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 hyperthermophili