J Inorg Biochem, 2002 Jul 25, 91(1), 46 - 58 Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin; Ming LJ et al.; Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms . This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+) . Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently . This antibiotic has been suggested to influence cell functioning through more than one route . Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance . In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed. Gene, 2002 Jun 12, 292(1-2), 205 - 11 Identification and characterization of a site-specific tyrosine recombinase within the variable loci of Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae; Ron Y et al.; Three highly mutable loci of the wall-less pathogens Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae undergo high-frequency genomic rearrangements and generate extensive antigenic variation of major surface lipoproteins . Adjacent to each locus, an open reading frame exists as a single chromosomal copy and is predicted to encode a site-specific DNA recombinase exhibiting high homology to the recombinases XerD of Escherichia coli and CodV of Bacillus subtilis . Each of the mycoplasmal proteins are members of the lambda integrase family of tyrosine site-specific recombinases and likely mediates site-specific DNA inversions observed within the adjacent, variable loci. J Food Prot, 2002 Jul, 65(7), 1134 - 41 Immobilized bacterial spores for use as bioindicators in the validation of thermal sterilization processes; Serp D et al.; Spores of Bacillus subtilis ATCC 6051 and Bacillus stearothermophilus NCTC 10003 were immobilized in monodisperse alginate beads (diameter, 550 microm +/- 5%), and the capacity of the immobilized bioindicators to provide accurate and reliable F-values for sterilization processes was studied . The resistance of the beads to abrasion and heat was strong enough to ensure total retention of the bioindicators in the beads in a sterilization cycle . D- and z-values for free spores were identical to those for immobilized spores, which shows that immobilization does not modify the thermal resistance of the bioindicators . A D(100 degrees C) value of 1.5 min was found for free and immobilized B . subtilis spores heated in demineralized water, skimmed milk, and milk containing 4% fat, suggesting that a lipid concentration as low as 4% does not alter the thermal resistance of B . subtilis spores . Providing that the pH range is kept between 3.4 to 10 and that sufficiently low concentrations of Ca2+ competitors or complexants are present in the medium, immobilized bioindicators may serve as an efficient, accurate, and reliable tool with which to validate the efficiency of any sterilization process . The environmental factors (pH, media composition) affecting the thermoresistance of native contaminants are intrinsically reflected in the F-value, allowing for a sharper adjustment of the sterilization process . Immobilized spores of B . stearothermophilus were successfully used to validate a resonance and interference microwave system that is believed to offer a convenient alternative for the sterilization of temperature-sensitive products and medical wastes. J Comput Chem, 2002 Aug, 23(11), 1045 - 57 Modern protein force fields behave comparably in molecular dynamics simulations; Price DJ et al.; Several molecular dynamics simulations were performed on three proteins--bovine apo-calbindin D9K, human interleukin-4 R88Q mutant, and domain IIA of bacillus subtilis glucose permease--with each of the AMBER94, CHARMM22, and OPLS-AA force fields as implemented in CHARMM . Structural and dynamic properties such as solvent-accessible surface area, radius of gyration, deviation from their respective experimental structures, secondary structure, and backbone order parameters are obtained from each of the 2-ns simulations for the purpose of comparing the protein portions of these force fields . For one of the proteins, the interleukin-4 mutant, two independent simulations were performed using the CHARMM22 force field to gauge the sensitivity of some of these properties to the specific trajectory . In general, the force fields tested performed remarkably similarly with differences on the order of those found for the two independent trajectories of interleukin-4 with CHARMM22 . When all three proteins are considered together, no force field showed any consistent trend in variations for most of the properties monitored in the study . Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(5), 601 - 603 High Expression of Penicillin G Acylase Gene from Bacillus megaterium in Bacillus subtilis; Yang S et al.; The penicillin G acylase gene (pga) amplified by PCR from Bacillus megaterium was subcloned into an expressing vector pPZW103 (P43 as promoter) . The recombinant plasmid was transferred into Bacillus subtilis DB104 . Penicillin G acylase production in the B . subtilis transformant was 3-6 u/ml, higher than that of published recombinant strains . Penicillin G acylase production was induced by phenylacetic acid in B . megaterium, whereas the enzyme was produced constitutively in the B . subtilis transformant carrying B . megaterium pga . The recombinant strain showed high stability in antibiotic-free medium for 10 days . Enzyme in crude broth was purified by Al(2)O(3) chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography and the total yield is 79% . The purified enzyme with specific activity of 52 u/mg can be directly immobilized for use. Proteomics, 2002 Jun, 2(6), 649 - 55 Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp . strain 1 by a proteomic approach; Rabus R et al.; Pirellula sp . strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes . As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively . Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated . Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby) . Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells . Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp . strain 1 . This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B . subtilis . Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively . Thus the coding genes of three proteins expressed during growth of Pirellula sp . strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism. Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 211 - 6 Epub 2002 Apr 12. Construction of self-disruptive Bacillus megaterium in response to substrate exhaustion for polyhydroxybutyrate production; Hori K et al.; In order to establish a novel recovery system for polyhydroxyalkanoates, a self-disruptive strain of Bacillus megaterium that responds to substrate exhaustion was constructed . A gene cassette carrying the lysis system of Bacillus amyloliquefaciens phage - holin and endolysin - was inserted into the Escherichia coli- Bacillus subtilis shuttle vector pX under the control of a xylose-inducible expression system, xylR-xylA ' . In this system, the expression of a target gene is induced by xylose but inhibited by glucose, which acts as an anti-inducer . B . megaterium was transformed with pX conveying the phage lysis system, which was integrated into the amyE locus of chromosomal DNA of B . megaterium by homologous recombination . The lysis system caused self-disruption of the transformant cells effectively even when expression of the lysis genes was induced during stationary phase . For the production of polyhydroxybutyrate (PHB), the transformant was grown in a medium containing glucose as a substrate in the presence of xylose . When the glucose concentration approached zero, self-disruption was spontaneously induced, releasing intracellularly accumulated PHB into the culture broth . This system realizes timely cell disruption immediately after the PHB content in the cell reaches a maximum level. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(1), 74 - 76 Immobilized Tryptophanyl-tRNA Synthetase from Bacillus subtilis; Xu F et al.; In order to investigate the recognition mechanism and the relationship between structure and function of tRNA(Trp) with tryptophanyl-tRNA synthetase (TrpRS), TrpRS from Bacillus subtilis was purified and immobilized on CNBr-activated Sepharose 4B . Protein recovery and activity recovery of the immobilization were 95.5% and 31.3%, respectively . Properties of immobilized TrpRS were studied in detail . The thermal stability and the shelf stability of immobilized TrpRS were much higher than those of the native TrpRS . Besides these, the immobilized TrpRS, with good operation stability, had increased optimum temperature and optimum pH . A 56-base single-stranded RNA library containing 20 consecutive completely randomized bases was subjected to 3 successive rounds of immobilized TrpRS column selection with SELEX method, resulting in a sharp increase of the percentage of the RNA pool that could bind immobilized TrpRS from 4.3% for the first round pool to 14.7% for the third-round pool . After sequencing the third-round RNA pool, a RNA secondary structure resembling the structure of the acceptor stem in tRNA(Trp) was obtained though the selection . All the results indicated that immobilized TrpRS could be used as an affinity chromatography matrix and was qualified for the SELEX of a RNA pool simulating tRNA(Trp) molecule. FEMS Immunol Med Microbiol, 2002 Jul 12, 33(3), 179 - 89 Identification of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy; Burnie J et al.; The occurrence of an outbreak of septicaemias due to vancomycin-resistant Enterococcus faecium (VRE), in Manchester, UK, provided an opportunity to examine the antibody responses in patients infected by the same strain . Immunoblotting sera from 24 cases, six of whom died, showed an immunodominant cluster of antigens at 34, 54 and 97 kDa, with a statistically significant correlate between survival and immunoglobulin G to the 34 and 97 kDa bands (P<0.05) . Screening a genomic expression library of VRE with seropositive serum and peritoneal dialysate from a survivor gave a recombinant clone with two contiguous open reading frames, the derived amino acid sequences of which both showed sequence homologue with ABC transporters, with a Walker A and Walker B motif and the signature sequence LSGGQ . The first open reading frame (putative VRE ABC1) showed 57% homologue with YbxA from Bacillus subtilis . A partial sequence (putative VRE ABC2) was also obtained, in the same recombinant clone, of a second ABC transporter with 72% homologue with ybaE from B . subtilis . Affinity selection with the seropositive serum and peritoneal dialysate used to screen the library showed that the eluted antibody bound to the 97, 54, 34 and 30 kDa bands . Direct amino acid sequencing identified this as a possible ABC transporter . Rabbit antiserum against peptides representing Walker A and an area adjacent to the Walker B site cross-reacted with bands at 34, 54, 97, 110 kDa and at 30, 34 and 54 kDa respectively . This therefore appeared to be an immunodominant complex of ABC transporters of which the smallest was the 30 kDa antigen . Epitope mapping of this antigen with seropositive patients' sera delineated three linear epitopes (KVGIV, FGPKNF and RVAI) . The Walker A site represented by peptide 1 (GHNGSGKSTLAKTIN), epitope RVAI represented by peptides 2 (MRRVAIAGVLAMPRE) and 3 (ELSGGQMRRVAIAGV), epitope KVGIV represented by peptide 4 (LKPIRKKVGIVFQFP), and recombinant VRE ABC1 and VRE ABC2 expressed in Escherichia coli pBAD were then used to isolate human genetically recombinant antibodies from a phage antibody display library . An assessment of the protective potential of these antibodies was carried out in a mouse model of the infection . This study suggests that an ABC transporter homologue could be a target for antibody therapy against VRE infections. J Mol Evol, 2002 Aug, 55(2), 211 - 21 Analysis of the cellular functions of Escherichia coli operons and their conservation in Bacillus subtilis; de Daruvar A et al.; The common assumption of operons as composed of genes that cooperate in a biological process is confirmed here by showing that Escherichia coli operons tend to be composed of genes that belong to the same general class of cellular function . Furthermore, the comparison between the genomic organization of E . coli and that of Bacillus subtilis shows that the genes that are homologous to genes that belong to experimentally characterized E . coli operons tend to cluster in neighboring regions of the genome . This tendency is greater for the subset of E . coli operons whose genes belong to a single functional class . These observations indicate strong evolutionary pressure that, translated into functional constraints, leads to the inclusion of many essential functions in conserved operons and clusters in these two distant species. J Bacteriol, 2002 Aug, 184(15), 4316 - 20 Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase; Soldo B et al.; The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene . At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content . MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit. J Bacteriol, 2002 Aug, 184(15), 4288 - 95 Transcriptome and proteome analysis of Bacillus subtilis gene expression modulated by amino acid availability; Mader U et al.; A comprehensive study of Bacillus subtilis gene expression patterns in response to amino acid availability was performed by means of proteomics and transcriptomics . The methods of two-dimensional protein gel electrophoresis and DNA macroarray technology were combined to analyze cells exponentially grown in minimal medium with and without 0.2% Casamino Acids (CAA) . This approach revealed about 120 genes predominantly involved in amino acid biosynthesis, sporulation, and competence, which were downregulated in CAA-containing medium . Determination of sporulation frequencies confirmed the physiological relevance of the expression data. Microbiology, 2002 Jul, 148(Pt 7), 2079 - 87 tagO is involved in the synthesis of all anionic cell-wall polymers in Bacillus subtilis 168; Soldo B et al.; Sequence homologies suggest that the Bacillus subtilis 168 tagO gene encodes UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme responsible for catalysing the first step in the synthesis of the teichoic acid linkage unit, i.e . the formation of undecaprenyl-PP-N-acetylglucosamine . Inhibition of tagO expression mediated by an IPTG-inducible P(spac) promoter led to the development of a coccoid cell morphology, a feature characteristic of mutants blocked in teichoic acid synthesis . Indeed, analyses of the cell-wall phosphate content, as well as the incorporation of radioactively labelled precursors, revealed that the synthesis of poly(glycerol phosphate) and poly(glucosyl N-acetylgalactosamine 1-phosphate), the two strain 168 teichoic acids known to share the same linkage unit, was affected . Surprisingly, under phosphate limitation, deficiency of TagO precludes the synthesis of teichuronic acid, which is normally induced under these conditions . The regulatory region of tagO, containing two partly overlapping sigma(A)-controlled promoters, is similar to that of sigA, the gene encoding the major sigma factor responsible for growth . Here, the authors discuss the possibility that TagO may represent a pivotal element in the multi-enzyme complexes responsible for the synthesis of anionic cell-wall polymers, and that it may play one of the key roles in balanced cell growth. Mol Microbiol, 2002 Jul, 45(1), 179 - 90 Direct and indirect roles of CcpA in regulation of Bacillus subtilis Krebs cycle genes; Kim HJ et al.; Carbon catabolite repression of the Bacillus subtilis citrate synthase (citZ) and aconitase (citB) genes, previously known to be regulated by CcpC, was shown to depend on CcpA as well . Transcription of the citZ gene was partially derepressed in ccpA and ccpC single mutants and fully derepressed in a ccpA ccpC double mutant . DNase I footprinting studies showed that CcpA binds to a catabolite-responsive element (cre) site located at positions +80 to +97 with respect to the transcription start site, whereas CcpC binds at positions -14 to +6 and +16 to +36 . Mutations in the citZ cre site greatly altered CcpA binding and repression . A ccpA null mutation also caused partial derepression of citB . Disruption of citrate synthase activity, however, suppressed the effect of the ccpA mutation, suggesting that increased citrate accumulation in a ccpA mutant partially inactivates CcpC and causes partial derepression of citB . Therefore, CcpA controls expression of Krebs cycle genes directly by regulating transcription of citZ and in-directly by regulating availability of citrate, the inducer for CcpC. Mol Microbiol, 2002 Jul, 45(1), 123 - 9 Peptidyl-tRNA hydrolase in Bacillus subtilis, encoded by spoVC, is essential to vegetative growth, whereas the homologous enzyme in Saccharomyces cerevisiae is dispensable; Menez J et al.; Peptidyl-tRNA hydrolase in Escherichia coli, encoded by pth, is essential for recycling tRNA molecules sequestered as peptidyl-tRNA as a result of pre-mature dissociation from the ribosome during translation . Genes homologous to pth are present in other bacteria, yeast and man, but not in archaea . The homologous gene in Bacillus subtilis, spoVC, was first identified as a gene involved in sporulation . A second copy of spoVC, under the control of the xyl promoter, was integrated into B . subtilis at the amy locus . In this background, interruption of the original gene was possible provided that expression of the copy at the amy locus was induced . When spoVC was interrupted, both vegetative growth and sporulation were dependent on xylose, showing that SpoVC is essential . The role of SpoVC in sporulation is discussed and appears to be consistent with previous hypotheses that a relaxation of translational accuracy may occur during sporulation . The homologous gene in Saccharomyces cerevisiae, yHR189W, has been interrupted in both haploid and diploid strains . The mutant haploid strains remain viable, as do the yHR189W mutant spores obtained by tetrad dis-section, with either glucose or glycerol as carbon source, showing that the yHR189W gene product is dispensable for cell growth and for mitochondrial respiration. Mol Microbiol, 2002 Jul, 45(1), 73 - 87 Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis; Kadoya R et al.; Current views of bacterial chromosome segregation vary in respect of the likely presence or absence of an active segregation mechanism involving a mitotic-like apparatus . Furthermore, little is known about cis-acting elements for chromosome segregation in bacteria . In this report, we show that two separate DNA regions, a 3' coding region of dnaA and the AT-rich sequence between dnaA and dnaN (the initial opening site of duplex DNA during replication), are necessary for efficient segregation of the chromosome in Bacillus subtilis . When a plasmid replicon was integrated into argG, far from oriC, on the chromosome and then the oriC function was disrupted, the oriC-deleted mutant formed anucleate cells at 5% possibly because of defects in chromosome segregation . However, when the two DNA sequences were added near oriN, frequency of anucleate cells decreased to 1% . In these cells, the origin (argG) regions were localized near cell poles, whereas they were randomly distributed in cells without the two DNA sequences . These results suggest that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B . subtilis. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jul, 34(4), 457 - 62 Sequence-specific assignments of proton NMR resonance peaks and analysis of secondary structural elements of LC1, a novel antibacterial polypeptide; Shao CH et al.; LC1 is a type of novel antibacterial polypeptide secreted by a Bacillus subtilis strain . It consists of 47 residues . Using bioengineering, LC1 was expressed in E.coli DH5alpha by using recombinant plasmid PBVAB16 . By means of two-dimensional DQF-COSY, TOCSY and NOESY spectroscopies, protons of all 47 residues are identified . The studies show that the secondary structures of LC1 are principally anti-parallel beta sheets and extended conformations . It was speculated that there may be a hydrophobic core around Trp(23) in its three-dimensional structure. J Biochem (Tokyo), 2002 Jul, 132(1), 29 - 36 The importance of region 2.1 in sustaining the functional structure of the Bacillus subtilis sigma(A) factor; Liao CT et al.; Region 2.1 of the sigma factor is once proposed to be involved in core binding, and certain bulky hydrophobic amino acids in region 2.1 are thought to make contact with the conserved isoleucine residues in the promoter -10 binding region on the same protein . To examine the roles of the contact between these two regions in sigma(A) structure and function, sigma(A )factor with L145A, I149A, or Y153A was created, and the effects of each substitution on the growth of Bacillus subtilis and on the structural and functional properties of sigma(A) were analyzed . Our data revealed that the growth potential of B . subtilis was significantly affected by each of the substitutions of sigma(A) at elevated temperature . The growth defect was most pronounced with the strain containing L145A-sigma(A); it possessed a low growth potential even at 37 degrees C . In parallel, changes in the structural stability and core-binding activity of sigma(A) and in the promoter-binding and transcription activities of sigma(A)-RNA polymerase were observed for each of the substitutions, with the most drastic effects exerted by L145A . Clearly, region 2.1 of sigma(A) has extra functions, such as the binding of RNA polymerase to promoter DNA, other than the known core-binding ability . Moreover, the multiple effects of each of the substitutions on sigma(A) demonstrate that the contacts between the hydrophobic amino acids in region 2.1 and those in the promoter -10 binding region are critical to the maintenance of the functional sigma(A) structure and that L145 in region 2.1 plays an important role in this respect. Microbiology, 1997 Sep, 143(Pt 9), 2945 - 51 Activation of the CheA kinase by asparagine in Bacillus subtilis chemotaxis; Garrity LF et al.; Past experiments have shown that CheA and CheY are required to generate smooth swimming signals in Bacillus subtilis chemotaxis . This study, as anticipated from in vivo experiments, demonstrates in vitro that an attractant-bound chemoreceptor leads to an increase in CheA activity, which in turn leads to an increase in the CheY-P pool that ultimately causes a behavioural change in the bacteria . Asparagine has been found to increase the rate of CheY-P formation in the presence of McpB-containing membranes, CheA, and an excess of CheY . This asparagine effect requires the presence of both CheA and McpB, the latter of which has been shown to be the sole receptor for this attractant . Utilizing membranes from a number of B . subtilis null mutant strains, insight has also been gained into the potential roles of a number of unique chemotaxis proteins in the regulation of CheA activity in the presence and absence of this attractant. Biosci Biotechnol Biochem, 2002 May, 66(5), 986 - 95 Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108; Yoon HG et al.; The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size . The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6%, 48.0%, and 12.6% identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively . C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group . The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C . The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography . The half-life of the enzyme was 40 min at 90 degrees C . The purified protein was also highly stable, retaining above 50% residual activities during treatment with denaturants such as urea (8 M) and guanidine x HCl (4 M) at 37 degrees C for 30 min . The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product. Appl Environ Microbiol, 2002 Jul, 68(7), 3261 - 9 Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production; Wu SC et al.; To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis . Through a systematic study involving a series of B . subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA . Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner . The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B . subtilis strain, WB800HM{pEPP} . This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter . In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions . Secreted MH-1 SCA from WB800HM{pMH1, pEPP} could be affinity purified using a protein L matrix . It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody . This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems. Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8518 - 23 The rate-limiting step in the folding of a large ribozyme without kinetic traps; Fang XW et al.; A fundamental question in RNA folding is the nature of the rate-limiting step . Folding of large RNAs often is trapped by the need to undo misfolded structures, which precludes the study of the other, potentially more interesting aspects in the rate-limiting step, such as conformational search, metal ion binding, and the role of productive intermediates . The catalytic domain of the Bacillus subtilis RNase P RNA folds without a kinetic trap, thereby providing an ideal system to elucidate these steps . We analyzed the folding kinetics by using fluorescence and absorbance spectroscopies, catalytic activity, and synchrotron small-angle x-ray scattering . Folding begins with the rapid formation of early intermediates wherein the majority of conformational search occurs, followed by the slower formation of subsequent intermediates . Before the rate-limiting step, more than 98% of the total structure has formed . The rate-limiting step is a small-scale structural rearrangement involving prebound metal ions. J Biol Chem, 2002 Sep 6, 277(36), 32659 - 67 Epub 2002 Jun 24. Isolation and analysis of mutant alleles of the Bacillus subtilis HrcA repressor with reduced dependency on GroE function; Reischl S et al.; The hrcA gene of Bacillus subtilis codes for a transcriptional repressor protein that negatively regulates expression of the heptacistronic dnaK and the bicistronic groE operon by binding to an operator-element called CIRCE . Recently, we have published data suggesting that the activity of HrcA is modulated by the GroE chaperonin system . Biochemical analyses of the HrcA protein have been hampered so far by its strong tendency to aggregate . Here, a genetic method was used to isolate mutant forms of HrcA with increased activity under conditions of decreased GroE function . One of these mutant forms (HrcA114) containing five amino acid replacements exhibited enhanced solubility when overexpressed . HrcA114 purified under native conditions produced two retarded CIRCE-containing DNA fragments in band shift experiments . The amount of the larger fragment increased after addition of GroEL, GroES, and ATP but decreased when ATP was replaced by the nonhydrolyzable ATP analog ATPgammaS . DNase I footprinting experiments exhibited full protection of the CIRCE element and neighboring nucleotides in an asymmetric way . An in vitro binding assay using affinity chromatography showed direct and specific interaction between HrcA114 and GroEL . All these experimental data are in full agreement with our previously published model that HrcA needs the GroE chaperonin system for activation. J Bacteriol, 2002 Jul, 184(14), 4018 - 24 In vivo and in vitro studies of Bacillus subtilis ferrochelatase mutants suggest substrate channeling in the heme biosynthesis pathway; Olsson U et al.; Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway . The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known . Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have . The effects of these changes were studied in vivo and in vitro . S54 and Q63 are both located at helix alpha3 . The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure . None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure . The exchange S54A, but not Q63A, reduced the growth rate of B . subtilis and resulted in the accumulation of coproporphyrin III in the growth medium . This was in contrast to the in vitro activity measurements with the purified enzymes . The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V(max) . The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product. J Bacteriol, 2002 Jul, 184(14), 3923 - 30 RelA protein is involved in induction of genetic competence in certain Bacillus subtilis strains by moderating the level of intracellular GTP; Inaoka T et al.; We found that the ability to develop genetic competence of a certain relaxed (relA) aspartate-auxotrophic strain of Bacillus subtilis is significantly lower than that of the isogenic stringent (relA+) strain . Transcriptional fusion analysis utilizing a lacZ reporter gene indicated that the amount of the ComK protein, known as the key protein for competence development, is greatly reduced in the relaxed strain than in the stringent strain . We also found that the addition of decoyinine, a GMP synthetase inhibitor, induces expression of a competence gene (comG) in the relaxed strain, accompanied by a pronounced decrease in the level of intracellular GTP as measured by high-performance liquid chromatography . The transformation efficiency of the relaxed strain increased 100-fold when decoyinine was added at t0 (the transition point between exponential to stationary growth phase) . Conversely, supplementation of guanosine together with decoyinine completely abolished the observed effect of adding decoyinine on competence development . Furthermore, the impaired ability of the relaxed strain for competence development was completely restored by disrupting the codY gene, which is known to negatively control comK expression . Our results indicate that the RelA protein plays an essential role in the induction of competence development at least under certain physiological conditions by reducing the level of intracellular GTP and overcoming CodY-mediated regulation. J Bacteriol, 2002 Jul, 184(14), 3856 - 63 FtsA mutants of Bacillus subtilis impaired in sporulation; Kemp JT et al.; Spore formation in Bacillus subtilis involves a switch in the site of cell division from the midcell to a polar position . Both medial division and polar division are mediated in part by the actin-like, cytokinetic protein FtsA . We report the isolation of an FtsA mutant (FtsA(D265G)) that is defective in sporulation but is apparently unimpaired in vegetative growth . Sporulating cells of the mutant reach the stage of asymmetric division but are partially blocked in the subsequent morphological process of engulfment . As judged by fluorescence microscopy and electron microscopy, the FtsA(D265G) mutant produces normal-looking medial septa but immature (abnormally thin) polar septa . The mutant was unimpaired in transcription under the control of Spo0A, the master regulator for entry into sporulation, but was defective in transcription under the control of sigmaF, a regulatory protein whose activation is known to depend on polar division . An amino acid substitution at a residue (Y264) adjacent to D265 also caused a defect in sporulation . D265 and Y264 are conserved among endospore-forming bacteria, raising the possibility that these residues are involved in a sporulation-specific protein interaction that facilitates maturation of the sporulation septum and the activation of sigmaF. J Mol Biol, 2002 Jun 21, 319(5), 1035 - 48 RNA recognition by transcriptional antiterminators of the BglG/SacY family: mapping of SacY RNA binding site; Declerck N et al.; Transcriptional antiterminators of the BglG/SacY family are bacterial regulatory proteins able to prevent the premature arrest of transcription through specific binding to a ribonucleic antiterminator (RAT) sequence . The RNA recognition module of these regulators is made of the 55-amino acid long N-terminal domain which can by itself promote efficient antitermination activity in vivo and RNA binding in vitro . The structure of this domain, which was called CAT for co-antiterminator, has first been determined for SacY from Bacillus subtilis and the putative surface contacting RNA has been defined by NMR footprinting . Here we have performed a genetic mapping of the SacY-CAT RNA binding site by substituting 24 amino acid residues including those previously identified by NMR, the highly conserved residues in the 55 homologous antiterminators recognised in the databases and all the positively charged residues . A total of 57 SacY-CAT variants have been constructed and tested in vivo for their antitermination efficiency . A few of these variants were then purified in order to analyse their RNA binding properties by surface plasmon resonance and to check their structural integrity by NMR . The present study validates and clarifies the RNA interacting surface previously mapped by NMR . The residues that are the most intolerant to substitutions, Asn8, His9, Asn10, Gly25, Gly27, and Phe30, are aligned across the CAT dimer interface and form the core of the RNA binding site . Three highly conserved residues stand outside the interaction surface but are essential for maintaining the CAT dimeric structure (Phe47) or may play an important functional role in the full length protein (Glu20 and Lys32) . Interestingly, none of the twelve positively charged residues of SacY-CAT are crucial for the antitermination activity . By replacing three Lys residues and combining the Ala26-->Arg mutation that significantly enhanced the affinity for RNA, we engineered a SacY-CAT variant that should be suitable for NMR study of the complex . (c) 2002 Elsevier Science Ltd. Cell Physiol Biochem, 2002, 12(2-3), 127 - 34 DNA interacts with Bacillus subtilis mechano-sensitive channels in native membrane patches; Szabo I et al.; Much recent evidence indicates that systems devoted to the transmembrane transport of proteins and/or genetic material in bacteria comprise proteins capable of forming large pores as a key element . In several cases these pores have been observed in electrophysiological experiments after purification and reconstitution of the proteins in artificial bilayers . A comparison of their properties with those of large mechanosensitive channels observed by patch-clamping bacterial proto- or spheroplasts suggests that the latter may be formed by such transport machines . In support of this hypothesis, this paper reports that the properties of high-conductance channels in the membrane of Bacillus subtilis are altered by DNA through specific interactions . Thus, the previously demonstrated interaction between DNA and the same channels reconstituted in planar bilayers, which in that system results in the transmembrane translocation of the genetic material, takes place also in the native membrane . Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1168 - 74 Epub 2002 Jun 20. Alternate conformations observed in catalytic serine of Bacillus subtilis lipase determined at 1.3 A resolution; Kawasaki K et al.; Bacillus subtilis extracellular lipase (BsL) has an exceptionally low molecular weight (19.4 kDa) for a member of the lipase family . A crystallographic study was performed on BsL in order to design and produce mutant BsL that will be more suitable for industrial uses based on analysis of the three-dimensional structure . Recently, the crystal structure of BsL has been determined at 1.5 A resolution {van Pouderoyen et al . (2001) . J . Mol . Biol . 309, 215-226} . In the present study, a new crystal form of BsL which provides diffraction data to higher resolution was obtained and its structure was determined at 1.3 A using the MAD method . It was found that the active-site residue Ser77 has alternate side-chain conformations . The O(gamma) atom of the first conformer forms a hydrogen bond to the N(epsilon) atom of His155, a member of the catalytic triad . In contrast, the second conformer is constructed with a hydrogen bond to the side-chain atom of the adjacent His76 . These two conformers presumably correspond to the active and inactive states, respectively . Similar alternate conformations in the catalytic serine residue have been observed in Fusarium solani cutinase determined at 1.0 A resolution and Penicillium purpurogenum acetylxylan esterase at 0.9 A resolution . In addition, a glycerol molecule, which was used as a cryoprotectant, is found to be located in the active site . On the basis of these results, a model for substrate binding in the reaction-intermediate state of BsL is proposed. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1138 - 46 Epub 2002 Jun 20. NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution; Symersky J et al.; The final step of NAD+ biosynthesis includes an amide transfer to nicotinic acid adenine dinucleotide (NaAD) catalyzed by NAD+ synthetase . This enzyme was co-crystallized in microgravity with natural substrates NaAD and ATP at pH 8.5 . The crystal was exposed to ammonium ions, synchrotron diffraction data were collected and the atomic model was refined anisotropically at 1 A resolution to R = 11.63% . Both binding sites are occupied by the NAD-adenylate intermediate, pyrophosphate and two magnesium ions . The atomic resolution of the structure allows better definition of non-planar peptide groups, reveals a low mean anisotropy of protein and substrate atoms and indicates the H-atom positions of the phosphoester group of the reaction intermediate . The phosphoester group is protonated at the carbonyl O atom O7N, suggesting a carbenium-ion structure stabilized by interactions with two solvent sites presumably occupied by ammonia and a water molecule . A mechanism is proposed for the second catalytic step, which includes a nucleophilic attack by the ammonia molecule on the intermediate. FEMS Microbiol Lett, 2002 Jun 4, 211(2), 219 - 23 AbrB is a regulator of the sigma(W) regulon in Bacillus subtilis; Qian Q et al.; The Bacillus subtilis global regulator AbrB was found to negatively control expression of sigW and genes of the sigma(W) regulon . AbrB bound to DNA regions in the autoregulatory sigW promoter and to some, but not all, of the other sigma(W)-dependent promoters in B . subtilis . Defects in antibiotic resistance properties caused by spo0A mutations are at least partially correlated with AbrB repression of the sigma(W) regulon. Acta Microbiol Immunol Hung, 2002, 49(1), 21 - 35 Stress related changes of cell surface hydrophilicity in Bacillus subtilis; Kovacs T et al.; The changes of cell surface hydrophilicity in Bacillus subtilis were analyzed in response to oxygen-limitation, heat shock, salt stress, pH-shock, phosphate- and carbon-limitation . Although cell surface hydrophilicity varied during growth phases, an increase of surface hydrophilicity was observed under several of these stress conditions . An observed drop in intracellular GTP and/or ATP may be an element of the signal transduction pathway leading to an increase in surface hydrophilicity in response to environmental stresses . Attachment of cells to soil particles under salt stress conditions is strongly influenced by the degS/degU two-component system, which thereby provides a mechanism for the bacteria to escape from the hostile environment. Appl Microbiol Biotechnol, 2002 Jun, 59(1), 9 - 14 Epub 2002 Apr 16. Biochemistry and molecular genetics of poly-gamma-glutamate synthesis; Ashiuchi M et al.; Current research into poly-gamma-glutamate (PGA) and its biosynthesis is reviewed . In PGA-producing Bacillus subtilis, glutamate racemase supplies abundant DL-glutamate, the substrate for PGA synthesis . The pgsBCA genes of PGA-producing B . subtilis, which encode the membrane-associated PGA synthetase complex PgsBCA, were characterized and the enzyme complex was suggested to be an atypical amide ligase based on its structure and function . A novel reaction mechanism of PGA synthesis is proposed. Mol Genet Genomics, 2002 May, 267(3), 391 - 400 Epub 2002 Apr 13. The beta-propeller protein YxaL increases the processivity of the PcrA helicase; Noirot-Gros MF et al.; The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli . These helicases have been extensively studied in vitro and their mode of unwinding are well characterised . However, little is known about the putative cellular partners of such helicases . To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library . Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA . The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro . YxaL enhanced the processivity of the PcrA helicase . A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller" . This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component. J Am Chem Soc, 2002 Jun 26, 124(25), 7324 - 30 Mimicry of host-defense peptides by unnatural oligomers: antimicrobial beta-peptides; Porter EA et al.; We have designed beta-amino acid oligomers that are helical, cationic, and amphiphilic with the intention of mimicking the biological activity of amphiphilic, cationic alpha-helical antimicrobial peptides found in nature (e.g., magainins) . We have previously identified a 17-residue beta-peptide (called beta-17) with antibiotic activity similar to that of a magainin derivative against four bacterial species, including two clinical isolates that are resistant to common antibiotics . This beta-peptide displays very low hemolytic activity against human red blood cells, which indicates selectivity for bacterial cells over mammalian cells . Here we examine some of the factors important for activity in this class of beta-peptides . An amphiphilic helix is necessary, because a nonamphiphilic isomer proved to be inactive . The ratio of cationic to hydrophobic residues is also important . Active beta-peptides induce the leakage of beta-galactosidase from treated Bacillus subtilis cells, as do alpha-helical antibiotic peptides, and this similarity suggests that the beta-peptide mode of action involves disruption of microbial membranes . This class of beta-peptides is not degraded by proteases, which bodes well for biological applications. Protein Expr Purif, 2002 Jun, 25(1), 149 - 59 Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli; Jin HJ et al.; The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to escape from the antibiotics' action, and this mechanism is predominantly adopted by microorganisms resistant to MLS antibiotics . ErmSF methyltransferase is one of the four gene products synthesized by Streptomyces fradiae NRRL 2338 to be resistant to its autogenous antibiotic, tylosin . In order to have a convenient source for the purification of milligram amounts, we expressed ErmSF in Escherichia coli using a T7 promoter-driven expression vector system, pET 23b, and the protein was expressed with a carboxy-terminal addition of six histidine residues in order to facilitate purification . Expression at 22 degrees C reduced the formation of insoluble aggregate, inclusion body, and resulted in accumulation of soluble hexahistidine-ErmSF up to 30% of total cell protein after 18 h . Metal-chelation chromatography yielded 126 mg of hexahistidine-ErmSF per liter of culture with a purity slightly greater than 95% . To examine the function of ErmSF in vivo and in vitro, its activity in E . coli (antibiotic susceptibility assay) andin vitro methyltransferase activity using in vitro-produced B . subtilis domain V, 434-, 257-, and 243-nt RNAs were investigated . The ErmSF in E . coli conferred resistance to erythromycin, whereas E . coli harboring an empty vector, pET23b, was susceptible . The purified recombinant protein successfully methylated domain V of 23S rRNA, which is known to contain all of the substrate elements recognized and to be methylated by erm proteins . However, the truncated substrates were methylated with decreased efficiencies . Almost all of domain V was monomethylated with less than 0.2 pM S-{methyl-(3)H}adenosylmethionine concentration . The roles of three structurally divided regions of domain V in recognition and methylation by ErmSF are proposed through kinetic studies using RNA substrates, in which each region is deleted, under the monomethylation condition . Biochemistry, 2002 Jun 25, 41(25), 8087 - 92 Three G.C base pairs required for the efficient aminoacylation of tRNATrp by tryptophanyl-tRNA synthetase from Bacillus subtilis; Xu F et al.; Acceptor stem is an essential region in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase . In this study, a library containing 20 nt random region and tryptophanyl-tRNA synthetase (TrpRS) from Bacillus subtilis were used for in vitro selection to find a new structural feature in the tRNA(Trp) acceptor stem sequence that is required for B . subtilis TrpRS recognition . After three rounds of selection, the TrpRS binding RNAs dominate the RNA pool . The aptamers share a common structure of three G.C base pairs, which was also found in the acceptor stem of wild-type B . subtilis tRNA(Trp) . A series of tRNA(Trp) variants was prepared by in vitro transcription, and their efficiencies of tryptophanylation (k(cat)/K(M)) were measured with the aid of TrpRS from B . subtilis . The mutants that possess the three G.C base pairs and G73 discriminator base exhibit almost the same aminoacylation efficiencies as B . subtilis tRNA(Trp), while the G73 discriminator base itself cannot confer efficient aminoacylation to the tRNA(Trp) molecule . Thus, these three base pairs (G2.C71, G3.C70, and G4.C69) in the B . subtilis tRNA(Trp) acceptor stem were established to be new identity elements, and their importance was between the previously characterized major element G73 and minor elements A1/U72 and G5/C68 . The minimum set of identity elements that is required to confer efficient aminoacylation by B . subtilis TrpRS included G73, G2.C71, G3.C70, and G4.C69. Mol Microbiol, 2002 Jun, 44(6), 1561 - 73 Specific activation of the Bacillus quorum-sensing systems by isoprenylated pheromone variants; Ansaldi M et al.; Natural genetic competence in Bacillus subtilis is controlled by quorum-sensing (QS) . The ComP- ComA two-component system detects the signalling molecule ComX, and this signal is transduced by a conserved phosphotransfer mechanism . ComX is synthesized as an inactive precursor and is then cleaved and modified by ComQ before export to the extracellular environment . The comQXP' loci of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum-sensing response . We have developed a simple purification method for the modified peptide signalling pheromones allowing the characterization of four distinct ComX molecules representing different pherotypes . Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the post-translational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various pherotypes . These results give new insights into peptidemediated quorum-sensing signalling in Gram-positive bacteria and emphasize the role of isoprenylation in bacterial signal transduction. Mol Microbiol, 2002 Jun, 44(6), 1455 - 67 Interaction surface of the Spo0A response regulator with the Spo0E phosphatase; Stephenson SJ et al.; Spo0A~P is the essential response regulator and transcription factor for sporulation initiation in Bacillus subtilis . The phosphorylation level of Spo0A in the cell is determined by the sensor kinase activity of the phosphorelay, donating phosphoryl groups, and the antagonistic effects of dephosphorylation mediated by the Rap and Spo0E families of phosphatases . In this study, spo0A mutations were generated that encoded proteins less sensitive to the activity of Spo0E than the wild-type protein . The Spo0A substitutions N12K, P60S, L62P and F88L are surface exposed and localize to the same face of the molecule as the active site and in its close proximity on the beta1-alpha1, beta3-alpha3 and beta4-alpha4 loops . The corresponding surface in the Spo0F response regulator was shown previously to be involved in the interaction with the RapB phosphatase, as well as the KinA histidine kinase and the Spo0B phosphotransferase . Thus, residues occupying the same position (N12:Q12, F88:Y84) and the same loops in Spo0A or Spo0F are involved in the interaction with the structurally unrelated Spo0E and RapB phosphatases, respectively, in addition to kinases and phosphotransferase . The specificity in phosphatase target recognition must be the result of side-chain variability within the response regulators and the interactions they promote . The residues involved in Spo0E interaction are identical in all Spo0A orthologues from spore-forming Bacilli encoding Spo0E phosphatases. EMBO J, 2002 Jun 17, 21(12), 3137 - 47 Essential bacterial helicases that counteract the toxicity of recombination proteins; Petit MA et al.; PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature . PcrA is encoded by Gram-positive bacteria and is essential for cell growth . Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable . To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation . Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation . Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant . We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent. Curr Biol, 2002 Jun 4, 12(11), R391 - 2 Bacterial sporulation: FtsZ rings do the twist; Margolin W; Formation of the polar Z ring is a crucial step in the establishment of cellular asymmetry during sporulation of Bacillus subtilis . New results suggest that the transition from medial to polar Z rings involves a dynamic FtsZ spiral structure that may transfer FtsZ from medial to polar sites. Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 8342 - 7 An expanded view of bacterial DNA replication; Noirot-Gros MF et al.; A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays . The network consists of 91 specific interactions linking 69 proteins . Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report . These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways . They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control . Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication. Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8701 - 6 Epub 2002 Jun 11. Evidence that subcellular localization of a bacterial membrane protein is achieved by diffusion and capture; Rudner DZ et al.; Bacteria lack an endoplasmic reticulum, a Golgi apparatus, and transport vesicles and yet are capable of sorting and delivering integral membrane proteins to particular sites within the cell with high precision . What is the pathway by which membrane proteins reach their proper subcellular destination in bacteria? We have addressed this question by using green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVFB) that is involved in the process of sporulation in the bacterium Bacillus subtilis . SpoIVFB-GFP localizes to a region of the sporulating cell known as the outer forespore membrane, which is distinct from the cytoplasmic membrane . Experiments are presented that rule out a mechanism in which SpoIVFB-GFP localizes to all membranes but is selectively eliminated from the cytoplasmic membrane by proteolytic degradation and argue against a model in which SpoIVFB-GFP is selectively inserted into the outer forespore membrane . Instead, the results are most easily compatible with a model in which SpoIVFB-GFP achieves proper localization by insertion into the cytoplasmic membrane followed by diffusion to, and capture in, the outer forespore membrane . The possibility that diffusion and capture is a general feature of protein localization in bacteria is discussed. Biochem J, 2002 Sep 15, 366(Pt 3), 929 - 36 PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity; Iwanicki A et al.; Bacillus subtilis is a Gram-positive bacterium with a relatively large number of protein phosphatases . Previous studies have shown that some Ser/Thr phosphatases play an important role in the life cycle of this bacterium {Losick and Stragier (1992) Nature (London) 355, 601-604; Yang, Kang, Brody and Price (1996) Genes Dev . 10, 2265-2275} . In this paper, we report the biochemical properties of a putative, previously uncharacterized phosphatase, PrpE, belonging to the PPP family . This enzyme shares homology with other PPP phosphatases as well as with symmetrical diadenosine tetraphosphatases related to ApaH (symmetrical Ap(4)A hydrolase) from Escherichia coli . A His-tagged recombinant PrpE was purified from E . coli and shown to have Ni(2+)-dependent and okadaic acid-resistant phosphatase activity against a synthetic phosphorylated peptide and hydrolase activity against diadenosine 5',5"'-tetraphosphate . Unexpectedly, PrpE was able to remove phosphate from phosphotyrosine, but not from phosphothreonine or phosphoserine. J Bacteriol, 2002 Jul, 184(13), 3664 - 70 Multiple pathways of Spx (YjbD) proteolysis in Bacillus subtilis; Nakano S et al.; ATP-dependent proteases degrade denatured or misfolded proteins and are recruited for the controlled removal of proteins that block activation of regulatory pathways . Among the ATP-dependent proteases, those of the Clp family are particularly important for the growth and development of Bacillus subtilis . Proteolytic subunit ClpP, together with regulatory ATPase subunit ClpC or ClpX, is required for the normal response to stress, for development of genetic competence, and for sporulation . The spx (formally yjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred by clpP and clpX . The level of Spx in wild-type cells grown in competence medium is low, and that in clpP mutants is high . This suggests that the Spx protein is a substrate for ClpP-containing proteases and that accumulation of Spx might be partly responsible for the observed pleiotropic phenotype resulting from the clpP mutation . In this study we examined, both in vivo and in vitro, which ClpP protease is responsible for degradation of Spx . Western blot analysis showed that Spx accumulated in clpX mutant to the same level as that observed in the clpP mutant . In contrast, a very low concentration of Spx was detected in a clpC mutant . An in vitro proteolysis experiment using purified proteins demonstrated that Spx was degraded by ClpCP but only in the presence of one of the ClpC adapter proteins, MecA or YpbH . However, ClpXP, either in the presence or in the absence of MecA and YpbH, was unable to degrade Spx . Transcription of spx, as measured by expression of spx-lacZ, was slightly increased by the clpX mutation . To exclude a possible effect of clpX and clpP on spx transcription, the spx gene was placed under the control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible Pspac promoter . In this strain, Spx accumulated when ClpX or ClpP was absent, suggesting that ClpX and ClpP are required for degradation of Spx . Taken together, these results suggest that Spx is degraded by both ClpCP and ClpXP . The putative proteolysis by ClpXP might require another adapter protein . Spx probably is degraded by ClpCP under as yet unidentified conditions . This study suggests that the level of Spx is tightly controlled by two different ClpP proteases. Biochemistry, 2002 Jun 18, 41(24), 7763 - 70 Plant chromosomal HMGB proteins efficiently promote the bacterial site-specific beta-mediated recombination in vitro and in vivo; Stemmer C et al.; In the presence of an accessory DNA bending protein, the bacterial site-specific beta recombinase catalyzes resolution and DNA inversion . Five different maize high mobility group B (HMGB) proteins were examined for their potential to facilitate beta recombination in vitro using DNA substrates with different intervening distances (73-913 bp) between two directly oriented recombination (six) sites . All analyzed HMGB proteins (HMGB1 to HMGB5) could promote beta recombination, but depending on the DNA substrate with different efficiencies . The HMGB1 protein displayed an activity comparable to that of the natural promoting protein Hbsu, whereas the other HMGB proteins were less effective . Phosphorylation of the HMGB1 protein resulted in an increased efficiency of HMGB1 to promote beta recombination . Analyses of DNA substrates with closely spaced six sites demonstrated that in the presence of HMGB1 the recombination rate was correlated to the distance between the six sites, but independent of the helical orientation of the six sites . Using a Bacillus subtilis strain defective in Hbsu, the coexpression of beta recombinase and HMGB1 (or a truncated HMGB1 derivative) revealed that a plant HMG-box domain protein is sufficient for assisting beta to catalyze recombination in vivo . Our results using beta recombination as a model system suggest that the various plant HMGB proteins (and their posttranslationally modified versions) have the potential of forming a repertoire of different DNA structures, which is compatible with the idea that the HMGB proteins can act as architectural factors in a variety of nucleoprotein structures. Biochemistry, 2002 Jun 18, 41(24), 7659 - 69 Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution; Anand R et al.; Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide . We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate . The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins . Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication . Each oxalate decarboxylase cupin domain contains one manganese binding site . Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide . Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar . Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues . The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase . We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate . Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25 . The function of the other domain in oxalate decarboxylase is not yet known. Microbiology, 2002 Jun, 148(Pt 6), 1805 - 11 HPr kinase/phosphatase of Bacillus subtilis: expression of the gene and effects of mutations on enzyme activity, growth and carbon catabolite repression; Hanson KG et al.; HPr kinase/phosphatase (HPrK/P) is the key protein in regulation of carbon metabolism in Bacillus subtilis and many other Gram-positive bacteria . Whether this enzyme acts as a kinase or phosphatase is determined by the nutrient status of the cell . Mutational analysis of residues in a Walker A box nucleotide-binding motif revealed that it is not only important for kinase but is also involved in phosphatase activity . In addition, a signature sequence specifically conserved among HPrK/P orthologues is required for phosphatase activity and may be involved in interaction with HPr/HPr-(Ser46)-P . Carbon catabolite repression was abolished in a B . subtilis strain expressing a mutant form of HPrK/P deficient in kinase and phosphatase activities . The growth characteristics of this strain were similar to those of the wild-type . In contrast, B . subtilis strains expressing HPrK/P with partial kinase and no phosphatase activities showed growth impairment but exhibited catabolite repression. Microbiology, 2002 Jun, 148(Pt 6), 1795 - 803 A 5' stem-loop and ribosome binding but not translation are important for the stability of Bacillus subtilis aprE leader mRNA; Hambraeus G et al.; The Bacillus subtilis aprE leader is a determinant of extreme mRNA stability . The authors examined what properties of the aprE leader confer stability on an mRNA . The secondary structure of the aprE leader mRNA was analysed in vitro and in vivo, and mutations were introduced into different domains of an aprE leader-lacZ fusion . The half-lives of the corresponding transcripts were determined and beta-galactosidase activities were measured . Removal of a stem-loop structure at the 5' end or diminishing the strength of the RBS reduced the half-lives from more than 25 min to about 5 min . Interfering with translation by abolishing the start codon or creating an early stop codon had no or little effect on mRNA stability . The authors conclude that a 5' stem-loop and binding of ribosomes are necessary for the stability of aprE leader mRNA . The present results, together with a number of other data, suggest that translation of a B . subtilis mRNA is generally not important for its stability; the situation seems different in Escherichia coli . It is further concluded that the calculated strength of a B . subtilis RBS cannot be used to predict the stability of the corresponding transcript. Microbiology, 2002 Jun, 148(Pt 6), 1785 - 94 Swarming motility in Bacillus cereus and characterization of a fliY mutant impaired in swarm cell differentiation; Senesi S et al.; This report describes a new behavioural response of Bacillus cereus that consists of a surface-induced differentiation of elongated and hyperflagellated swarm cells exhibiting the ability to move collectively across the surface of the medium . The discovery of swarming motility in B . cereus paralleled the isolation of a spontaneous non-swarming mutant that was found to carry a deletion of fliY, the homologue of which, in Bacillus subtilis, encodes an essential component of the flagellar motor-switch complex . However, in contrast to B . subtilis, the fliY mutant of B . cereus was flagellated and motile, thus suggesting a different role for FliY in this organism . The B . cereus mutant was completely deficient in chemotaxis and in the secretion of the L2 component of the tripartite pore-forming necrotizing toxin, haemolysin BL, which was produced exclusively by the wild-type strain during swarm-cell differentiation . All the defects in the fliY mutant of B . cereus could be complemented by a plasmid harbouring the B . cereus fliY gene . These results demonstrate that the activity of fliY is required for swarming and chemotaxis in B . cereus, and suggest that swarm-cell differentiation is coupled with virulence in this organism. Biochem Biophys Res Commun, 2002 May 31, 294(1), 71 - 5 Isoprene synthase activity parallels fluctuations of isoprene release during growth of Bacillus subtilis; Sivy TL et al.; Isoprene is a volatile metabolite of uncertain function in plants, animals, and bacteria . Here, we demonstrate that the isoprene-producing bacterium, Bacillus subtilis, contains an isoprene synthase activity that catalyzes dimethylallyl diphosphate-dependent isoprene formation . Although the enzyme was very labile, it was demonstrated in both permeabilized cells and in partially purified cell extracts . Its activity was optimal at pH 6.2, required low levels of a divalent cation, and appears distinct from chloroplast isoprene synthases . When grown in a bioreactor, B . subtilis cells released isoprene in three distinct phases; using permeabilized cells, it was shown that isoprene synthase activity rose and fell in parallel with each phase . These results suggest that isoprene synthesis is highly regulated in B . subtilis and further research in this model system may shed light on the role of isoprene formation in biological systems. Biochem Biophys Res Commun, 2002 May 3, 293(2), 857 - 61 A novel and potent ribonuclease from fruiting bodies of the mushroom Pleurotus pulmonarius; Ye XY et al.; A ribonuclease (RNase), with an N-terminal sequence different from those of ribonucleases from the mushrooms Irpex lacteus, Lentinus edodes, Pleurotus ostreatus, Pleurotus tuber-regium, and Volvariella volvacea, was purified from fruiting bodies of the edible mushroom Pleurotus pulmonarius . The N-terminal sequence of P . pulmonarius RNase manifested homology to a portion of the sequences of ribosome inactivating protein abrin-b, abrin-c, and abrin-d, and Bacillus subtilis transcriptional regulator . The ribonuclease was adsorbed on Affi-gel blue gel, CM-Sepharose, and Mono S . It displayed a molecular mass of 14.4 kDa in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75 . The ribonuclease exhibited an activity of 25 114 U/mg on yeast tRNA . The highest ribonucleolytic activity was demonstrated toward poly C, followed by poly A, and then by poly G . There was no activity toward poly U . The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C . It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 0.33 nM . Mutat Res, 2002 Jun 19, 503(1-2), 77 - 84 Base-change mutations induced by various treatments of Bacillus subtilis spores with and without DNA protective small, acid-soluble spore proteins; del Carmen Huesca Espitia L et al.; Previous work has shown that spores of wild-type Bacillus subtilis are more resistant to killing by dry and wet heat, low vacuum lyophilization and hydrogen peroxide than are spores lacking the majority of their DNA protective alpha/beta-type small, acid-soluble spore proteins (SASP) (termed alpha(-)beta(-) spores) . These four treatments kill alpha(-)beta(-) spores in large part by DNA damage with accompanying mutagenesis, but only dry heat kills wild-type spores by DNA damage and mutagenesis . DNA sequence analysis of nalidixic acid-resistant (nal(r)) mutants generated by these treatments has now shown that the nal(r) mutations are base changes in the gyrA gene that encodes one subunit of DNA gyrase . Analysis of the DNA sequence of the gyrA gene in a large number of nal(r) mutants also indicates that: (1) base changes induced by hydrogen peroxide and wet heat in alpha(-)beta(-) spores are similar to those in spontaneous nal(r) mutants with only a few notable differences; (2) base changes induced by dry heat in wild-type spores and low vacuum lyophilization of alpha(-)beta(-) spores are similar, and include a high level of a tandem base change seen previously only in spores treated with very high vacuum and (3) base changes induced by lyophilization and dry heat are very different from those in spontaneous mutants in wild-type and alpha(-)beta(-) spores, which exhibit only one significant difference . While the initial DNA damage generated in spores by dry heat, lyophilization or high vacuum is almost certainly different than that generated by hydrogen peroxide or wet heat, the precise nature of the DNA damage remains to be determined. Arch Biochem Biophys, 2002 Jun 1, 402(1), 1 - 13 The 4-oxalocrotonate tautomerase family of enzymes: how nature makes new enzymes using a beta-alpha-beta structural motif; Whitman CP; 4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers . The enzyme is part of a plasmid-encoded pathway, which enables bacteria harboring the plasmid to use various aromatic hydrocarbons as their sole sources of carbon and energy . Among isomerases and enzymes in general, 4-OT is unusual for two reasons: it has one of the smallest known monomer sizes (62 amino acids) and the amino-terminal proline functions as the catalytic base . In addition to Pro-1, three other residues (Arg-11, Arg-39, and Phe-50) have been identified as critical catalytic residues by kinetic analysis, site-directed mutagenesis, chemical synthesis, NMR, and crystallographic studies . Arginine-39 functions as the general acid catalyst (assisted by an ordered water molecule) in the reaction while Arg-11 plays a role in substrate binding and facilitates catalysis by acting as an electron sink . Finally, the hydrophobic nature of the active site, which lowers the pK(a) of Pro-1 to approximately 6.4 and provides a favorable environment for catalysis, is largely maintained by Phe-50 . 4-OT is also the title enzyme of the 4-OT family of enzymes . The chromosomal homologues in this family are composed of monomers ranging in size from 61 to 79 amino acids, which code a beta-alpha-beta structural motif . The homologues all retain Pro-1 and generally have an aromatic or hydrophobic amino acid at the Phe-50 position . Characterization of representative members has uncovered mechanistic and structural diversity . A new activity, a trans-3-chloroacrylic acid dehalogenase, has been identified in addition to the previously known tautomerase and isomerase activities . Two new structures have also been found, along with the 4-OT hexamer . The dehalogenase functions as a heterohexamer while the Escherichia coli homologue, designated YdcE, functions as a dimer . Moreover, both 4-OT and the Bacillus subtilis homologue, designated YwhB, exhibit low-level dehalogenase activity . Amplification of this activity could have produced the full-fledged dehalogenase . The sum of these observations indicates that Nature uses the beta-alpha-beta structural motif as a building block in a variety of manners to create new enzymes. FEMS Microbiol Lett, 2002 May 7, 210(2), 193 - 9 Identification and characterization of the Bacillus subtilis D-glucarate/galactarate utilization operon ycbCDEFGHJ; Hosoya S et al.; In the course of the Bacillus subtilis functional genomics project, an open reading frame called ycbG whose product is classified as a transcriptional regulatory protein with a helix-turn-helix motif in the putative D-glucarate/galactarate utilization operon (ycbCDEFGHJ) was initially screened as the gene disruptant that exhibits a defect that blocked the early stage of sporulation . However, the transcription of ycbCDEFG was extremely highly induced in response to nutrient exhaustion by the disruption of ycbG, but inactivation of the transcription from upstream ycbC in the ycbG mutant restored the sporulation efficiency, suggesting that the inappropriate over-production of the ycbCDEFG gene products inhibits efficient sporulation . We further analyzed the role of the ycbCDEFGHJ cluster and found that (i) a unit of ycbCDEFGHJ was induced by either D-glucarate or D-galactarate, and (ii) the cell growth was inhibited by the mutation of the ycbF and ycbH genes, that respectively encode the putative proteins, D-glucarate dehydratase and D-galactarate dehydratase on plates supplemented with D-glucarate and D-galactarate, respectively, as the sole carbon source . Our results indicate that the ycbCDEFGHJ genes are involved in the utilization of D-glucarate and D-galactarate in B . subtilis. FEMS Microbiol Lett, 2002 May 7, 210(2), 173 - 9 Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis; Rothkamp A et al.; Two novel Brachyspira hyodysenteriae-specific DNA fragments, designated as Bh100 and Bh400, were identified using representational difference analysis . To isolate the fragments the combined DNA of the Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens reference strains was subtracted from the genome of B . hyodysenteriae strain B204 . Both fragments were present in a single copy and mapped to different positions on the genome of B . hyodysenteriae B78(T) . Larger fragments encompassing the continuous open reading frames (ORF) of Bh100 and Bh400 were cloned and analysed . Whereas the ORF of 2130 bp encompassing Bh100 did not show homology to any known bacterial protein, Bh400 was part of a putative operon with significant homology to the phosphotransferase system of Bacillus subtilis. J Antimicrob Chemother, 2002 Jun, 49(6), 917 - 24 Molecular characterization of the genes encoding DNA gyrase and topoisomerase IV of Listeria monocytogenes; Lampidis R et al.; The genes encoding subunits A and B of DNA gyrase and subunits C and E of topoisomerase IV of Listeria monocytogenes, gyrA, gyrB, parC and parE, respectively, were cloned and sequenced . Compared with the sequences of quinolone-susceptible bacteria, such as Escherichia coli and Bacillus subtilis, the quinolone resistance-determining region (QRDR) of DNA gyrase subunit A was altered; the deduced amino acid sequences revealed the substitutions Ser-84-->Thr and Asp/Glu-88-->Phe, two amino acid variations at hot spots, commonly associated with resistance to quinolones . No relevant divergences from QRDR consensus sequences were observed in GyrB or both topoisomerase IV subunits . Thus, it could be argued that the amino acid substitutions in GyrA would explain the intrinsic resistance of L . monocytogenes to nalidixic acid . In order to analyse the actual role of the GyrA alterations, a plasmid-encoded gyrA allele was mutated and transformed into L . monocytogenes . However, these heterodiploid strains were not affected in their resistance to nalidixic acid . The effects of the mutant plasmids on ciprofloxacin and sparfloxacin susceptibility were only modest. Appl Environ Microbiol, 2002 Jun, 68(6), 3172 - 5 Mechanisms of induction of germination of Bacillus subtilis spores by high pressure; Paidhungat M et al.; Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa . However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination . Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid . These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination. Anal Chem, 2002 May 15, 74(10), 2233 - 9 Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry; Soga T et al.; A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described . To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable . A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface . Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0 . The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas . The concentration detection limits for these metabolites were between 0.3 and 6.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3 . This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified. Adv Space Res, 2000, 26(12), 1995 - 2003 Spore dosimetry of solar UV radiation: applications to monitoring of daily irradiance and personal exposure; Munakata N et al.; Environmental UV radiation can be quantified using spore dosimetry, which measures the inactivation of repair-deficient Bacillus subtilis spores dried on a membrane filter . The system exhibits highly selective sensitivity to UV radiation, not being affected by various environmental adversities, such as high and low temperature and humidity . Biologically-effective dose rate and cumulative dose of ambient radiation are measurable under various conditions at various places on the earth, including tropical, temperate, and polar sites . Applications to monitor the exposure at the surface of organisms including humans and plants have also been advanced . c2001 COSPAR Published by Elsevier Science Ltd . All rights reserved. Biosci Biotechnol Biochem, 2002 Apr, 66(4), 892 - 6 Med, a cell-surface localized protein regulating a competence transcription factor gene, comK, in Bacillus subtilis; Ogura M et al.; Med was found as a positive regulator for comK, a master regulator for late competence genes . It was found by Western analysis that the ComK level was decreased in a med mutant . Experiments using an alkaline phosphatase fusion with Med and Western analysis of Med were done because a putative lipo-modification signal is found at the N-terminus of Med . The results obtained are consistent with the localization of Med at the cell surface . An implication of the cell-surface localization of Med is discussed in terms of comK regulation. J Chem Ecol, 2002 Apr, 28(4), 755 - 68 Activation of soil respiration and shift of the microbial population balance in soil as a response to Lavandula stoechas essential oil; Vokou D et al.; Lavandula stoechas, a native plant of Greece, is rich in essential oil and fenchone is its major constituent . We examined the effect of the essential oil and its main constituents on soil metabolism and microbial growth . Addition of the essential oil or fenchone to soil samples induced a remarkable increase in soil respiration . This was accompanied by an increase in the soil bacterial population of three orders of magnitude . This sizable population was not qualitatively similar to that of the control soil samples . One bacterial strain dominated soil samples treated with L . stoechas essential oil or fenchone . By use of the disk diffusion assay, we evaluated the capacity of three bacterial strains that we isolated from the soil samples, as well as Escherichia coli and Bacillus subtilis (reference strains), to grow in the presence of the essential oil and three of its main constituents (fenchone, cineol, alpha-pinene) . The substances tested did not inhibit the growth of the strain found to dominate the bacterial populations of treated soil samples; they severely inhibited B . subtilis . The other two isolated strains could also grow in liquid cultures in the presence of different quantities of essential oil or fenchone . Addition of fenchone at the end of the exponential phase increased the cell numbers of the strain that dominated the bacterial populations of treated soil samples, indicating use of the substrate added . On the basis of these results, we propose a scheme of successional stages during the decomposition process of the rich-in-essential-oil litter of aromatic plants that abound in the Mediterranean environment. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 687 - 690 Homology Modeling of Bacillus subtilis Tryptophanyl-tRNA Synthetase; Chen L et al.; Tryptophanyl-tRNA synthetase (TrpRS) plays a pivotal role in protein synthesis . However, till now no stereostructural data of Bacillus subtilis TrpRS were reported . Here, by using the homology modeling using Bacillus stearothermophilus TrpRS as a template, it is demonstrated that the synthetase has 16alpha helices and 5beta sheets . The only tryptophan Trp(92) is located on the interface of subunits . Also the modeling presents the ligand binding site, active site and the putative binding of tRNA(Trp). Nucleic Acids Res, 2002 Jun 1, 30(11), 2280 - 9 Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase, G40P, interacts with forked DNA; Ayora S et al.; SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication . Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner . The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors . Nuclease protection studies suggest that G40P protects the 5' tail of a forked molecule, and the duplex region at the junction against exonuclease attack . G40P does not protect the 3' tail of a forked molecule from exonuclease attack . By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring . Our results show that hexameric G40P DNA helicase encircles the 5' tail, interacts with the duplex DNA at the ss-double-stranded DNA junction and excludes the 3' tail of the forked DNA. Biophys Chem, 2002 May 2, 96(2-3), 173 - 90 Thermodynamics of a diffusional protein folding reaction; Perl D et al.; The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory . For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration . Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters . After viscosity correction the activation barriers for folding become less enthalpic and more entropic . The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction . It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity . Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding . The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state . As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB. Microb Ecol, 2001 Feb, 41(4), 301 - 309 Development of gfp Vectors for Expression in Listeria monocytogenes and Other Low G+C Gram Positive Bacteria; Qazi SN et al.; The gfp (green fluorescent protein) gene has previously been used to construct a variety of reporter plasmids for Gram-positive bacteria for bacterial localization and gene expression studies . When a native red-shifted gfp variant (gfp3) was cloned into an expression vector using the Pxyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy . When the Pxyn promoter was replaced with the PxylA promoter, with accompanying modification of the translation initiation region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained . When expressed in other Gram-positive organisms, such as Staphylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacterial population . High-level expression of these reporter constructs in L . monocytogenes was evaluated to determine if it had any detrimental biological effect during intracellular infection of eukaryotic cell lines . The gfp3+ Listeria were found to invade equally as well as the wild-type cells; showing that these expression systems can be used to monitor the bacterium in natural environments . Based on these results, similar translationally enhanced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L . monocytogenes and therefore can be used as a sensitive monitor of gene expression. Biochim Biophys Acta, 2002 Jun 7, 1576(1-2), 30 - 8 A mutation in GerE that affects cotC promoter activation in Bacillus subtilis; Crater DL et al.; The DNA-binding protein GerE acts as both a repressor and an activator of transcription of genes transcribed by sigma(K)-RNA polymerase (RNA-P) during the later stages of endospore formation in Bacillus subtilis . GerE represses transcription from the sigK promoter, and activates transcription from other promoters, including cotC and cotX . Two different regions of GerE (AR1 and AR2) are required for activation of cotC and cotX, respectively . We used a genetic screen to seek mutations that would define additional regions of GerE required for promoter activation . We found that a substitution of proline for leucine at position 12 of GerE (L12P) decreased cotC promoter activity but did not interfere with GerE-dependent repression of the sigK promoter or with activation of the cotX promoter in vivo . We also found that the L12P substitution had no effect on binding to cotC in vitro . However, the L12P-substituted GerE failed to stimulate cotC transcription in vitro, whereas it stimulated transcription from PcotX . The crystal structure of GerE suggests that L12 is not exposed on the surface of the molecule . Therefore, we propose that the L12P substitution reduces the flexibility of the N-terminal arm, preventing an interaction of AR1 with RNA-P that is essential for activation of the cotC promoter. Arch Microbiol, 2002 Jun, 177(6), 433 - 40 Epub 2002 Apr 03. Control of transcription termination in bacteria by RNA-binding proteins that modulate RNA structures; Stulke J; During the past few years, our knowledge of gene regulation by RNA-binding proteins has greatly increased . RNA-binding proteins are involved in processes such as protection of RNAs from RNase degradation, prevention of ribosome binding to mRNA, control of formation of secondary structures of the mRNA that permit or prevent translation initiation, and termination/antitermination of transcription in response to external signals . Modulation of transcription termination by RNA-binding proteins involves the formation of alternative structures . One of the structures can act as a transcriptional terminator, while adoption of the alternative structure prevents formation of the terminator and does thus result in transcript elongation . Which of the two structures prevails under a given condition depends on two factors: the intrinsic stability of the alternative structures and the stabilization of one of both by an RNA-binding regulatory protein . Binding of a protein to the nascent mRNA may result in transcript elongation, as is the case for cold-shock proteins or in several catabolic operons . The RNA-binding ability of the RNA-binding proteins is modulated by direct interaction with the inducer, by protein-protein interactions with sensor proteins or by protein phosphorylation . In contrast, in the pyrimidine or tryptophan biosynthetic operons of Bacillus subtilis, the transcriptional terminators are stabilized by RNA-binding proteins resulting in the absence of expression of these operons. J Bacteriol, 2002 Jun, 184(12), 3276 - 86 Regulation of the Bacillus subtilis fur and perR genes by PerR: not all members of the PerR regulon are peroxide inducible; Fuangthong M et al.; PerR is a ferric uptake repressor (Fur) homolog that functions as the central regulator of the inducible peroxide stress response in Bacillus subtilis . PerR has been previously demonstrated to regulate the mrgA, katA, ahpCF, hemAXCDBL, and zosA genes . We now demonstrate that PerR also mediates both the repression of its own gene and that of fur . Whereas PerR-mediated repression of most target genes can be elicited by either manganese or iron, repression of perR and fur is selective for manganese . Genetic studies indicate that repression of PerR regulon genes by either manganese or iron requires PerR and is generally independent of Fur . Indeed, in a fur mutant, iron-mediated repression is enhanced . Unexpectedly, repression of the fur gene by manganese appears to require both PerR and Fur, but only PerR binds to the fur regulatory region in vitro . The fur mutation appears to act indirectly by affecting cellular metal ion pools and thereby affecting PerR-mediated repression . While many components of the perR regulon are strongly induced by hydrogen peroxide, little, if any, induction of fur and perR could be demonstrated . Thus, not all components of the PerR regulon are components of the peroxide stimulon . We suggest that PerR exists in distinct metallated forms that differ in DNA target selectivity and in sensitivity to oxidation . This model is supported by the observation that the metal ion composition of the growth medium can greatly influence the transcriptional response of the various PerR regulon genes to hydrogen peroxide. J Bacteriol, 2002 Jun, 184(12), 3232 - 41 Transcription analysis of the Bacillus subtilis PucR regulon and identification of a cis-acting sequence required for PucR-regulated expression of genes involved in purine catabolism; Beier L et al.; The PucR protein of Bacillus subtilis has previously been suggested to regulate the expression of 15 genes, pucABCDE, pucFG, pucH, pucI, pucJKLM, pucR, and gde, all of which encode proteins involved in purine catabolism . When cells are grown under nitrogen-limiting conditions, the expression of these genes is induced and intermediary compounds of the purine catabolic pathway affect this expression . By using pucR deletion mutants, we have found that PucR induces the expression of pucFG, pucH, pucI, pucJKLM, and gde while it represses the expression of pucR and pucABCDE . Deletions in the promoters of the five induced operons and genes combined with bioinformatic analysis suggested a conserved upstream activating sequence, 5'-WWWCNTTGGTTAA-3', now named the PucR box . Potential PucR boxes overlapping the -35 and -10 regions of the pucABCDE promoter and located downstream of the pucR transcription start point were also found . The positions of these PucR boxes are consistent with PucR acting as a negative regulator of pucABCDE and pucR expression . Site-directed mutations in the PucR box upstream of pucH and pucI identified positions that are essential for the induction of pucH and pucI expression, respectively . Mutants with decreased pucH or increased pucR expression obtained from a library of clones containing random mutations in the pucH-to-pucR intercistronic region all contained mutations in or near the PucR box . The induction of pucR expression under nitrogen-limiting conditions was found to be mediated by the global nitrogen-regulatory protein TnrA . In other gram-positive bacteria, we have found open reading frames that encode proteins similar to PucR located next to other open reading frames encoding proteins with similarity to purine catabolic enzymes . Hence, the PucR homologues are likely to exert the same function in other gram-positive bacteria as PucR does in B . subtilis. Lett Appl Microbiol, 2002, 34(6), 394 - 7 Simultaneous inactivation of the wprA and dltB genes of Bacillus subtilis reduces the yield of alpha-amylase; Stephenson K et al.; AIMS: In Gram-positive bacteria, signal peptide-bearing secretory proteins are translocated through the cytoplasmic membrane and fold into their native conformation on the outside of the cell . The products of the Bacillus subtilis wprA and dltB genes separately influence post-translocational stages of the secretion process by mediating proteolytic degradation and folding of secretory proteins . Inactivation of either wprA or dltB in B . subtilis increases the yield of secretory proteins released into the culture medium in an intact and biologically active conformation . The aim of this work was to study the combined influence of these genes . METHODS AND RESULTS: A wprA/dltB double mutant was constructed, but did not have an additive effect on secretion and caused a significant reduction in the yield of alpha-amylase . CONCLUSIONS AND SIGNIFICANCE: The activities of the wprA gene and the dlt operon interact in a negative way to influence the growth cycle and protein secretion . The mechanism by which this may occur, and its potential significance for the secretion of native and non-native proteins from B . subtilis and related bacteria, is discussed. Mol Microbiol, 2002 Jun, 44(5), 1341 - 9 Spx (YjbD), a negative effector of competence in Bacillus subtilis, enhances ClpC-MecA-ComK interaction; Nakano MM et al.; ComK, a key transcriptional regulator in the development of competence in Bacillus subtilis, is required for its own transcription as well as that of the late competence genes encoding proteins involved in DNA uptake . ComK is sequestered in a complex with ClpC and MecA until a peptide, ComS, accumulates in cells . ComS releases ComK from the inhibitory complex, thus allowing ComK to carry out its function as a transcriptional activator . Spx (formerly YjbD), a negative effector of competence, accumulates in clpP mutants . High concentrations of Spx may be responsible for the inability of clpP mutants to become competent because spx mutations are able to restore competence in the clpP mutant . In this paper, we showed, based on in vitro experiments, that Spx forms a quaternary complex with ClpC, MecA and ComK and enhances ComK binding to ClpC-MecA . Two ComS alanine substitution mutants (I13A and W43A), previously shown to be defective in vivo, were less efficient in releasing ComK from ClpC-MecA . The I13A mutant with a weaker binding affinity to MecA was inefficient in releasing ComK regardless of whether Spx was present . In contrast, the defect of the W43A mutant in dissociating ComK was more readily observed in the presence of Spx . Spx is a highly conserved protein among Gram-positive bacteria, in which it may function closely with the protease adaptor protein, MecA. J Nat Prod, 2002 May, 65(5), 730 - 3 New metabolites from sponge-derived fungi Curvularia lunata and Cladosporium herbarum; Jadulco R et al.; The fungus Curvularia lunata, isolated from the marine sponge Niphates olemda, yielded the new 1,3,8-trihydroxy-6-methoxyanthraquinone, which we named lunatin (1), the known modified bisanthraquinone cytoskyrin A (2), and the known plant hormone (+)-abscisic acid (3) . Both anthraquinones were found to be active against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli . Two strains of the fungus Cladosporium herbarum, isolated from the sponges Aplysina aerophoba and Callyspongia aerizusa, respectively, yielded two new alpha-pyrones, herbarin A (4) and herbarin B (5), the known compound citreoviridin A (6), and the new phthalide herbaric acid (7) . All structures were unambiguously established by 1D and 2D NMR and MS data. Drug Dev Ind Pharm, 2002 Mar, 28(3), 329 - 37 Characterization of khaya gum as a binder in a paracetamol tablet formulation; Odeku OA et al.; The influence of khaya gum, a binding agent obtained from Khaya grandifolia (Meliaceae family), on the bulk, compressional, and tabletting characteristics of a paracetamol tablet formulation was studied in comparison with the effects of two standard binders: polyvinylpyrrolidone (PVP; molecular weight 40,000) and gelatin . The relative ability of khaya gum to destroy any residual microbial contamination in the binder or in the formulation during tabletting was also studied using Bacillus subtilis spores as a model . Formulations containing khaya gum exhibited more densification than formulations containing PVP and gelatin during die filling, but less densification due to rearrangement at low pressures . The mean yield pressure of the formulation particles obtained from Heckel plots, and another pressure term, also inversely related to plasticity, obtained from Kawakita plots, showed dependence on the nature and concentration of the binder, with formulations containing khaya gum exhibiting the lowest and highest values respectively . The values of the pressure terms suggest that the yield pressure relates to the onset of plastic deformation during compression, while the Kawakita pressure relates to the total amount of plastic deformation occurring during the compression process . Tablets made from formulations containing khaya gum had the lowest tensile strength values but also the lowest tendency to laminate or cap, as indicated by their lowest brittleness . All the tablets had friability values < 1% at higher concentrations of the three binders . In addition, khaya gum demonstrated a comparable ability to destroy microorganisms in the formulation during tabletting as the two binders . The characterization of the formulations suggests that khaya gum can be developed into a commercial binding agent for particular tablets. Mikrobiologiia, 2002 Mar-Apr, 71(2), 255 - 7 {Soil strain of Bacillus subtilis harboring a large plasmid that mediates high-frequency conjugal mobilization}; Lotareva OV et al.; The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcus plasmid, pUB110, was studied . The latter plasmid was transferred to the recipient cells of Bacillus subtilis 168 at a high frequency (about 10(-2) per recipient cell) both on filter surface and in liquid medium . Mobilization was initiated 40 to 50 min after the beginning of the contact between donor and recipient cells. FEMS Microbiol Lett, 2002 Apr 23, 210(1), 157 - 64 Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene; Kaito C et al.; A protein encoded by the Staphylococcus aureus dnaC gene has 44% and 58% homology with Escherichia coli DnaB and Bacillus subtilis DnaC replicative DNA helicases, respectively . We identified five mutant strains whose temperature-sensitive colony formation phenotypes were complemented by the dnaC gene . DNA replication in these mutants has a fast-stop phenotype, indicating that the S . aureus dnaC gene encodes the replicative DNA helicase required for the elongation step . These mutants were also sensitive to UV irradiation, suggesting that the dnaC gene is involved in DNA repair . The number of viable mutant cells decreased at a non-permissive temperature, suggesting that S . aureus DnaC helicase is a promising target for antibiotics providing bactericidal effects. BMC Microbiol . 2002 Apr 25;2(1):8. The methionine salvage pathway in Bacillus subtilis; Sekowska A et al.; BACKGROUND: Polyamine synthesis produces methylthioadenosine, which has to be disposed of . The cell recycles it into methionine through methylthioribose (MTR) . Very little was known about MTR recycling for methionine salvage in Bacillus subtilis . RESULTS: Using in silico genome analysis and transposon mutagenesis in B . subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins . The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression . Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling . CONCLUSIONS: A complete methionine salvage pathway exists in B . subtilis . This pathway is chemically similar to that in K . pneumoniae, but recruited different proteins to this purpose . In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway . A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs . In addition to methionine salvage, this pathway protects B . subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane). Mol Microbiol, 2002 May, 44(3), 685 - 94 The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP beta-lactamase; Filee P et al.; The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP beta-lactamases by beta-lactam antibiotics occurs according to similar processes . In both bacteria, the products of the blaI and blaRl genes share a high degree of sequence homology and act as repressors and penicillin-sensory transducers respectively . It has been shown in S . aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer . In the present study,we followed the fate of BlaI during beta-lactamase induction in B . licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B . licheniformis blaP, blaI and blaRl genes . In contrast to the situation in B . licheniformis 749/I, beta-lactamase induction in B.subtilis 168/pDML995 was not correlated with the proteolysis of BlaI . To exclude molecular variations undetectable by SDS-PAGE, two-dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B . subtilis 168/pDML995cells . No variation in the Blal isoelectric point was observed in induced cells, whereas the DNA-binding property was lost . Cross-linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B . subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions . This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non-covalent modification by a co-activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon . In addition to the presence of this co-activator, our results show that the presence of penicillin stress is also required for full induction of beta-lactamase biosynthesis. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 338 - 40 {Molecular cloning and expression of Nattokinase gene in Bacillus subtilis}; Liu BY et al.; In order to characterize biochemically the nattokinase,the nucleotide sequence of the nattokinase gene was amplified from the chromosomal DNA of B.subtilis (natto) by PCR . The expression plasmid pBL NK was constructed and was used to transform Bacillus subtilis containing a chromosomal deletion in its subtilisin gene . The supernatant of the culture was collected after 15 h culture . The target proteins were identified by SDS-PAGE . Nattokinase was purified by a method including ultrafiltration, Sephacryl S-100 gel filtration and S-Sepharose ion-exchange chromatography, and 100 mg of purified nattokinase was obtained from one liter of culture . The purity of the protein and the specific activity were 95% and 12 000 u/mg (compared to tPA), respectively. Appl Biochem Biotechnol, 2002 Spring, 98-100, 803 - 13 Development of continuous surfactin production from potato process effluent by Bacillus subtilis in an airlift reactor; Noah KS et al.; The biosurfactant surfactin has the potential to aid in the recovery of subsurface organic contaminants (environmental remediation) or crude oils (oil recovery) . However, high medium and purification costs limit its use in these high-volume applications . In previous work, we showed that surfactin can be produced from an inexpensive low-solids (LS) potato process effluent with minimal amendments or pretreatments . Previous research has also shown that 95% or more of the surfactin in Bacillus subtilis cultures can be recovered by foam fractionation . In this work, we present the results of research to integrate surfactin production with foam fractionation . Experiments were performed in an airlift reactor, with continuous collection of the foam through a tube at the top of the column . Preliminary results using both purified potato starch and unamended low-solids potato process effluent as substrates for surfactin production indicate that the process is oxygen limited and that recalcitrant indigenous bacteria in the potato process effluent may hamper continuous surfactin production. Appl Biochem Biotechnol, 2002 Spring, 98-100, 539 - 51 The effect of composition of parenteral solution on the thermal resistance of Bacillus stearothermophilus and Bacillus subtilis spores; Penna TC et al.; Large-volume parenteral solutions were submitted to heat treatments after being inocul