification of its structural gene, pssBS . When an Escherichia coli mutant lacking its own phosphatidylserine synthase harbored a plasmid carrying this allele, the Mn(2+)-requiring Bacillus-type synthase activity, as assayed in vitro, was at least six-fold lower than that with the wild-type pssBS gene and the cellular phosphatidylethanolamine content was similarly lowered, indicating that the altered region of the enzyme is critically important for its activity . In contrast to the E . coli counterpart, amplification of the wild-type Bacillus enzyme increased both the relative and absolute contents of phosphatidylethanolamine and impaired cell growth . However, amplification of the mutant enzyme of the same level was much less toxic, implying that E . coli cells are more sensitive to the unbalanced accumulation of phosphatidylethanolamine than that of the hydrophobic enzyme molecules . Possible roles of the conserved region of the enzyme in its activity and the wild-type phospholipid composition in the proper membrane function are discussed.

Int J Food Microbiol, 1996 Apr, 29(2-3), 321 - 33
Control of ammonia formation during Bacillus subtilis fermentation of legumes; Allagheny N et al.; The control of ammonia formation during the Bacillus subtilis fermentation of autoclaved, roasted soybean cotyledons (Glycine max) and of autoclaved African locust bean cotyledons (Parkia spp.) was investigated . Addition of NaCl, 1.5 mol (kg wet cotyledons)-1, part way through the fermentation inhibited ammonia formation and softening of the cotyledons . Addition of glycerol, 1.7 mol (kg wet cotyledons)-1 part way through the fermentation inhibited alkalinisation and ammonia formation while allowing enzymic activity and softening of the cotyledons to continue . Restriction of the oxygen supply by incubating the cotyledons in a sealed container also prevented excessive ammonia production and increase in pH value . Fermentations conducted in sealed containers with an air to cotyledons ratio of approximately 130-175 ml air (g wet cotyledons)-1 supported good microbial growth and proteolysis without the formation of detectable ammonia aroma.

Zhonghua Liu Xing Bing Xue Za Zhi, 1996 Apr, 17(2), 95 - 8
{Preliminary study of microbiocide effect and its mechanism of electrolyzed oxidizing water}; Li XW et al.; Electrolyzed Oxidizing water (EO Water) is characterized by possessing higher oxidizing reduction potential (ORP), lower pH value and oxidizing potential . Under conditions of free organic matter, it was tested for microbiocide efficacy in laboratory . The results showed that EO water could completely kill all of the staphylococcus aureus and E . coli within 15 seconds, while for completely killing of spores of Bacillus subtilis Var . niger it would take 10 min . When it was used to destroy the antigenicity of HBsAg, 30 seconds was needed . The ORP and pH values of EO water were not obviously changed when stored in room-temperature with, airtight and light-free conditions for three weeks . Distilled water and physiological saline had little influence on the ORP and pH value of EO water, but organic matters and phosphates had greater influence upon the two values.

J Hosp Infect, 1996 Apr, 32(4), 283 - 94
Sporicidal activity of sodium dichloroisocyanurate, peroxygen and glutaraldehyde disinfectants against Bacillus subtilis; Coates D; The activity of sodium dichloroisocyanurate (NaDCC), peroxygen and glutaraldehyde disinfectants against spores of Bacillus subtilis NCTC 10073 was evaluated using suspension tests . The activity of aqueous solutions of NaDCC disinfectants increased with the level of available chlorine (av.Cl) but was considerably reduced by low levels of blood . Five percent Titan Sanitizer (1200 ppm av.Cl) achieved a > 10(5)-fold reduction in spore count (kill) in 3 h in the absence of blood but no kill in 3 h with 2% blood present . One percent Presept (3180 ppm av.Cl) achieved a > 10(5)-fold kill in 1 h in the absence of blood and in 2 h with 2% blood present . One percent Haz-Tab (5750 ppm av.Cl) achieved > 10(5)-fold kill in 5 min in the absence of blood and in 30 min with 2% blood present . One percent Virkon (peroxygen) achieved a 10(5)-fold kill in 2-3 h in the absence of blood but little kill in 3 h with 2% blood present . Nu-Cidex (peracetic acid) was rapid in action and tolerant of organic matter . A 24 h old solution achieved a > 10(5)-fold kill in 5 min with 10% serum present . Cidex Long-Life (glutaraldehyde) worked much slower; a 28-day-old solution achieved a > 10(5)-fold kill in 2 h with 4% blood present . Neat sporicidin (glutaraldehyde-phenate) was slightly superior to Cidex Long-Life but in a 1 in 8 dilution exhibited markedly reduced activity; 30-day-old solution achieved a 10(4)-fold kill in 10 h in the absence of blood but only a 10(2)-fold kill in 10 h with 2% blood present.

Mol Microbiol, 1996 Apr, 20(2), 339 - 50
Bacillus subtilis operon under the dual control of the general stress transcription factor sigma B and the sporulation transcription factor sigma H; Varon D et al.; The sigma B transcription factor of Bacillus subtilis is activated in response to a variety of environmental stresses, including those imposed by entry into the stationary-growth phase, and by heat, salt or ethanol challenge to logarithmically growing cells . Although sigma B is thought to control a general stress regulon, the range of cellular functions it directs remains largely unknown . Our approach to understand the physiological role of sigma B is to characterize genes that require this factor for all or part of their expression, i.e . the csb genes . In this study, we report that the transposon insertion csb40::Tn917lac identifies an operon with three open reading frames, the second of which resembles plant proteins induced by desiccation stress . Primer-extension and operon-fusion experiments showed that the csb40 operon has a sigma B-dependent promoter which is strongly induced by the addition of salt to logarithmically growing cells . The csb40 operon also has a second, sigma H-dependent promoter that is unaffected by salt addition . These results provide support for the hypothesis that sigma B controls a general stress regulon, and indicate that the sigma B and sigma H regulons partly overlap . We suggest that in addition to its acknowledged role in the sporulation process, sigma H is also involved in controlling a subclass of genes that are broadly involved in a general stress response.

Arch Microbiol, 1996 Apr, 165(4), 243 - 51
Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8; Tsuge K et al.; Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1 . A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8 . When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced . However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides . Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame . The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B . subtilis), lpa-14 (from iturin A- and surfactin-producing B . subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin . The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides.

J Bacteriol, 1996 Apr, 178(8), 2450 - 4
Autogenous regulation of the Bacillus subtilis glnRA operon; Brown SW et al.; Purified Bacillus subtilis GlnR was shown to bind with high affinity to a specific region that overlaps with the glnRA promoter site . The GlnR binding site includes four copies of a repeated sequence that may be the recognition site for the protein . GlnR inhibited transcription from the glnRA promoter in vitro.

J Bacteriol, 1996 Apr, 178(8), 2375 - 82
Properties of a Bacillus subtilis polynucleotide phosphorylase deletion strain; Wang W et al.; The pnpA gene of Bacillus subtilis, which codes for polynucleotide phosphorylase (PNPase), has been cloned and employed in the construction of pnpA deletion mutants . Growth defects of both B . subtilis and Escherichia coli PNPase-deficient strains were complemented with the cloned pnpA gene . RNA decay characteristics of the B . subtilis pnpA mutant were studied, including the in vivo decay of bulk mRNA and the in vitro decay of either poly(A) or total cellular RNA . The results showed that mRNA decay in the pnpA mutant is accomplished despite the absence of the major, Pi-dependent RNA decay activity of PNPase . In vitro experiments suggested that a previously identified, Mn2+ -dependent hydrolytic activity was important for decay in the pnpA mutant . In addition to a cold-sensitive-growth phenotype, the pnpA deletion mutant was found to be sensitive to growth in the presence of tetracycline, and this was due to an increased intracellular accumulation of the drug . The pnpA deletion strain also exhibited multiseptate, filamentous growth . It is hypothesized that defective processing of specific RNAs in the pnpA mutant results in these phenotypes.

J Bacteriol, 1996 Apr, 178(8), 2351 - 5
Characterization of cis-acting mutations in the first attenuator region of the Bacillus subtilis pyr operon that are defective in pyrimidine-mediated regulation of expression; Ghim SY et al.; A transcriptional attenuation mechanism for the regulation of pyr operon expression in Bacillus subtilis in which the PyrR regulatory protein binds pyr mRNA at three sites with similar sequences to cause transcription termination in response to elevated pyrimidine nucleotide pools has been proposed (R . J . Turner, Y . Lu, and R . L . Switzer, J . Bacteriol . 176:3708-3722, 1994) . Twenty-seven mutants with cis-acting defects in the repression by pyrimidines of beta-galactosidase expression of a pyr-lacZ fusion-integrant were isolated as blue colonies on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) agar plates containing uracil and uridine after UV irradiation or treatment with mutagens or following mutD mutagenesis . These mutants showed normal repression of the chromosomal pyr operon by exogenous pyrimidines . Sequence analysis revealed 12 unique sites of mutation, which occurred in the conserved putative PyrR binding sequence (10 of the 12) or in the stem of the transcriptional terminator structure . These mutants strongly support the proposed model for regulation of the pyr operon.

J Bacteriol, 1996 Apr, 178(8), 2343 - 50
A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis; Daniel RA et al.; We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B . The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli . Both B . subtilis genes are shown to be nonessential . The third B . subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E . coli . The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence . The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene . The operon is transcribed throughout growth at a low level.

J Bacteriol, 1996 Apr, 178(8), 2204 - 10
Analysis of the relationship between the decrease in pH and accumulation of 3-phosphoglyceric acid in developing forespores of Bacillus species; Magill NG et al.; Analysis of the pH decrease and 3-phosphoglyceric acid (3PGA) accumulation in the forespore compartment of sporulating cells of Bacillus subtilis showed that the pH decrease of 1 to 1.2 units at approximately 4 h of sporulation preceded 3PGA accumulation, as observed previously in B . megaterium . These data, as well as analysis of the forespore pH decrease in asporogenous mutants of B . subtilis, indicated that sigma G-dependent forespore transcription, but not sigma K-dependent mother cell transcription, is required for the forespore pH decrease . Further analysis of these asporogenous mutants showed an excellent correlation between the forespore pH decrease and the forespore's accumulation of 3PGA . These latter results are consistent with our previous suggestion that the decrease in forespore pH results in greatly decreased activity of phosphoglycerate mutase in the forespore, which in turn leads to 3PGA accumulation . In further support of this suggestion, we found that (i) elevating the pH of developing forespores of B . megaterium resulted in rapid utilization of the forespore's 3PGA depot and (ii) increasing forespore levels of PGM approximately 10-fold in B . subtilis resulted in a large decrease in the spore's depot of 3PGA . The B . subtilis strain with a high phosphoglycerate mutase level sporulated, and the spores germinated and went through outgrowth normally, indicating that forespore accumulation of a large 3PGA depot is not essential for these processes.

J Bacteriol, 1996 Apr, 178(7), 2150 - 3
A CUC triplet confers leucine-dependent regulation of the Bacillus subtilis ilv-leu operon; Marta PT et al.; Regulation of the ilv-leu operon probably involves interaction of a tR NA(GAG) with leader region mRNA . Conversion of a CUC (Leu) triplet located within the leader region to UUC (Phe), CGC (Arg), or UAC (Tyr) converted reporter gene expression to control by corresponding amino acids . Conversion of the CUC triplet to CUU (Leu) decreased expression and disrupted regulation . The results suggested that other tRNAs can substitute for tRNA(Leu) but that interactions in addition to pairing of the anticodon with the CUC triplet are important for proper control.

J Bacteriol, 1996 Apr, 178(7), 2079 - 85
Phenotypes of Bacillus subtilis mutants lacking multiple class A high-molecular-weight penicillin-binding proteins; Popham DL et al.; Examination of Bacillus subtilis strains containing multiple mutations affecting the class A high-molecular-weight penicillin-binding proteins (PBPs) 1, 2c, and 4 revealed a significant degree of redundancy in the functions of these three proteins . In rich media, loss of PBPs 2c and 4 resulted in no obvious phenotype . The slight growth and cell morphology defects associated with loss of PBP 1 were exacerbated by the additional loss of PBP 4 but not PBP 2c . Loss of all three of these PBPs slowed growth even further . In minimal medium, loss of PBPs 2c and 4 resulted in a slight growth defect . The decrease in growth rate caused by loss of PBP 1 was accentuated slightly by loss of PBP 2c and greatly by loss of PBP 4 . Again, a lack of all three of these PBPs resulted in the slowest growth . Loss of PBP 1 resulted in a 22% reduction in the cell radius . Cultures of a strain lacking PBP 1 also contained some cells that were significantly longer than those produced by the wild type, and some of the rod-shaped cells appeared slightly bent . The additional loss of PBP 4 increased the number of longer cells in the culture . Slow growth caused by a mutation in prfA, a gene found in an operon with the gene encoding PBP 1, was unaffected by the additional loss of PBPs 2c and 4, whereas loss of both prfA and PBP 1 resulted in extremely slow growth and the production of highly bent cells.

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