Bacillus subtilis

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 inoculated with Bacillus stearothermophilus ATCC 7953 (Tr = 121 degrees C) and Bacillus subtilis ATCC 9372 (Tr = 104.5 degrees C) spores . The average decimal reduction time for B . stearothermophilus ranged from a D121 degrees C value of 1.31 to 3.14 min, in glucophysiologic and Ringer's solutions respectively . For B . subtilis, D104.5 degrees C value increased from 0.69 to 1.37 min, in Ringer's (pH=5.91) and 50% glucose (pH 3.05) solutions respectively . The z value ranged from 7.95 degrees C (20% mannitol solution) to 13.14 degrees C (50% glucose solution), corresponding to an activation energy (Ea) of 81.48 and 49.30 kcal/mol, respectively.

Dev Cell, 2002 May, 2(5), 519 - 21
Unexpected twist to the Z ring; Lutkenhaus J; Development in Bacillus subtilis involves a switch in the location of the cytokinetic Z ring from midcell to the pole . Time lapse photography of an FtsZ-GFP fusion reveals that this switch involves a spiral intermediate and allows identification of the specific sporulation functions involved.

J Biol Chem, 2002 Jul 12, 277(28), 25356 - 62 Epub 2002 May 13.
Bacillus subtilis CheD is a chemoreceptor modification enzyme required for chemotaxis; Kristich CJ et al.; The chemotaxis machinery of Bacillus subtilis is similar to that of the well characterized system of Escherichia coli . However, B . subtilis contains several chemotaxis genes not found in the E . coli genome, such as cheC and cheD, indicating that the B . subtilis chemotactic system is more complex . In B . subtilis, CheD is required for chemotaxis; the cheD mutant displays a tumbly phenotype, has abnormally methylated chemoreceptors, and responds poorly to most chemical stimuli . Homologs of B . subtilis CheD have been found in chemotaxis-like operons of a large number of bacteria and archaea, suggesting that CheD plays an important role in chemotactic sensory transduction for many organisms . However, the molecular function of CheD has remained unknown . In this study, we show that CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B . subtilis chemoreceptor McpA . In addition, we present evidence that CheD deamidates other B . subtilis chemoreceptors including McpB and McpC . Previously, deamidation of B . subtilis receptors was thought to be catalyzed by the CheB methylesterase, as is the case for E . coli receptors . Because cheD mutant cells do not respond to most chemoattractants, we conclude that deamidation by CheD is required for B . subtilis chemoreceptors to effectively transduce signals to the CheA kinase.

J Appl Microbiol, 2002, 92(6), 1105 - 15
Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures; Melly E et al.; AIMS: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition . METHODS AND RESULTS: Bacillus subtilis spores prepared at temperatures from 22 to 48 degrees C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures . As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures . Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox . However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v . radiation and dry heat . The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures . In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased . However, this latter change was not due to a reduction in cotA gene expression at higher temperatures . CONCLUSIONS: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition . SIGNIFICANCE AND IMPACT OF THE STUDY: The precise conditions for the formation of B . subtilis spores have a large effect on many spore properties.

J Appl Microbiol, 2002, 92(6), 1051 - 7
Carton sterilization by u.v.-C excimer laser light: recovery of Bacillus subtilis spores on vegetable extracts and food simulation matrices; Warriner K et al.; AIMS: To determine the recovery of Bacillus subtilis spores loaded onto preformed cartons and irradiated with u.v.-excimer laser (248 nm) light . METHODS AND RESULTS: Bacillus subtilis spores irradiated with u.v.-excimer laser light retained phase brightness, but were blocked at various stages of germination . In the presence of germinant, the majority of spores began to lose phase brightness but only after an extended lag period (ca 90 min) . After 6 h ca 9% of the spores had elongated but failed to form new cells, approx . 12% had undergone partial phase darkening (grey spores), 15% remained phase bright whilst the remainder had turned fully phase dark but failed to elongate . No enhanced recovery of u.v.-treated spores (with intact or permeabilized coats) occurred in media containing hen egg white lysozyme or vegetable extracts (celery, carrot, swede or turnip) . However, recovery did occur when irradiated spores were incubated for 26 d, semiaerobically, within cartons containing nutrient broth or milk . CONCLUSIONS: The germination ability of B . subtilis spores is altered following u.v.-excimer laser treatment . Recovery of treated spores was found in liquid systems but not on agar plates supplemented with vegetable extracts or lysozyme . SIGNIFICANCE AND IMPACT OF THE STUDY: The potential recovery of u.v.-excimer laser-treated spores in a range of carton-packed food systems requires further investigation.

Biochemistry, 2002 May 21, 41(20), 6218 - 25
Insights into the functioning of Bacillus subtilis HPr kinase/phosphatase: affinity for its protein substrates and role of cations and phosphate; Lavergne JP et al.; In Bacillus subtilis, carbon catabolite repression is mediated by the HPr kinase/phosphatase (HprK/P) which catalyzes both an ATP-dependent phosphorylation and a dephosphorylation on Ser-46 of either HPr (histidine-containing protein) or Crh (catabolite repression HPr) . By using a surface plasmon resonance approach, it was shown here that the presence of magnesium is a prerequisite for the interaction of HprK/P with either HPr or Crh . HprK/P binds both protein substrates with a similar affinity (K(D) of about 40 nM), and addition of nucleotides increases by about 10-fold its affinity for each substrate . In addition, the specificity and the concentration of the cation required for the binding of protein substrates are different from that exhibited by the cation-binding site involved in the nucleotide binding, suggesting the presence of two cation-binding sites on HprK/P . The effects of phosphate on enzymatic activities of HprK/P were also investigated . Phosphate was able to unmask the phosphatase activity, especially in the presence of ATP or both ATP and fructose 1,6-bisphosphate whereas it was shown to inhibit the kinase activity of HprK/P . An apparent competition between phosphate and a fluorescent analogue of nucleotide led to the suggestion that phosphate mediates its effect by binding directly to the ATP-binding site of the enzyme.

Metab Eng, 2002 Apr, 4(2), 159 - 69
Metabolic network analysis of Bacillus clausii on minimal and semirich medium using (13)C-labeled glucose; Christiansen T et al.; Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium . The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis . Two futile cycles in the pyruvate metabolism were included in the metabolic network . A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose . The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations . It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose . In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium . Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose . The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium.

Arch Pharm Res, 2002 Apr, 25(2), 154 - 7
Antimicrobial constituents of Foeniculum vulgare; Kwon YS et al.; A phenyl propanoid derivative, dillapional(1) was found to be a antimicrobial principle of the stems of Foeniculum vulgare (Umbelliferae) with MIC values of 125, 250 and 125/ against Bacillus subtilis, Aspergillus niger and Cladosporium cladosporioides, respectively . A coumarin derivative, scopoletin(2) was also isolated as marginally antimicrobial agent along with inactive compounds, dillapiol(3), bergapten(4), imperatorin(5) and psolaren(6) from this plant . The isolates 1-6 were not active against the Escherichia coli.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 237 - 41
Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis; Braaz R et al.; Purification of poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75) from Paucimonas lemoignei is complicated because the bacterium produces several isoenzymes which are difficult to separate from each other . The phaZ5 gene of P . lemoignei encoding extracellular poly(3-hydroxybutyrate) depolymerase A was functionally expressed from the constitutive P43 promoter of pWB980 in a multiple protease-negative mutant of Bacillus subtilis (strain WB800) and secreted to the culture medium . The depolymerase (apparent M(r), 42 kDa; 1.9 mg purified protein per liter culture) was purified from cell-free culture fluid to homogenity by applying only one chromatography step in comparison to at least two necessary steps if poly(3-hydroxybutyrate) depolymerases are purified from P . lemoignei . The recombinant depolymerase lacked any carbohydrate content in contrast to the glycosylated depolymerase of the wild-type . Glycosylation was not essential for activity but enhanced the thermal stability of the enzyme at high temperature . Overexpression of poly(3-hydroxybutyrate) depolymerase in B . subtilis is more efficient than in Escherichia coli.

J Ethnopharmacol, 2002 May, 80(2-3), 131 - 5
Interaction of lectin-like proteins of South African medicinal plants with Staphylococcus aureus and Bacillus subtilis; Gaidamashvili M et al.; Lectin-like proteins from seven medicinal plant species of South Africa possessing well-documented antibacterial effects were examined for the interaction with two Gram-positive bacterial pathogens Staphylococcus aureus and Bacillus subtilis . Agglutinins elicited selective aggregation reactions with bacterial strains . Hypoxis hemerocallidea and Combretum mkhuzense agglutinins aggregated S . aureus at 4 and 5 microg ml(-1) of protein concentrations, respectively . B . subtilis was aggregated by C . mkhuzense, Kniphofia spp . and Tulbaghia violacea agglutinins at relatively high concentrations . The inhibition of bacterial growth at the initial stages of multiplication was observed in the presence of particular plant agglutinins . Suggestions are given about possible employment of agglutinins from medicinal plants in clinical microbiology . The possible contribution of lectin-like proteins to pharmacological effects associated with microbial infections is presumed.

FEMS Microbiol Lett, 2002 Mar 19, 209(1), 23 - 30
Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes; Lapidus A et al.; We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium . In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved . These regions constitute approximately 60% of the compared chromosomes . Sequencing of the competence genes of B . licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems . A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B . licheniformis strain.

Cell, 2002 Apr 19, 109(2), 257 - 66
Asymmetric cell division in B . subtilis involves a spiral-like intermediate of the cytokinetic protein FtsZ; Ben-Yehuda S et al.; A fundamental feature of development in the spore-forming bacterium Bacillus subtilis is the switch from medial to asymmetric division . The switch is brought about by a change in the location of the cytokinetic Z ring, which is composed of the tubulin-like protein FtsZ, from the cell middle to the poles during sporulation . We report that the medial Z ring is replaced by a spiral-like filament of FtsZ that grows along the long axis of the cell . We propose that the filament mediates the switch by redeploying FtsZ to the poles . Spiral formation and the switch to polar Z rings are largely caused by a sporulation-specific increase in transcription of the gene for FtsZ and activation of the gene for the FtsZ-associated protein SpoIIE.

RNA, 2002 Mar, 8(3), 296 - 306
Archaeal RNase P has multiple protein subunits homologous to eukaryotic nuclear RNase P proteins; Hall TA et al.; Although archaeal RNase P RNAs are similar in both sequence and structure to those of Bacteria rather than eukaryotes, and heterologous reconstitution between the Bacillus subtilis RNase P protein and some archaeal RNase P RNAs has been demonstrated, no archaeal protein sequences with similarity to any known bacterial RNase P protein subunit have been identified, and the density of Methanothermobacter thermoautotrophicus RNase P in Cs2SO4 (1.42 g/mL) is inconsistent with a single small bacterial-like protein subunit . Four hypothetical open reading frames (MTH11, MTH687, MTH688, and MTH1618) were identified in the genome of M . thermoautotrophicus that have sequence similarity to four of the nine Saccharomyces cerevisiae RNase P protein subunits: Pop4p, Pop5p, Rpp1p, and Rpr2p, respectively . Polyclonal antisera generated to recombinant Mth11p, Mth687p, Mth688p, and Mth1618p each recognized a protein of the predicted molecular weight in western blots of partially purified M . thermoautotrophicus RNase P, and immunoprecipitated RNase P activity from the same partially purified preparation . RNase P in Archaea is therefore composed of an RNA subunit similar to bacterial RNase P RNA and multiple protein subunits similar to those in the eukaryotic nucleus.

Life Sci Space Res, 1979, 17, 123 - 8
Dosimetric and biological results from the Bacillus subtilis Biostack experiment with the Apollo-Soyuz Test Project; Facius R et al.; The evaluation of the Bacillus subtilis experiment has been completed . The biological and the physical results for this part of the Apollo-Soyuz Test Project (ASTP) Biostack experiment are given . This comprises dosimetric data for the cosmic radiation at that orbit as well as biological findings from two types of plastic detectors . Further, the frequency distributions of the physical quantities atomic number, energy and energy loss of the heavy ions within the sample of spores hit are presented . The biological hazard presented by cosmic HZE-particles has been much underestimated.

Life Sci Space Res, 1973, 11, 295 - 305
The Biostack experiment on Apollo 16; Bucker H et al.; The object of the Biostack experiment is to study the biological effects of high ZE particles of cosmic radiation in order to obtain information on the mechanism of these particles in biological matter . For this purpose individual local evaluation methods have been developed which allow one to identify each biologically effective particle and to correlate the individual hitting particle with the biological effect produced . The Biostack experimental package contains a series of monolayers of selected biological objects (Bacillus subtilis spores, Arabidopsis thaliana seeds, Vicia faba radiculae, Artemia salina eggs) with each layer sandwiched between several different cosmic ion track detectors (nuclear emulsions, cellulose nitrate, polycarbonate) . By this arrangement a variety of biological effects due to a single penetrating particle can be analysed . Influence on cellular and tissue development, nuclear damages, and mutation induction are the main investigated effects . These space flight findings will be completed by results of balloon flight and accelerator experiments.

Environ Sci Technol, 2002 Apr 1, 36(7), 1546 - 53
Hg(II) adsorption by bacteria: a surface complexation model and its application to shallow acidic lakes and wetlands in Kejimkujik National Park, Nova Scotia, Canada; Daughney CJ et al.; The fate and environmental threat posed by mercury in aquatic systems is controlled, in part, bythe transport of Hg(II) from oxic to anoxic zones in lakes and its subsequent transformation to organic mercury . The transport of Hg(II) in aquatic systems can be affected by its partitioning between the dissolved and particulate phases . In this study, batch experiments were performed to quantify Hg(II) adsorption to Bacillus subtilis as bacteria-to-metal ratio, pH, chloride concentration, growth phase, and reaction time were independently varied . The laboratory data were well described by a surface complexation model (SCM) considering the adsorption of neutral Hg(II) hydroxide and chloride complexes by specific functional groups on the bacterial surface . To evaluate its applicability to complex aquatic systems, the SCM was used to predict the distributions of Hg(II) in 36 shallow acidic lakes and wetlands in Kejimkujik National Park, Nova Scotia, Canada . The lab-derived SCM provided a statistically accurate (r2 = 0.615, P < 0.01) fit to the field data when it was expanded to consider Hg(II) complexation by dissolved organic matter . Inclusion of Hg(II)-mineral adsorption reactions did not improve the fit of the model . The quality of fit provided by the expanded SCM suggested that the major assumptions implicit in applying a lab-derived model to the field were justifiable . Our study has demonstrated that SCMs are powerful tools for dynamic prediction of the sorption of environmental contaminants to biocolloids at the regional scale.

Shokuhin Eiseigaku Zasshi, 2002 Feb, 43(1), 44 - 8
{Spore rec-assay by dry sheet medium culture plate for bacterial counts}; Ueno S et al.; A simple and rapid method for spore rec-assay by utilizing dry sheet medium culture (Compactdry TC, CTC) for determining numbers of bacteria, instead of the spore agar plate, was developed . One mL of spore suspension (2 x 10(6)/mL) of Bacillus subtilis strain M45 Rec- or H17 Rec+ was inoculated in the center of the CTC plate . In the case of metabolic activation, 1 mL of mixed solution (spore suspension of M45 or H17: 9,000 x g supernatant of rat-liver homogenate treated with Aroclor 1254 = 19:1) was used . The spore suspension spreads over the whole sheet in seconds and gels . A paper disk impregnated with 20-40 microL of the sample solution and 20 microL of the cofactor solution was placed on the surface of CTC plate . For the assay of samples that do not require metabolic activation, use of the cofactor solution can be omitted . After 48 hr incubation at 37 degrees C, 0.01% MTT {3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide} aqueous solution (0.5 mL) was dropped uniformly on the plate . The plate was left for 5 min, and the diameter of the inhibition circle was measured with slide calipers . The samples for which the difference in inhibition zone between M45 and H17 was more than 2 mm were judged positive . Under these conditions, the DNA damaging activities of sodium sulfite, sodium benzoate and citric acid, used as food additives, were investigated by the proposed method . Sodium sulfite and sodium benzoate gave positive results and citric acid gave a negative result with or without metabolic activation, in agreement with the results obtained by the conventional method.

J Microbiol Methods, 2002 Jul, 50(2), 215 - 23
Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups; Shaver YJ et al.; Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp . subtilis, B . subtilis subsp . spizizenii, and B . atrophaeus were compared . ISR sequences of the B . subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation . However, RFLP of rRNA operons of the B . subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g . the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group) . The more distantly related B . atrophaeus was distinct from both B . subtilis subspecies in terms of ISR sequence and rRNA operon number and organization . RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence . However, ISR sequence defines the relatedness of B . subtilis to other species (e.g . B . atrophaeus) within the genus Bacillus.

Chemosphere, 2002 Apr, 47(1), 81 - 5
Genotoxicological effects of some phenoxyherbicides and their metabolites on Bacillus subtilis M45 Rec- and H17 Rec+ strains; Grabinska-Sota E et al.; Because they are used in a number of commercial preparations phenoxyacetic acids and their salts can occur in wastewater . During their degradation genotoxic substances may be created . The results of investigations of biodegradability and genotoxicity of some phenoxyherbicides are presented . Commercial formulations of 2,4-D (Aminopielik 720) and MCPA (Chwastox Extra) were the substrates studied . Biodegradation tests were conducted according to OECD guidelines for testing of chemicals--confirmatory test (OECD Method 303 A) . Genotoxicity tests were conducted with Bacillus subtilis strains according to the method of {Chemical Mutagens, vol . 6, Plenum Press . New York, 1980, p . 149} . Genotoxicity of biodegradation products was also studied . Both commercial formulations were biodegradable . Aminopielik 720 was potentially genotoxic but only at great concentrations while Chwastox Extra was not genotoxic . Biodegradation products of neither compound were genotoxic.

Mol Microbiol, 2002 May, 44(3), 663 - 74
The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components; Robson SA et al.; The bacterial septum appears to comprise a macromolecular assembly of essential cell division proteins (the 'septasome') that are responsible for physically dividing the cell during cytokinesis . FtsL and DivIC are essential components of this division machinery in Bacillus subtilis . We used yeast two-hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC . We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome . However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC . We propose that the observed interdependence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.

Mol Microbiol, 2002 May, 44(3), 601 - 6
Regulation of autolysins in teichuronic acid-containing Bacillus subtilis cells; Calamita HG et al.; Bacillus subtilis cells grown under phosphate starvation induce teichuronic acid (TUA) synthesis while simultaneously repressing teichoic acid synthesis (TA) . The turnover rates of TA-containing and TUA-containing walls are similar, indicating that autolysin function is similar and suggesting that modulation of autolytic function may be similar . In this study, it is demonstrated, utilizing fluorescein isothiocyanate (FITC)-dextran to probe the wall pH, that a low pH exists in the wall matrix . A second probe, cationized ferritin (CF), was used to observe cell surface protonation . Suspensions of B . subtilis cells containing either TA or TUA were aggregated with CF only after the addition of a proton-motive-force-dissipating agent . Respiring B . subtilis TUA-containing cells labelled with FITC-dextran exhibited little fluorescence . Conversely, fluorescence intensities exhibited by cells de-energized with nitrogen gas were significantly greater . The effects of protonmotive force on autolytic activity were studied by adding cell wall protein extract containing concentrated autolysin to exponentially growing TA-containing and TUA-containing B . subtilis cells . Both TUA-containing and TA-containing cells were lysed only after the addition of sodium azide . These data suggest that during normal growth the wall of TUA-containing B . subtilis cells is protonated, and proton-motive force influences autolytic regulation in both TUA-containing and TA-containing B . subtilis cells.

Can J Microbiol, 2002 Mar, 48(3), 230 - 8
Isolation of plant-growth-promoting Bacillus strains from soybean root nodules; Bai Y et al.; Endophytic bacteria reside within plant tissues and have often been found to promote plant growth . Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max . (L.) Merr.) root nodules . These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB) . Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B . japonicum alone . In the absence of B . japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth . All three isolates were Gram-positive spore-forming rods . While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species . Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.

Microbiology, 2002 May, 148(Pt 5), 1593 - 602
Regulatory interactions between the Pho and sigma(B)-dependent general stress regulons of Bacillus subtilis; Pragai Z et al.; When Bacillus subtilis is subjected to phosphate starvation, the Pho and sigma(B)-dependent general stress regulons are activated to elicit, respectively, specific and non-specific responses to this nutrient-limitation stress . A set of isogenic mutants, with a beta-galactosidase reporter gene transcriptionally fused to the inactivated target gene, was used to identify genes of unknown function that are induced or repressed under phosphate limitation . Nine phosphate-starvation-induced (psi) genes were identified: yhaX, yhbH, ykoL and yttP were regulated by the PhoP-PhoR two-component system responsible for controlling the expression of genes in the Pho regulon, while ywmG (renamed csbD), yheK, ykzA, ysnF and yvgO were dependent on the alternative sigma factor sigma(B), which controls the expression of the general stress genes . Genes yhaX and yhbH are unique members of the Pho regulon, since they are phosphate-starvation induced via PhoP-PhoR from a sporulation-specific sigma(E) promoter or a promoter that requires the product of a sigma(E)-dependent gene . Null mutations in key regulatory genes phoR and sigB showed that the Pho and sigma(B)-dependent general stress regulons of Bacillus subtilis interact to modulate the levels at which each are activated.

Proteomics, 2002 May, 2(5), 591 - 602
Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach; Antelmann H et al.; Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome . In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis . Thus, the extracellular proteome of the multiple protease-deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase . In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover . Next, we analyzed if proteins that can be extracted from B . subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant . Thus, the cell wall proteome of B . subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing . The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome . We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.

Mol Microbiol, 2002 Jan, 43(2), 399 - 410
Regulation of the bacillus subtilis ccpC gene by ccpA and ccpC; Kim HJ et al.; Bacillus subtilis CcpC, a LysR-type transcriptional regulator, represses the transcription of genes for citrate synthase (citZ) and aconitase (citB) in response to citrate availability . Transcription of ccpC was shown to initiate at two promoters, P1, located just upstream of the ccpC gene, and P2, located within or upstream of the neighbouring ykuL gene . Expression from the ccpC-specific promoter (P1) was negatively regulated by CcpC but independent of the carbon source in the medium . Gel shift and DNase I footprinting experiments revealed that CcpC binds to an interrupted dyad sequence that surrounds the ccpC transcriptional start point . Transcription of ccpC from the upstream promoter (P2) was repressed by glucose in a CcpA-dependent manner . A putative CcpA binding site (cre) was identified upstream of the -35 region of the P1 promoter . Transcriptional fusion studies demonstrated that glucose repression of ccpC expression from the P2 promoter depends on this cre site . In addition, DNase I footprinting experiments showed that CcpA specifically binds to this cre site and that the introduction of mutations (cre*) into this site abolished the binding . These results suggest that CcpA may control CcpC synthesis by acting as a road-block to readthrough transcription from the P2 promoter.

Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6690 - 5 Epub 2002 Apr 30.
The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic acid derivative; Fuangthong M et al.; Reactive oxygen species induce the expression of detoxification and repair genes critical for life in an aerobic environment . Bacterial factors that sense reactive oxygen species use either thiol-disulfide exchange reactions (OxyR, RsrA) or redox labile 2Fe-2S clusters (SoxR) . We demonstrate that the reduced form of Bacillus subtilis OhrR binds cooperatively to two adjacent inverted repeat sequences in the ohrA control region and thereby represses transcription . In the presence of organic hydroperoxides, OhrR is inactivated by the reversible oxidation of a single conserved cysteine residue to the corresponding cysteine-sulfenic acid, and perhaps to higher oxidation states.

Life Sci Space Res, 1968, 6, 115 - 22
Microbiological studies on the radiation environment of the ionosphere and stratosphere; Petras E et al.; Rocket, balloon and laboratory experiments have been performed in order to study the survival chances of microorganisms, which exist under the environmental conditions of ionosphere and stratosphere . The main results are: 1 . Not only near the earth, but also in the stratosphere and even in the ionosphere, microorganisms are endangered primarily by UV- and EUV-light irradiation . 2 . The observed effect of more penetrating kinds of radiation was relatively unimportant . High-vacuum and temperature effects have not been observed at all . Even membrane filters and thin protein layers protected the exposed spores of Bacillus subtilis var . niger (= Bac . globigii) in a clear-cut manner . 3 . UV-light with a wavelength between 200 and 300 nm reduces the number of cells able to divide much quicker, than EUV-light of the same energy level does, but damages caused by EUV-light can not be reversed by photoreactivation . 4 . Microbes which have been damaged by solar radiation, can be photoreactivated to a degree . Photoreactivation is high after exposure near the Earth and significant after exposure within the stratosphere . 5 . After exposure to ionospheric irradiations no changes in the antigenic behavior of E . coli cells could be detected.

J Bacteriol, 2002 May, 184(10), 2845 - 9
Ribonuclease M5 has few, if any, mRNA substrates in Bacillus subtilis; Condon C et al.; In Bacillus subtilis, maturation of 5S rRNA is catalyzed by an enzyme called RNase M5 . We searched for potential mRNA substrates for RNase M5 by gene array technology, based on the premise that most endonucleolytic cleavages have an effect on the stability of RNA and hence on steady-state levels of expression . Only a handful of genes had significantly altered expression in rnmV mutants compared to wild-type strains that could subsequently be confirmed by Northern blotting . The effect of RNase M5 on the expression of the best candidates, the odhAB and sucCD operons, is indirect, by a mechanism we do not yet understand . We show that an effect of RNase M5 on the expression of the remaining candidate, ctsR, is due to the failure to process the 5S rRNA contained in the rrnW lying directly upstream . We thus conclude that RNase M5 has very few or possibly no mRNA substrates in B . subtilis.

J Bacteriol, 2002 May, 184(10), 2780 - 8
The E1beta and E2 subunits of the Bacillus subtilis pyruvate dehydrogenase complex are involved in regulation of sporulation; Gao H et al.; The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1alpha and E1beta), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH) . There are two promoters: one for the entire operon and an internal one in front of the pdhC gene . The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1alpha and E1beta . Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1alpha is essential . The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium . The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level . This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity . Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations . Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III) . This analysis was confirmed by fluorescent membrane staining . The E1beta and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.

J Bacteriol, 2002 May, 184(10), 2740 - 7
An antisense RNA-mediated transcriptional attenuation mechanism functions in Escherichia coli; Brantl S et al.; Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501 . In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids . In this work we asked whether such a mechanism can be supported by Escherichia coli . The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host . In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough . Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism . Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E . coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect . Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis . Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.

J Bacteriol, 2002 May, 184(10), 2654 - 63
BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti; Boscari A et al.; Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%) . Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids) . After heterologous expression of betS in E . coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock . Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport . Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl . BetS activity appears to be Na(+) driven . In an S . meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress . beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed . Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation . Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S . meliloti subjected to osmotic upshock.

Appl Environ Microbiol, 2002 May, 68(5), 2624 - 8
Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis; Kaltwasser M et al.; Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein . These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences . Fusion of the tagging sequences occurs by PCR amplification of the 3' terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene . Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein(+), yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell . The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.

Appl Environ Microbiol, 2002 May, 68(5), 2133 - 9
Identification of a salt-induced primary transporter for glycine betaine in the methanogen Methanosarcina mazei Gö1; Roessler M et al.; The salt adaptation of the methanogenic archaeon Methanosarcina mazei Go1 was studied at the physiological and molecular levels . The freshwater organism M . mazei Go1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations . Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in M . mazei Go1, and inhibitor studies argue for a primary transport device . Analysis of the genome of M . mazei Go1 identified a homolog of known primary glycine betaine transporters . This gene cluster was designated Ota (osmoprotectant transporter A) . Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of Bacillus subtilis . Northern blot analysis of otaC revealed a salt-dependent transcription of this gene . Ota is the first identified salt-induced transporter for compatible solutes in ARCHAEA:

Life Sci Space Res, 1967, 5, 1 - 6
The survival of micro-organisms in space . Further rocket and balloon-borne exposure experiments; Hotchin J et al.; This report describes the results of survival studies of terrestrial micro-organisms exposed directly to the space environment on two balloons and in two rocket flights . The work is part of a program to develop techniques for the collection of micro-organisms in the size range of micrometeorite particles in space or non-terrestrial atmospheres, and their return to earth in a viable state for further study . Previous survival studies were reported (J . Hotchin, P . Lorenz and C . Hemenway, Nature 206 (1965) 442) in which a few relatively large area samples of micro-organisms were exposed on millipore filter cemented to aluminum plates . In the present series of experiments, newly developed techniques have resulted in a 25-fold miniaturization resulting in a corresponding increase in the number of experiments performed . This has enabled a statistical evaluation of the results to be made . A total of 756 separate exposure units (each approximately 5 x 5 mm in size) were flown in four experiments, and organisms used were coliphage T1, penicillium roqueforti (THOM) mold spores, poliovirus type I (Pfizer attenuated Sabin vaccine strain), and bacillus subtilis spores . The organisms were deposited either by spraying directly upon the vinyl-coated metal units, or by droplet seeding into shallow depressions in the millipore filter membrane-coated units . Groups of units were prepared comprising fully exposed, inverted (screened by 2 mm of Al), and filter-protected organisms . All of these were included in the flight set, the back up set, and a laboratory control set . The altitude of the exposures varied from 35 km in the balloon experiments to 150 km in the rocket experiments . Times of exposures at altitude were approximately 6 hours for the balloon flights and about 3 minutes for the rocket experiments.

Mol Microbiol, 2002 Apr, 44(2), 403 - 16
Dual control of subtilin biosynthesis and immunity in Bacillus subtilis; Stein T et al.; The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated . Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized . Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization . Primer extension experiments and beta-galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI . beta-Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid-exponential growth . Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production . The lacZ activity was dependent on co-expression with the two-component regulatory system spaRK . The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions . This suggests a transcriptional autoregulation according to a quorum-sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK . Additionally, spaR expression was found to be under positive control of the alternative sigma factor H . Deletion of sigma H strongly decreased subtilin production . Full subtilin production could be restored after in-trans complementation of spaR . Deletion of the major B . subtilis transition state regulator AbrB strongly increased subtilin production . These results show that the spaRK two-component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.

Nucleic Acids Res, 2002 May 1, 30(9), 1935 - 43
Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort; Landthaler M et al.; We have recently described three group I introns inserted into a single gene, orf142, of the staphylococcal bacteriophage Twort and suggested the presence of at least two additional self-splicing introns in this phage genome . Here we report that two previously uncharacterized introns, 429 and 1087 nt in length, interrupt the Twort gene coding for the large subunit of ribonucleotide reductase (nrdE) . Reverse transcription-polymerase chain reaction (RT-PCR) of RNA isolated from Staphylococcus aureus after phage infection indicates that the introns are removed from the primary transcript in vivo . Both nrdE introns show sequence similarity to the Twort orf142 introns I2 and I3, suggesting either a common origin of these introns or shuffling of intron structural elements . Intron 2 encodes a DNA endonuclease, I-TwoI, with similarity to homing endonucleases of the HNH family . Like I-HmuI and I-HmuII, intron-encoded HNH endonucleases in Bacillus subtilis phages SPO1 and SP82, I-TwoI nicks only one strand of its DNA recognition sequence . However, whereas I-HmuI and I-HmuII cleave the template strand in exon 2, I-TwoI cleaves the coding strand in exon 1 . In each case, the 3' OH created on the cut strand is positioned to prime DNA synthesis towards the intron, suggesting that this reaction contributes to the mechanism of intron homing . Both nrdE introns are inserted in highly conserved regions of the ribonucleotide reductase gene, next to codons for functionally important residues.

Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics, 1999 Jun, 59(6), 7036 - 41
Modeling of spatiotemporal patterns in bacterial colonies; Lacasta AM et al.; A diffusion-reaction model for the growth of bacterial colonies is presented . The often observed cooperative behavior developed by bacteria which increases their motility in adverse growth conditions is here introduced as a nonlinear diffusion term . The presence of this mechanism depends on a response which can present hysteresis . By changing only the concentrations of agar and initial nutrient, numerical integration of the proposed model reproduces the different patterns shown by Bacillus subtilis OG-01.

Nat Genet, 2002 May, 31(1), 69 - 73 Epub 2002 Apr 22.
Regulation of noise in the expression of a single gene; Ozbudak EM et al.; Stochastic mechanisms are ubiquitous in biological systems . Biochemical reactions that involve small numbers of molecules are intrinsically noisy, being dominated by large concentration fluctuations . This intrinsic noise has been implicated in the random lysis/lysogeny decision of bacteriophage-lambda, in the loss of synchrony of circadian clocks and in the decrease of precision of cell signals . We sought to quantitatively investigate the extent to which the occurrence of molecular fluctuations within single cells (biochemical noise) could explain the variation of gene expression levels between cells in a genetically identical population (phenotypic noise) . We have isolated the biochemical contribution to phenotypic noise from that of other noise sources by carrying out a series of differential measurements . We varied independently the rates of transcription and translation of a single fluorescent reporter gene in the chromosome of Bacillus subtilis, and we quantitatively measured the resulting changes in the phenotypic noise characteristics . We report that of these two parameters, increased translational efficiency is the predominant source of increased phenotypic noise . This effect is consistent with a stochastic model of gene expression in which proteins are produced in random and sharp bursts . Our results thus provide the first direct experimental evidence of the biochemical origin of phenotypic noise, demonstrating that the level of phenotypic variation in an isogenic population can be regulated by genetic parameters.

J Appl Microbiol, 2002, 92(4), 675 - 80
Studies on the mechanisms of the sporicidal action of ortho-phthalaldehyde; Cabrera-Martinez RM et al.; AIMS: To determine the mechanism of killing of spores of Bacillus subtilis by ortho-phthalaldehyde (OPA), an aromatic dialdehyde currently in use as an antimicrobial agent . METHODS AND RESULTS: OPA is sporicidal, although spores are much more OPA resistant than are vegetative cells . Bacillus subtilis mutants deficient in DNA repair, spore DNA protection and spore coat assembly have been used to show that (i) the coat appears to be a major component of spore OPA resistance, which is acquired late in sporulation of B . subtilis at the time of spore coat maturation, and (ii) B . subtilis spores are not killed by OPA through DNA damage but by elimination of spore germination . Furthermore, OPA-treated spores that cannot germinate are not recovered by artificial germinants or by treatment with NaOH or lysozyme . CONCLUSIONS: OPA appears to kill spores by blocking the spore germination process . SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to, and spore killing by, the disinfectant, OPA.

Biochem J, 2002 Jul 15, 365(Pt 2), 547 - 53
Embryonic extracts derived from the nematode Caenorhabditis elegans remove uracil from DNA by the sequential action of uracil-DNA glycosylase and AP (apurinic/apyrimidinic) endonuclease; Shatilla A et al.; DNA bases continuously undergo modifications in response to endogenous reactions such as oxidation, alkylation or deamination . The modified bases are primarily removed by DNA glycosylases, which cleave the N-glycosylic bond linking the base to the sugar, to generate an apurinic/apyrimidinic (AP) site, and this latter lesion is highly mutagenic . Previously, no study has demonstrated the processing of these lesions in the nematode Caenorhabditis elegans . Herein, we report the existence of uracil-DNA glycosylase and AP endonuclease activities in extracts derived from embryos of C . elegans . These enzyme activities were monitored using a defined 5'-end (32)P-labelled 42-bp synthetic oligonucleotide substrate bearing a single uracil residue opposite guanine at position 21 . The embryonic extract rapidly cleaved the substrate in a time-dependent manner to produce a 20-mer product . The extract did not excise adenine or thymine opposite guanine, although uracil opposite either adenine or thymine was processed . Addition of the highly specific inhibitor of uracil-DNA glycosylase produced by Bacillus subtilis to the extract prevented the formation of the 20-mer product, indicating that removal of uracil is catalysed by uracil-DNA glycosylase . The data suggest that the 20-mer product was generated by a sequential reaction, i.e., removal of the uracil base followed by 5'-cleavage of the AP site . Further analysis revealed that product formation was dependent upon the presence of Mg(2+), suggesting that cleavage of the AP site, following uracil excision, is carried out by a Mg(2+)-dependent AP endonuclease . It would appear that these activities correspond to the first two steps of a putative base-excision-repair pathway in C . elegans.

Biochem J, 2002 Aug 1, 365(Pt 3), 749 - 56
Two essential regions for tRNA recognition in Bacillus subtilis tryptophanyl-tRNA synthetase; Jia J et al.; Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) is a homodimeric enzyme . A model for its ability to recognize tRNA(Trp) in B . subtilis was proposed by using computer modelling . This was based on the the fact that there is high homology among bacterial TrpRSs {Chen, Jiang, Jin and Wang (2001) Acta Biochim . Biophys . Sinica 33, 687-690}, in which the enzyme dimer binds to two tRNA(Trp) molecules and each tRNA(Trp) is bound to two different domains across the surface of the dimer . In this work, three deletion mutants of TrpRS were constructed and their products were purified . After determining the kinetic parameters of the mutants in the two-step reaction, it was found that the relative activities of wild-type and mutant enzymes had changed little in the ATP-pyrophosphate exchange reaction . In contrast, the activities of three mutant proteins were much decreased in the tRNA(Trp) aminoacylation assay . Deletion of residues 108-122 and residues 234-238 caused 44% and 80% reductions in the activity, respectively . When both regions were deleted, the aminoacylation activity of the TrpRS mutant was too low to be determined using tRNA(Trp) at the limiting concentration . Gel-retardation assays showed that the acceptor minihelix and the anticodon microhelix were recognized by the domains of TrpRS spanning residues 108-122 and residues 234-238 respectively . In addition, the deletion of amino acids 234-238 affected the normal induced expression of TrpRS at 37 degrees C . In conclusion, residues 108-122 and 234-238 were found essential for tRNA(Trp) recognition.

Life Sci Space Res, 1978, 16, 151 - 6
Radiobiological results from the Bacillus subtilis Biostack experiments within the Apollo and the ASTP space flights; Facius R et al.; In order to check the results of earlier Biostack experiments, new experimental techniques were developed for the Biostack III experiment in the Apollo-Soyuz test project (ASTP) . These techniques resulted in an increased accuracy of localization down to 0.2 micrometers for the determination of the impact parameter, accompanied by an increase in the sample size available for biological investigation . In addition, colony forming ability, metabolic mutations, and mutations affecting UV- and x-ray sensitivity were rendered observable by these methods . The biological and physical results obtained so far from the evaluation of the Bacillus subtilis experiment within Biostack III confirm and extend the findings of the previous Biostack experiments . They also add to the questions about the mechanisms of action of the radiation field under investigation, since the observed effects cannot be interpreted in terms of standard concepts.

Cell Mol Life Sci, 2002 Mar, 59(3), 434 - 44
Assembly and genetics of spore protective structures; Takamatsu H et al.; The sporulation program in Bacillus subtilis ends in the formation of a highly resistant endospore that can withstand extremes of heat, mechanical disruption, ultraviolet irradiation, lytic enzymes and chemical attack . These properties are attributed mainly to the unique structure of spore coat and cortex, as well as to the physical state of the spore cytoplasm . The outermost layer of the spore, called the coat, has two morphologically distinct sublayers: an electron-dense outer coat and an electron-translucent inner coat . The coat is composed of more than 2 dozen proteins of varying size . Many coat genes and coat proteins have been isolated and characterized in detail, and studies of these have identified proteins with important roles in coat assembly, resistance and spore germination . We describe here characteristics of the coat proteins and propose a model for coat assembly based on recent work.

Cell Mol Life Sci, 2002 Mar, 59(3), 392 - 402
Bacillus subtilis sporulation and stationary phase gene expression; Phillips ZE et al.; Bacillus subtilis cells entering stationary phase due to nutrient deprivation have a number of options . Complex interconnected regulatory circuits govern differential gene expression patterns that channel the cell along the path it has sensed is most advantageous for survival in the environment . The actual choice depends upon the activity of an elaborate signal transduction network (the phosphorelay) that ultimately affects the activity of two key transcription factors, SpoOA and AbrB . Should the cell commit to sporulation, a temporally and spatially controlled cascade of RNA polymerase sigma factors leads to the development and release of an endospore from within the terminally differentiated, apoptotic mother cell.

Cell Mol Life Sci, 2002 Mar, 59(3), 389 - 91
Overview: Development in bacteria: spore formation in Bacillus subtilis; Driks A; Like eukaryotes, bacteria possess complex developmental programs that drive environmental adaptation and morphological differentiation . In some species, these morphological changes are quite elaborate and result in major changes in cell appearance, including the formation of ornate appendages . The ease with which some bacteria can be manipulated makes them highly attractive model systems for developmental analysis . In this set of reviews, we tackle the best studied of these systems, spore formation in Bacillus subtilis . Construction of a spore initiates in response to starvation, takes each cell about 8 h and is directed by a tightly controlled genetic program . First, the cell creates an internal protoplast with its own copy of the chromosome . Over the next several hours, development continues as proteins synthesized within the protoplast as well as in the surrounding cell cytoplasm coalesce into the various complex structures that comprise the spore . The resulting cell is metabolically dormant and as close to indestructible as any cell found on earth . Nonetheless, the spore retains the ability to revive almost immediately when nutrient returns to the environment . Here, we review the genetic control of spore formation, the structure and assembly of several major spore components, the process of germination, and the environmental and disease implications of spores . As these reviews document, spore formation in B . subtilis has been among the most productive systems for understanding both the broad themes and the molecular basis of development . Not only does this system continue to add to our understanding of these questions, but it provides a particularly powerful means to address the cell biological dimension of development.

Appl Biochem Biotechnol, 2001 Spring, 91-93, 551 - 61
Influence of culture conditions on lipopeptide production by Bacillus subtilis; Akpa E et al.; Bacillus subtilis produces various families of lipopeptides with different homologous compounds . To produce "new molecules" with improved activities and to select strains that produced a reduced number of homologs or isomers, we studied the effects of different media on the nature of the synthesis of fatty acid chains for each lipopeptide family . This study focused on two B . subtilis strains cultivated in flasks . Optimized medium for lipopeptide production and Landy medium modified by replacing glutamic acid with other alpha-amino acids were used . We found that the intensity of production of homologous compounds depends on the strain and the culture medium . Analysis of these lipopeptides by high-performance liquid chromatography showed that the strain B . subtilis NT02 yielded various homologous compounds when cultivated in Landy medium (L-Glu), but primarily one homologous product in high relative amounts when cultivated in the optimized medium . Mass spectrometric analysis and determination of the amino acid composition of this molecule enabled us to identify it as Bacillomycine L c15.

Appl Biochem Biotechnol, 2001 Spring, 91-93, 487 - 501
The effect of pretreatments on surfactin production from potato process effluent by Bacillus subtilis; Thompson DN et al.; Pretreatments of low-solids potato process effluent were tested for their potential to increase surfactin yield . Pretreatments included heat, removal of starch particulates, and acid hydrolysis . Elimination of contaminating vegetative cells was necessary for surfactin production . After autoclaving, 0.40 g/L of surfactin was produced from the effluent in 72 h, vs 0.24 g/L in the purified potato starch control . However, surfactin yields per carbon consumed were 76% lower from process effluent . Removal of starch particulates had little effect on the culture . Acid hydrolysis decreased growth and surfactant production, except 0.5 wt% acid, which increased the yield by 25% over untreated effluent.

Genes Dev, 2002 Apr 15, 16(8), 1007 - 18
A sporulation membrane protein tethers the pro-sigmaK processing enzyme to its inhibitor and dictates its subcellular localization; Rudner DZ et al.; The developmental transcription factor sigmaK is derived from the inactive precursor protein pro-sigmaK by regulated proteolysis during the process of sporulation in the bacterium Bacillus subtilis . The putative pro-sigmaK processing enzyme SpoIVFB is a member of a family of membrane-embedded metalloproteases and is held inactive by two other integral membrane proteins, SpoIVFA and BofA . Herein we show that the processing enzyme and its two regulators exist in a multimeric complex that localizes to the membrane surrounding the developing spore (the forespore) . We further show that one of the regulators, SpoIVFA, plays a central role in both the formation of this complex and its subcellular localization . Evidence is presented in support of a model in which SpoIVFA acts as a platform for bringing BofA and SpoIVFB together, whereby BofA inhibits pro-sigmaK processing until a signal has been received from the forespore.

Antimicrob Agents Chemother, 2002 May, 46(5), 1310 - 8
Purification, characterization, and identification of novel inhibitors of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus; He X et al.; Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections . The rise of multidrug-resistant strains of S . aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) . This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability . We have identified only one fabH in the genome of S . aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH) . Additional genomic sequence analysis revealed that this S . aureus FabH (saFabH) is not mutated in certain methicillin-resistant S . aureus (MRSA) and vancomycin-resistant S . aureus (VRSA) strains . saFabH was expressed in E . coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH . The apparent K(m) for malonyl-ACP was 1.76 +/- 0.40 microM, and the enzyme was active with acetyl-CoA (k(cat), 16.18 min(-1); K(m), 6.18 +/- 0.9 microM), butyryl-CoA (k(cat), 42.90 min(-1); K(m), 2.32 +/- 0.12 microM), and isobutyryl-CoA (k(cat), 98.0 min(-1); K(m), 0.32 +/- 0.04 microM) . saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration {IC50}, >100 microM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-{1,2}-dithiol-3-one (IC50, 1.87 +/- 0.10 microM) and 4-phenyl-5-phenylimino-{1,2,4}dithiazolidin-3-one (IC50, 0.775 +/- 0.08 microM).

Antimicrob Agents Chemother, 2002 May, 46(5), 1226 - 30
Antibacterial activity of licochalcone A against spore-forming bacteria; Tsukiyama R et al.; Licochalcone A was isolated from the roots of licorice, Glycyrrhiza inflata, which has various uses in the food and pharmaceutical industries; isolation was followed by extraction with ethanol and column chromatography with silica gel . In this study, the activities of licochalcone A against some food contaminant microorganisms were evaluated in vitro . The vegetative cell growth of Bacillus subtilis was inhibited in a licochalcone A concentration-dependent manner and was completely prevented by 3 micrograms of licochalcone A/ml . Licochalcone A showed a high level of resistance to heating at 80 to 121 degrees C for 15 min . Licochalcone A did not inhibit the germination of heat-treated spores of B . subtilis induced by L-alanine . Licochalcone A showed effects against all gram-positive bacteria tested and especially was effective against all Bacillus spp . tested, with MICs of 2 to 3 micrograms/ml, but was not effective against gram-negative bacteria or eukaryotes at 50 micrograms/ml . Although the cationic antimicrobial peptides protamine and epsilon-poly-L-lysine resulted in the loss of antimicrobial activity in the presence of either 3% (wt/vol) NaCl or protease at 20 micrograms/ml, the antibacterial activity of licochalcone A was resistant to these conditions . Thus, licochalcone A could be a useful compound for the development of antibacterial agents for the preservation of foods containing high concentrations of salts and proteases, in which cationic peptides might be less effective.

FEMS Microbiol Lett, 2002 Mar 5, 208(2), 215 - 8
Identification of a promoter for the crystal protein-encoding gene cry1Ia from Bacillus thuringiensis subsp . kurstaki; Tounsi S et al.; Expression of cry1Ia, one of the insecticidal protein genes of Bacillus thuringiensis subsp . kurstaki, was studied at the transcriptional level . By primer extension analysis, we have identified for the first time, the transcription start point of cry1Ia . Upstream from the cry1Ia transcription start point, was found a nucleotide sequence partially homologous to the consensus sequence for the E sigma(E) holoenzyme of Bacillus subtilis . Thus, it was strongly suggested that the identified cry1Ia promoter was under the control of sigma(35), the B . thuringiensis homolog of sigma(E) . The transcriptional activity from the promoter was detected only for the sporulating cells at T2 and T5 stages.

FEBS Lett, 2002 Apr 10, 516(1-3), 245 - 52
Trans-translation mediated by Bacillus subtilis tmRNA; Ito K et al.; Trans-translation, in which a ribosome switches between translation of an mRNA and a tmRNA, produces a chimera polypeptide of an N-terminal truncated polypeptide and a C-terminal tag-peptide encoded by tmRNA . One of the tmRNA binding proteins, a ribosomal protein S1, has not been found in a group of Gram-positive bacteria . In this study, the trans-translation reaction with tmRNA from Bacillus subtilis belonging to this group was examined . When a truncated gene lacking a termination codon was expressed in B . subtilis, a 15-amino acid tag-peptide derived from tmRNA was identified in the C-termini of the trans-translation products . An identical tag-peptide was also found at the C-termini of the products from a truncated gene, when it was coexpressed with B . subtilis tmRNA in Escherichia coli . B . subtilis tmRNA was functional, although much less efficiently, in the in vitro poly(U)-dependent tag-peptide synthesis system of E . coli . A comparison of two bacterial tmRNAs suggests that the rule for determining the tag-initiation point on tmRNA may be the same in Gram-positive and Gram-negative bacteria.

Biochim Biophys Acta, 2002 Feb 20, 1574(1), 93 - 9
1H NMR studies of a 17-mer DNA duplex; Liu W et al.; Transcription factor 1 (TF1), encoded by the Bacillus subtilis bacteriophage SPO1, is a DNA-binding protein of the HU family . In preparation for a determination of the structure of the DNA-TF1 complex, we have studied the conformation of one core 17-mer duplex d(5'-CACTACTCTTTGTAGTG-3')-d(5'-CACTACAAAGAGTAGTG-3') . NOESY, DQF-COSY and TOCSY spectroscopy provide resonance assignments of non-exchangeable and exchangeable protons, internucleotide and interstrand proton-proton distances, and dihedral angle constraints . Restrained molecular dynamics calculations yield a family of NMR solution structures for which the RMSD is 0.7 A (all atoms) . The helical twist is 34.9 degrees for the central 15 bp . Bends toward the major groove are located between the second and fourth base pairs from each end . The G12 x C23 base pair, which is bounded on each side by consecutive A x T pairs, causes a local disturbance to the DNA helix that makes the conformations of the two end segments unsymmetrical . The pyrimidine rings at T9, T10 and T11 experience more extensive rotational movement than the rest of the structure.

J Bacteriol, 2002 May, 184(9), 2521 - 8
Characterization of the interaction of Bacillus subtilis PyrR with pyr mRNA by site-directed mutagenesis of the protein; Savacool HK et al.; The Bacillus subtilis PyrR protein regulates transcriptional attenuation of the pyrimidine nucleotide (pyr) operon by binding in a uridine nucleotide-dependent manner to specific sites on pyr mRNA and stabilizing a secondary structure of the downstream RNA that favors termination of transcription . The high-resolution structure of unliganded PyrR was used to guide site-directed mutagenesis of 12 amino acid residues that were thought likely to be involved in the binding of RNA . Missense mutations were constructed and evaluated for their effects on regulation of pyr genes in vivo and their uracil phosphoribosyltransferase activity, which is catalyzed by wild-type PyrR . A substantial fraction of the mutant PyrR proteins did not have native structures, but eight PyrR mutants were purified and characterized physically, for their uracil phosphoribosyltransferase activity and for their ability to bind pyr RNA in vitro . On the basis of these studies Thr-18, His-22, Arg-141, and Arg-146 were implicated in RNA binding . Arg-27 and Lys-152 were also likely to be involved in RNA binding, but Gln substitution mutations in these residues may have altered their subunit-subunit interactions slightly . Arg-19 was implicated in pyr regulation, but a specific role in RNA binding could not be demonstrated because the R19Q mutant protein could not be purified in native form . The results confirm a role in RNA binding of a positively charged face of PyrR previously identified from the crystallographic structure . The RNA binding residues lie in two sequence segments that are conserved in PyrR proteins from many species.

J Bacteriol, 2002 May, 184(9), 2500 - 20
Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis; Eymann C et al.; The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches . Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences . According to their altered synthesis patterns in response to DL-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes) . Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes . However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis . The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp . The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.) . Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG) . On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins . A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

J Bacteriol, 2002 May, 184(9), 2344 - 51
Whole-genome analysis of genes regulated by the Bacillus subtilis competence transcription factor ComK; Ogura M et al.; The Bacillus subtilis competence transcription factor ComK is required for establishment of competence for genetic transformation . In an attempt to study the ComK factor further, we explored the genes regulated by ComK using the DNA microarray technique . In addition to the genes known to be dependent on ComK for expression, we found many genes or operons whose ComK dependence was not known previously . Among these genes, we confirmed the ComK dependence of 16 genes by using lacZ fusions, and three genes were partially dependent on ComK . Transformation efficiency was significantly reduced in an smf disruption mutant, although disruption of the other ComK-dependent genes did not result in significant decreases in transformation efficiency . Nucleotide sequences similar to that of the ComK box were found for most of the newly discovered genes regulated by ComK.

Biochem Biophys Res Commun, 2002 Apr 12, 292(4), 789 - 93
Amino acids activated by fengycin synthetase FenE; Shu HY et al.; Fengycin is a lipopeptidic antibiotic produced nonribosomally by Bacillus subtilis F29-3 . Synthesis of this antibiotic requires five fengycin synthetases encoded by fenC, fenD, fenE, fenA, and fenB . In this study, we analyze the functions of the enzyme encoded by fenE, which contains two amino acid activation modules, FenE1 and FenE2 . ATP-PP(i) exchange assay revealed that FenE1 activates l-Glu and FenE2 activates l-Ala, l-Val, and l-2-aminobutyric acid, indicating that FenE activates the fifth and the sixth amino acids in fengycin . Furthermore, l-Val is a better substrate than l-Ala for FenE2 in vitro, explaining why B . subtilis F29-3 normally produces twice as much of fengycin B than fengycin A, which contains d-Val and d-Ala at the sixth amino acid position, respectively . Results presented herein suggest that fengycin synthetase genes and amino acids in fengycin are colinear . (c)2002 Elsevier Science (USA).

Mikrobiol Z, 2002 Jan-Feb, 64(1), 57 - 9
{Bsu50441--isoschizomer of endonuclease restriction AsuI}; Reva OM et al.; Endonuclease of restriction of type II has been found in Bacillus subtilis B-5044 during testing of endophytic strains of cotton plants bacilli . The restrictase Bsu5044I hydrolysed DNA of M13mp18 phage in 4 sites; pUC19 DNA in 5 sites; pBR322 in 15 sites . There were a lot of sites restriction in DNAs of phages T7 and lambda . A comparison of electrophoretical divisions of Bsu5044I and Cfr13I fragments of phages and plasmid DNAs showed their identity . Thus, the found restrictase Bsu5044I is an isoschisomer of AsuI.

Curr Opin Microbiol, 2002 Apr, 5(2), 208 - 10
Regulation of the desaturation of fatty acids and its role in tolerance to cold and salt stress; Sakamoto T et al.; The expression of cold-inducible genes is regulated by a two-component system in Synechocystis and Bacillus subtilis . The cold sensors are membrane-bound histidine kinases and it seems likely that they sense and transduce changes in the fluidity of membranes . Desaturation of fatty acids in membrane lipids has been implicated in tolerance to cold and salt stress.

Curr Opin Microbiol, 2002 Apr, 5(2), 142 - 8
Molecular recognition of bacterial phosphorelay proteins; Varughese KI; The transfer of the phosphoryl group from a histidine kinase to a response regulator forms the basis of bacterial signal transduction . The critical question of how a component of a signal transduction system specifically associates with its partner to produce the ideal environment for phosphotransfer is addressed in this review in the light of the structure of the Spo0F-Spo0B complex in Bacillus subtilis.

FEMS Microbiol Lett, 2002 Feb 19, 208(1), 105 - 9
Investigation of the yvgW Bacillus subtilis chromosomal gene involved in Cd(2+) ion resistance; Solovieva IM et al.; Analysis of the complete genome sequence of Bacillus subtilis has identified the gene yvgW encoding a protein of 703 amino acids with sequence similarity to the cadmium resistance determinant CadA from the Staphylococcus aureus plasmid pI258 . Deletion of yvgW (designated cadA) resulted in increased sensitivity of the strain to cadmium . The cadA gene is expressed from its own promoter, and its expression is induced by cadmium . Northern hybridization analysis showed that cadmium induces the synthesis of a 2.2-kb cadA transcript . These results indicate that cadA is the chromosomal determinant to cadmium resistance in B . subtilis.

J Mol Microbiol Biotechnol, 2002 May, 4(3), 323 - 9
Structure-function relationship and regulation of two Bacillus subtilis DNA-binding proteins, HBsu and AbrB; Klein W et al.; Microorganisms use a number of small basic proteins for organization and compaction of their DNA . By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability . It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences . This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate . In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins.

J Mol Microbiol Biotechnol, 2002 May, 4(3), 315 - 21
CcpA-independent carbon catabolite repression in Bacillus subtilis; Dahl MK; The past decade has witnessed an exiting unveiling of numerous molecular mechanisms that characterize signal transduction by protein-protein interaction . The recent findings encouraged an increasing effort to understand the sequential metabolism of different sugars available as energy sources at the same time . It seems probable that at least three principle mechanisms which act together or separately, mediate carbon catabolite repression (CCR) depending on the system which is under metabolic control: i) by the main signal transducing chain via the ATP-dependent HPr-kinase, HPr(Ser46-P) or alternatively Crh via the central component CcpA and its interaction with cre, ii) by signals sensed from the specific regulators directly or via phosphorylation by HPr, iii) by inducer exclusion based on the concurrence of the enzyme IIA(Glc) domain of the glucose permease, and other PTS-dependent permeases composed only of the B and C domains and lacking the enzyme IIA domain.

Mol Microbiol, 2002 Feb, 43(4), 1039 - 52
Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P-GlpK dephosphorylation control Bacillus subtilis glpFK expression; Darbon E et al.; The Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol-3-P and repressed by rapidly metabolizable sugars . Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK . This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co-repressors, P-Ser-HPr or P-Ser-Crh . HPr is a component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and Crh is an HPr homologue . The hprK-encoded HPr kinase phosphorylates HPr and Crh at Ser-46 . But in neither ccpA nor hprK mutants was expression of a glpF'-lacZ fusion relieved from CCR, as a second, CcpA-independent CCR mechanism implying the terminator tglpFK, whose formation is prevented by the glycerol-3-P-activated antiterminator GlpP, is operative . Deletion of tglpFK led to elevated expression of the glpF'-lacZ fusion and to partial relief from CCR . CCR completely disappeared in DeltatglpFK mutants carrying a disruption of ccpA or hprK . The tglpFK-requiring CCR mechanism seems to be based on insufficient synthesis of glycerol-3-P, as CCR of glpFK was absent in ccpA mutants growing on glycerol-3-P or synthesizing H230R mutant GlpK . In cells growing on glycerol, glucose prevents the phosphorylation of GlpK by P-His-HPr . P-GlpK is much more active than GlpK, and the absence of P~GlpK formation in DeltaptsHI strains prevents glycerol metabolism . As a consequence, only small amounts of glycerol-3-P will be formed in glycerol and glucose-exposed cells (inducer exclusion) . The uptake of glycerol-3-P via GlpT provides high concentrations of this metabolite in the ccpA mutant and allows the expression of the glpF'-lacZ fusion even when glucose is present . Similarly, despite the presence of glucose, large amounts of glycerol-3-P are formed in a glycerol-exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P-GlpK.

J Agric Food Chem, 2002 Apr 10, 50(8), 2454 - 8
Antioxidative activity and safety of the 50 ethanolic extract from red bean fermented by Bacillus subtilis IMR-NK1; Chung YC et al.; This study aimed at evaluating the antioxidative activities and the safety of 50% ethanolic extract from red bean fermented by Bacillus subtillis IMR-NK1 . The antioxidative activities, including alpha,alpha-diphenyl-beta-picryl-hydrazyl (DPPH) radicals scavenging effects, Fe(2+)-chelating ability, and reducing power, were studied in vitro . It was found that the antioxidative activity increased with the concentrations of the extract to a certain extent and then leveled off as the concentration further increased . As compared to the commercial antioxidants, the fermented red bean extract showed less scavenging effect on the DPPH radical and reducing power than alpha-tocopherol and BHT, but better Fe(2+)-chelating ability . No mutagenicity or toxicity effect toward all tester strains was found in the 50% ethanolic extract of fermented red bean by means of the Ames test . The results suggested that the 50% ethanolic extract was safe in genotoxicity.

J Org Chem, 2002 Apr 5, 67(7), 2087 - 92
Investigation of the binding of epimer A of the covalent hydrate of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine to a recombinant F22W Bacillus subtilis lumazine synthase mutant by (15)N{(19)F} REDOR NMR; Mehta AK et al.; The two epimeric covalent hydrates A and B of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine are metabolically stable analogues of hypothetical intermediates proposed in the reactions catalyzed by riboflavin synthase and lumazine synthase . To confirm the stereochemical assignments previously based solely on results for epimer B, a (15)N{(19)F} REDOR NMR study was performed on the complex formed from epimer A and a recombinant, uniformly (15)N-labeled F22W mutant of Bacillus subtilis lumazine synthase . The results indicate that the fluorines of the ligands are closer to the side chain nitrogens of Arg127 and farther away from the side chain nitrogens of Lys135 in epimer B than in epimer A . These results are consistent with the assignment of the earlier 7R configuration of epimer A and the 7S configuration of epimer B.

Protein Expr Purif, 2002 Apr, 24(3), 357 - 65
Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion; Wu SC et al.; A major attraction in using Bacillus subtilis as an expression host for heterologous protein production is its ability to secrete extracellular proteins into the culture medium . To take full advantage of this system, an efficient method for recovering the target protein is crucial . For secretory proteins which cannot be purified by a simple scheme, in vitro biotinylation using biotin ligase (BirA) offers an effective alternative for their purification . The availability of large amounts of quality BirA can be critical for in vitro biotinylation . We report here the engineering and production of an Escherichia coli BirA and its application in the purification of staphylokinase, a fibrin-specific plasminogen activator, from the culture supernatant of Bacillus subtilis via in vitro biotinylation . BirA was tagged with both a chitin-binding domain and a hexahistidine tail to facilitate both its purification and its removal from the biotinylated sample . We show in this paper how, in a unique way, we solved the problem of protein aggregation in the E . coli BirA production system to achieve a yield of soluble functional BirA hitherto unreported in the literature . Application of this novel BirA to protein purification via in vitro biotinylation in general will also be discussed . Biotinylated staphylokinase produced in the study not only can act as an intermediate for easy purification, it can also serve as an important element in the creation of a blood clot targeting and dissolving agent .

Protein Expr Purif, 2002 Apr, 24(3), 348 - 56
Secretory production and purification of functional full-length streptavidin from Bacillus subtilis; Wu SC et al.; Streptavidin is a versatile molecule for many in vitro and in vivo applications . To optimize the production of the full-length streptavidin in a soluble and functional form via secretion using Bacillus subtilis as the expression host, three different strategies were used . These strategies include the construction of a synthetic streptavidin gene, the installation of a transcription terminator, and the use of a sporulation mutant strain . In comparison with the wild-type streptavidin gene in expression studies, a combination of these approaches resulted in a 2.3-fold increase in streptavidin production . The production yields in complex and semidefined media were 94 and 24 mg/liter, respectively . A simple purification scheme which requires only a single ion-exchange matrix was designed to purify streptavidin to homogeneity directly from the culture supernatant . Purified streptavidin was in full length with good biotin binding capacity (3.2 binding sites available per tetramer) . A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional streptavidin for characterizations and practical applications .

J Biol Chem, 2002 Jun 7, 277(23), 20483 - 9 Epub 2002 Mar 28.
Biochemical characterization of aspartyl phosphate phosphatase interaction with a phosphorylated response regulator and its inhibition by a pentapeptide; Ishikawa S et al.; The RapA and RapB proteins are aspartyl phosphate phosphatases that specifically dephosphorylate the Spo0F approximately P intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis . The approximately 48-kDa His-tag derivative proteins were purified by metal affinity chromatography, and their molecular and biochemical characteristics were studied . RapA and RapB were found to be dimers in solution . Enzymatic activity was strongly dependent upon maintaining reducing conditions during purification and storage . RapA phosphatase activity on Spo0F approximately P is inhibited in vivo by a pentapeptide generated from the phrA gene . Native gel assays demonstrated that the RapA dimer forms a stable complex with two molecules of Spo0F approximately P or with its PhrA pentapeptide inhibitor . The pentapeptide was shown to displace Spo0F approximately P from a preformed complex with RapA . The structural organization of Rap phosphatases in tetratricopeptide repeats provides insights on the mechanisms of RapA interaction with its substrate and its inhibitor.

J Mass Spectrom, 2002 Mar, 37(3), 259 - 64
Structural characterization of lipopeptide biomarkers isolated from Bacillus globigii; Williams BH et al.; Spectra obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of Bacillus globigii (Bacillus subtilis niger) spores, vegetative cells and the culture supernatant show a cluster of biomarkers centered at a molecular mass of 1478 Da . Three biomarkers were isolated from the cell-free culture supernatant by solid-phase extraction and reversed-phase high-performance liquid chromatography, and characterized using various kinds of mass spectrometry . A Fourier transform mass spectrometer with a MALDI source was used to determine the monoisotopic protonated masses at 1463.8, 1477.8, and 1505.8 Da in order of elution . The mass differences of 14 and 28 Da suggest that they are homologous molecules . Alkaline hydrolysis of each species showed that it contained a lactone linkage . Strong acid hydrolysis released a fatty acid from an amide bond, consistent with a lipopeptide . A quadrupole time-of-flight instrument with a nanospray source was used to sequence the hydrolyzed forms of the three biomarkers . The cyclic lipopeptides were found to have amino acid sequences identical with those in fengycins and plipastatins, antimicrobial compounds with phospholipase inhibitor activity, previously identified in related species of Bacillus subtilis and Bacillus cereus .

Biotechnol Bioeng, 2002 May 5, 78(3), 339 - 45
Engineering of baker's yeasts, E . coli and Bacillus hosts for the production of Bacillus subtilis Lipase A; Sanchez M et al.; Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes . The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively . Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S . cerevisiae strains . An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive .

Mol Microbiol, 2002 Mar, 43(5), 1331 - 45
Microarray analysis of the Bacillus subtilis K-state: genome-wide expression changes dependent on ComK; Berka RM et al.; In Bacillus subtilis, the competence transcription factor ComK activates its own transcription as well as the transcription of genes that encode DNA transport proteins . ComK is expressed in about 10% of the cells in a culture grown to competence . Using DNA microarrays representing approximately 95% of the protein-coding open reading frames in B . subtilis, we compared the expression profiles of wild-type and comK strains, as well as of a mecA mutant (which produces active ComK in all the cells of the population) and a comK mecA double mutant . In these comparisons, we identified at least 165 genes that are upregulated by ComK and relatively few that are downregulated . The use of reporter fusions has confirmed these results for several genes . Many of the ComK-regulated genes are organized in clusters or operons, and 23 of these clusters are preceded by apparent ComK-box promoter motifs . In addition to those required for DNA uptake, other genes that are upregulated in the presence of ComK are probably involved in DNA repair and in the uptake and utilization of nutritional sources . From this and previous work, we conclude that the ComK regulon defines a growth-arrested state, distinct from sporulation, of which competence for genetic transformation is but one notable feature . We suggest that this is a unique adaptation to stress and that it be termed the 'K-state'.

Mol Microbiol, 2002 Mar, 43(5), 1319 - 29
Endonuclease cleavage of messenger RNA in Bacillus subtilis; Drider D et al.; A deletion derivative of the ermC gene was constructed that expresses a 254-nucleotide mRNA . The small size of this mRNA facilitated the detection of processing products that did not differ greatly in size from the full-length transcript . In the presence of erythromycin, which induces ribosome stalling near the 5' end of ermC mRNA, the 254-nucleotide mRNA was cleaved endonucleolytically at the site of ribosome stalling . Only the downstream product of this cleavage was detectable; the upstream product was apparently too unstable to be detected . The downstream cleavage product accumulated at times after rifampicin addition, suggesting that the stalled ribosome at the 5' end conferred stability to this RNA fragment . Neither Bs-RNase III nor RNase M5, the two known narrow-specificity endoribonucleases of Bacillus subtilis, was responsible for this cleavage . These results indicate the presence in B . subtilis of another specific endoribonuclease, which may be ribosome associated.

Genes Cells, 2002 Mar, 7(3), 343 - 50
Detection of tmRNA-mediated trans-translation products in Bacillus subtilis; Fujihara A et al.; BACKGROUND: Bacterial tmRNA (10Sa RNA) is involved in a trans-translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes . To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo trans-translation . RESULTS: The wild-type tmRNA gene (ssrA) in the genome was replaced by a variant ssrA encoding a tag-peptide sequence containing six histidine residues (His-tag) and two aspartic acids at the C-terminus . The His-tagged proteins that accumulated in the cells without degradation were fractionated by Ni2+-NTA column and gel electrophoresis and were detected by Western blotting with an anti-His-tag antibody . The results showed that the trans-translation occurred more frequently at a high temperature (50 degrees C) than at a low temperature (37 degrees C) . Two-dimensional (2D) gel electrophoresis of the products revealed many distinct spots, which represent specific target proteins for the trans-translation reaction . Furthermore, the 2D gel patterns of the products from cells cultured at high and low temperatures were apparently different . Several tagged proteins were identified by the N-terminal amino acid sequences of the products . CONCLUSION: Trans-translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures.

Nucleic Acids Res, 2002 Apr 1, 30(7), 1593 - 605
Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator; Polard P et al.; The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage phiX174 and plasmid pBR322 . Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks . A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis . To characterise this protein, designated PriA(Bs), we constructed priA(Bs) mutants . These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation . Replication of pAMbeta1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMbeta1 are strongly affected in the mutants . The purified PriA(Bs) protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSB(Bs)) . PriA(Bs) also binds stably and specifically to an artificial D-loop structure in vitro . These data show that PriA(Bs) recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them . PriA(Bs) also displays a single-stranded DNA-dependent ATPase activity, which is reduced in the presence of SSB(Bs), unless the ssiA sequence is present on the ssDNA substrate . Finally, PriA(Bs) is shown to be an active helicase . Altogether, these results demonstrate a clear functional identity between PriA(Ec) and PriA(Bs) . However, PriA(Bs) does not complement an E.coli priA null mutant strain . This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes.

Appl Environ Microbiol, 2002 Apr, 68(4), 1760 - 71
Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures; Dauner M et al.; Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis . Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone . The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures . In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields . Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes . The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway . The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold . The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone . The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.

Appl Environ Microbiol, 2002 Apr, 68(4), 1541 - 7
Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene; Okada Y et al.; Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained . The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) {collectively designated (p)ppGpp} synthetase during stringent response . The mutant showed a deficiency in (p)ppGpp accumulation . In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift . The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L . monocytogenes was introduced into the mutant . After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth . The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl . Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L . monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.

J Mol Biol, 2002 Mar 15, 317(1), 131 - 44
Solution structure and dynamics of Crh, the Bacillus subtilis catabolite repression HPr; Favier A et al.; The solution structure and dynamics of the Bacillus subtilis HPr-like protein, Crh, have been investigated using NMR spectroscopy . Crh exhibits high sequence identity (45 %) to the histidine-containing protein (HPr), a phospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system, but contains no catalytic His15, the site of PEP-dependent phosphorylation in HPr . Crh also forms a mixture of monomers and dimers in solution whereas HPr is known to be monomeric . Complete backbone and side-chain assignments were obtained for the monomeric form, and 60 % of the dimer backbone resonances; allowing the identification of the Crh dimer interface from chemical-shift mapping . The conformation of Crh was determined to a precision of 0.46(+/-0.06) A for the backbone atoms, and 1.01(+/-0.08) A for the heavy atoms . The monomer structure is similar to that of known HPr 2.67(+/-0.22) A (C(alpha) rmsd), but has a few notable differences, including a change in the orientation of one of the helices (B), and a two-residue shift in beta-sheet pairing of the N-terminal strand with the beta4 strand . This shift results in a shortening of the surface loop present in HPr and consequently provides a flatter surface in the region of dimerisation contact, which may be related to the different oligomeric nature of these two proteins . A binding site of phospho-serine(P-Ser)-Crh with catabolite control protein A (CcpA) is proposed on the basis of highly conserved surface side-chains between Crh and HPr . This binding site is consistent with the model of a dimer-dimer interaction between P-Ser-Crh and CcpA . (15)N relaxation measured in the monomeric form also identified differential local mobility in the helix B which is located in the vicinity of this site .

Acta Crystallogr D Biol Crystallogr, 2002 Apr, 58(Pt 4), 687 - 9 Epub 2002 Mar 22.
Crystallization and preliminary X-ray diffraction analysis of fatty-acid hydroxylase cytochrome P450BSbeta from Bacillus subtilis; Lee DS et al.; Cytochrome P450 isolated from Bacillus subtilis (P450BSbeta; MW 48 kDa) catalyzes the hydroxylation of long-chain fatty acids at the alpha and beta positions using H(2)O(2) as an oxidant . Crystals of the substrate-free form of P450BSbeta belonging to the trigonal space group P3(2)21 or P3(1)21 were obtained by the sitting-drop vapour-diffusion method using a precipitate solution consisting of 10%(w/v) PEG 4000 and 50 mM MES pH 6.8 . Another crystal form, belonging to the rhombohedral space group R3 or R32, was obtained from precipitate solution consisting of 10% PEG 4000, 0.15 mM magnesium acetate and 50 mM MES pH 6.5 in the presence of 2 mM myristic acid (substrate) . Using synchrotron radiation, both P450BSbeta crystals diffracted to 2.5 A resolution . Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem iron.

J Bacteriol, 2002 Apr, 184(8), 2314 - 8
Prediction of gene function in methylthioadenosine recycling from regulatory signals; Murphy BA et al.; The S-box transcription termination control system, first identified in Bacillus subtilis, is used for regulation of gene expression in response to methionine availability . The presence of the S-box motif provided the first indication that the ykrTS and ykrWXYZ genes could play a role in recycling of 5'-methylthioadenosine, a by-product of polyamine biosynthesis that can be converted to methionine . In this study we demonstrate a role for the ykrTS and ykrWXYZ gene products in this pathway.

J Bacteriol, 2002 Apr, 184(8), 2310 - 3
A MecA paralog, YpbH, binds ClpC, affecting both competence and sporulation; Persuh M et al.; ComK, the master regulator of competence, is degraded by the general stress-related protease ClpCP but must be targeted to this protease by binding to the adapter protein MecA . The genome of Bacillus subtilis contains a paralog of mecA, ypbH . We show in the present study that YpbH, like MecA, binds ClpC and that its elimination or overproduction affects competence and sporulation.

J Bacteriol, 2002 Apr, 184(8), 2148 - 54
Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase; Fisher SH et al.; Expression of the two Bacillus subtilis genes encoding L-asparaginase is controlled by independent regulatory factors . The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional L-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor . Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter . The expression of the ansA gene, which encodes the second L-asparaginase, was found to be induced by asparagine . The ansA repressor, AnsR, was shown to negatively regulate its own expression.

Life Sci Space Res, 1975, 13, 161 - 6
Results of the Bacillus subtilis unit of the Biostack II experiment: physical characteristics and biological effects of individual cosmic HZE particles; Bucker H et al.; The effectiveness of cosmic HZE-particles on unicellular procaryotic, organisms was studied on Bacillus subtilis spores, which were accommodated in the Biostack I and II experiments on board Apollo 16 and 17 . Identification of the spores that were hit was achieved by using the Biostack sandwich construction and by precise microscopical measurements of tracks of particles . Germination, outgrowth and the rate of cellular elongation were investigated . A method was developed to determine the charge of each individual HZE particle that penetrated a spore and its energy loss in the region of hit . An attempt was made to establish a connection between these physical characteristics and the biological effects produced.

Life Sci Space Res, 1975, 13, 153 - 9
Radiobiological results of the Biostack experiment on board Apollo 16 and 17; Graul EH et al.; After penetrating the Biostack capsule, some of the HZE particles hit the biological objects carried: bacterial spores (Bacillus subtilis), seeds (Arabidopsis thaliana and Vicia faba), and shrimp eggs (Artemia salina) . The different biological objects were affected by heavy ions in widely varying ways . A broad range of radiobiological investigations has been carried out in regard to the objects' response to HZE particles . The most sensitive biological objects in the Biostack experiments proved to be the shrimp eggs . The development of 500 eggs hit by heavy cosmic ions was investigated . This differed significantly from the flight controls (eggs flown in the Biostack but not hit by heavy ions) and from the ground controls . From this it has been concluded that penetration on the part of a single heavy ion may injure the encysted blastula . This damage was found to influence gastrula formation and even the hatching process of the nauplius . Abnormalities (increased by a factor of 10) in the orthonauplius were observed during the development of the hit eggs; they consisted, for example, of shortened extremities or an abnormal thorax or abdomen . In addition, eggs of Tribolium confusum and Carausius morosus, which were included in Biostack 2 (Apollo 17), have been investigated, and the influence of single heavy ions on the development process of these highly organized insects has been studied.

Life Sci Space Res, 1975, 13, 143 - 9
Effects of solar ultraviolet radiations on Bacillus subtilis spores and T7 bacteriophage; Spizizen J et al.; Spores of Bacillus subtilis HA 101 and the DNA polymerase I-defective mutant HA 101 (59) F were exposed to selected wavelengths of solar ultraviolet light and space vacuum during the return of Apollo 16 . In addition, coliphage T7 suspensions were exposed to solar ultraviolet radiation as part of the Microbial Response to Space Environment Experiment . Optical filters were employed to provide different energy levels at wavelengths 254 nm and 280 nm . Dose-response curves for lethal and mutagenic effects were compared with ground-based data . A close parallel was observed between the results of solar radiation and ground tests with spores of the two strains . However, significantly greater inactivation of T7 bacteriophage was observed after exposure to solar ultraviolet radiation.

Life Sci Space Res, 1974, 12, 75 - 83
Microbial studies in the Biostack experiment of the Apollo 16 mission: germination and outgrowth of single Bacillus subtilis spores hit by cosmic HZE particles; Horneck G et al.; Bacillus subtilis spores were flown in the Biostack experiment aboard the Apollo 16 command module . The spores embedded in plastic foils were stacked between physical track detectors . The energy loss spectrum of the heavy particles of cosmic radiation was determined . Biological studies were restricted to the high-energy loss component of these particles . Spores that had received single hits whose positions were determined with a typical accuracy of +/- 1 micrometers, were investigated for radiation effects on germination and outgrowth . It was found that germination was not influenced by a hit by an HZE particle, but outgrowth was reduced significantly.

Life Sci Space Res, 1974, 12, 209 - 13
Viability of Bacillus subtilis spores exposed to space environment in the M-191 experiment system aboard Apollo 16; Bucker H et al.; During the Apollo 16 space flight, in the experiment system M-191, (microbial response to space environment) spores of Bacillus subtilis 168 were exposed to space vacuum or solar UV irradiation with a peak wavelength of 254 nm or both . The effects of these space factors on the colony-forming ability of the spores were studied . It was found (i) that space vacuum alone did not affect the survival of pre-dried spores; (ii) that space vacuum in combination with solar UV irradiation with a peak wavelength of 254 nm had a synergistic effect, which may by attributed to a UV supersensitivity of the spores during vacuum exposure . These results agreed with findings of simulation experiments on earth . It was concluded that air dried spores may survive exposure to space vacuum if shielded against solar UV irradiation.

Mikrobiologiia, 2002 Jan-Feb, 71(1), 66 - 74
{The colony architectonics in Bacillus subtilis 2335}; Puzyr' AP et al.; Colonies grown from vegetative B . subtilis 2335 cells had a standard structure, with bacillar cells occupying the whole colony volume . At the same time, the colonies of this bacterium grown from germinated spores had an abnormal structure characterized by the location of cells in a surface layer 100-200 microns thick at the colony boundary with the air . The glycocalyx of the colonies grown from spores was characterized by a wetting angle theta e of 120 degrees-160 degrees, whereas that of the colonies grown from vegetative cells had an angle theta e as low as 5 degrees-30 degrees . It is suggested that spores and vegetative cells follow different strategies of substrate colonization and that the architectonics of bacterial colonies is determined by the physicochemical properties of the glycocalyx.

Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3695 - 700
Metabolic efficiency and amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis; Akashi H et al.; Biosynthesis of an Escherichia coli cell, with organic compounds as sources of energy and carbon, requires approximately 20 to 60 billion high-energy phosphate bonds {Stouthamer, A . H . (1973) Antonie van Leeuwenhoek 39, 545-565} . A substantial fraction of this energy budget is devoted to biosynthesis of amino acids, the building blocks of proteins . The fueling reactions of central metabolism provide precursor metabolites for synthesis of the 20 amino acids incorporated into proteins . Thus, synthesis of an amino acid entails a dual cost: energy is lost by diverting chemical intermediates from fueling reactions and additional energy is required to convert precursor metabolites to amino acids . Among amino acids, costs of synthesis vary from 12 to 74 high-energy phosphate bonds per molecule . The energetic advantage to encoding a less costly amino acid in a highly expressed gene can be greater than 0.025% of the total energy budget . Here, we provide evidence that amino acid composition in the proteomes of E . coli and Bacillus subtilis reflects the action of natural selection to enhance metabolic efficiency . We employ synonymous codon usage bias as a measure of translation rates and show increases in the abundance of less energetically costly amino acids in highly expressed proteins.

Mol Microbiol, 1997 Jul, 25(1), 65 - 78
Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT; Stulke J et al.; Glucose is the preferred carbon and energy source of Bacillus subtilis . It is transported into the cell by the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI locus . We show here that these three genes (ptsG, ptsH, and ptsI) form an operon, the expression of which is inducible by glucose . In addition, ptsH and ptsl form a constitutive ptsHI operon . The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive . Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene . Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied . The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product . A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified . The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon . A deletion of the terminator alleviates the need for GlcT . The activity of GlcT is negatively regulated by the glucose permease.

Mol Microbiol, 1997 Jul, 25(1), 39 - 51
Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis; Dartois V et al.; Endospore formation in Bacillus subtilis is primarily dependent on the phosphorylation of the key transcription factor Spo0A by two major kinases, KinA and KinB, thought to be activated by distinct signals . Using a strategy designed to detect mutations that specifically affect the signalling pathway to KinB, we have isolated a Tn10 insertion mutant in one of two adjacent lrp-like genes coding for homologues of the Escherichia coli leucine-responsive regulatory protein (Lrp) and another mutant in the glyA gene encoding the serine hydroxymethyl transferase (SHMT) . SHMT catalyses interconversion of serine and glycine while transferring the resulting one-carbon unit into the C1 pool through methylene tetrahydrofolate . Sporulation experiments performed in a series of supplemented media indicated that the role of SHMT in the KinB pathway is to feed the pool of C1 units recruited for the biosynthesis of key metabolites, which include the methyl donor S-adenosyl-methionine (SAM) . The results of experiments using L-ethionine suggest that SAM is involved in post-synthetic methylation reactions or biosynthesis of metabolites that serve to activate KinB . Truncated LrpA and LrpB peptides that have retained the DNA-binding domain but have lost the C-terminal half of the protein appear to act as repressors of glyA transcription and KinB-dependent sporulation . However, deletions of lrpA, lrpB or lrpAB have little effect on glyA transcription or sporulation through KinB, suggesting that other effectors, such as additional Lrp homologues, may act in conjunction with LrpA and LrpB . Our results indicate that lrpA-lrpB together with the biosynthetic glyA gene lie on a common signalling pathway meant to activate the KinB sensor kinase.

J Membr Biol, 2002 Jan 1, 185(1), 9 - 16 Epub 2002 Feb 05.
Functional characterization of CitM, the Mg2+-citrate transporter; Li H et al.; The CitM transporter from Bacillus subtilis transports citrate as a complex with Mg2+ . In this study, CitM was functionally expressed and characterized in E . coli DH5a cells . In the presence of saturating Mg2+ concentrations, the Km for citrate in CitM was 274 mM, similar to previous studies using whole cells of B . subtilis . CitM has a high substrate specificity for citrate . Other di- and tricarboxylic acids including succinate, isocitrate, cis-aconitate and tricarballylic acid did not significantly inhibit the uptake of citrate in the presence of Mg2+ . However, CitM accepts complexes of citrate with metal ions other than Mg2+ . The highest rate of citrate transport was seen in the presence of Mg2+, followed in order of preference by Mn2+, Ba2+, Ni2+, Co2+ and Ca2+ . Citrate transport by CitM appears to be proton coupled . The transport was inhibited in transport buffers more alkaline than pH 7.5 and not affected by pH at acidic values . Transport was also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin . Valinomycin did not affect the uptake by CitM, suggesting that transport is electroneutral . In conclusion, the cloned CitM transporter from B . subtilis expressed in E . coli has properties similar to the transporter in intact B . subtilis cells . The results support a transport model with a coupling stoichiometry of one proton coupled to the uptake of one complex of (Mg2+-citrate)1-.

Biosens Bioelectron, 2002 May, 17(5), 373 - 82
Sterilization of enzyme glucose sensors: problems and concepts; von Woedtke T et al.; A useful method of enzyme glucose sensor sterilization has not only to ensure the needs of sterility assurance but has also to guarantee the functional stability of the sensors . The action of 2 or 3% alkalinized glutaraldehyde solution, as well as gamma irradiation with a dose of 25 kGy caused changes of the in vitro functionality and polymer material irritations, respectively . After a combined treatment by 0.6% hydrogen peroxide solution acting over 4 days with 7 kGy gamma irradiation only a slight loss of sensitivity must be registered . The combination of a specially designed universal homogeneous ultraviolet irradiation over 300 s with a 3 days lasting treatment by an inclusion compound of hydrogen peroxide with tensides in urea (0.15% effective hydrogen peroxide concentration) did not cause any influence on the glucose sensor function in vitro . With all methods tested here, a Bacillus subtilis spore reduction over 8 log(10) cycles from 10(6) to 10(-2) spores per test object on an average could be proved experimentally . In general, if non-thermal methods must be used it seems to be impossible to guarantee a sterility assurance level of 10(-6) as it is demanded by the pharmacopoeias . Consequently, effective concepts to produce sterile glucose biosensors for medical and biological applications should be based not only on final product treatments but should include germ reducing measures in every manufacturing step.

Biomacromolecules, 2002 Mar-Apr, 3(2), 333 - 41
Enzymatic synthesis of inulin-containing hydrogels; Ferreira L et al.; The Bacillus subtilis protease Proleather FG-F catalyzed the transesterification of inulin with vinyl acrylate (VA) in dimethylformamide (DMF) . The reaction conversion for different VA concentrations was greater than 57% after 96 h at 50 degrees C . The degree of substitution (DS, defined as the amount of acrylate groups per 100 inulin fructofuranoside residues) with acrylate moieties can be controlled by varying the molar ratio of VA to inulin . Reasonable yields were obtained (44-51%, 2 days) using a two-step purification methodology . Inulin derivatized with VA (Inul-VA) was characterized by gel permeation chromatography, and its structure was established by (1)H, (13)C, and (1)H-(1)H correlation spectroscopy and (1)H-(13)C heteronuclear multiple quantum coherence NMR . The main positional isomer was at the 6 position of the fructofuranoside residue and two other minor isomers were observed at the 3 and 4 positions . Thus, the enzymatic reaction was largely regioselective . Furthermore, the inulin fructose residues were monosubstituted . Gels with swelling ratios at equilibrium of up to ca . 20 were prepared by free radical polymerization of aqueous solutions of Inul-VA with different DS and monomer concentrations . Gel pore sizes were calculated from swelling experiments and range from 19 to 57 A . To our knowledge, this work reports the first successful enzymatic modification of a polysaccharide solubilized in 100% DMF solution.

Acta Astronaut, 1975 Mar-Apr, 2(3-4), 247 - 64
The biological effectiveness of HZE-particles of cosmic radiation studied in the Apollo 16 and 17 Biostack experiments; Bucker H et al.; The Biostack experiments I and II were flown on board the Apollo 16 and 17 command modules in order to obtain information on the biological damage produced by the bombardment of heavy high-energy (HZE) particles of cosmic radiation during spaceflight . Such data are required for estimating radiation hazards in manned spaceflight . Seven biological systems in resting state (Bacillus subtilis spores, Colpoda cucullus cysts, Arabidopsis thaliana seeds, and eggs of Artemia salina, Tribolium castaneum and of Carausius morosus) were accommodated in the two Biostacks . By using a special sandwich construction of visual track detectors and layers of biological objects, identification of each hit biological object was achieved and the possible biological damage correlated with the physical features of the responsible HZE-particle . In the different systems the degree of damage depended on whether the hit cell was replaceable or not . A high sensitivity to HZE-particle bombardment was observed on Artemia salina eggs; 90% of the embryos, which were induced to develop from hit eggs, died at different developmental stages . Malformations of the abdomen or the extremities of the nauplius were frequently induced . In contrast, the growth of hit Vicia faba radiculae and the germination of hit Arabidopsis thaliana seeds and hit Bacillus subtilis spores were not influenced remarkably . But there was an increase in multicaulous plants and a reduction in the outgrowth of the bacterial spores . In addition, information was obtained on the fluence of the HZE-particles, on their spectrum of charge and energy loss, and on the absorption by the Apollo spacecraft and the Biostack material itself . This will help to improve knowledge concerning radiation conditions inside of spacecrafts, necessary to secure a maximum possible protection to the astronauts.

Mol Microbiol, 2001 Dec, 42(5), 1211 - 21
Isolation and characterization of topological specificity mutants of minD in Bacillus subtilis; Karoui ME et al.; In rod-shaped bacteria such as Bacillus subtilis, division site selection is mediated by MinC and MinD, which together function as a division inhibitor . Topological specificity is imposed by DivIVA, which ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at mid-cell . MinD plays a central role in this process, as it positions and activates MinC and is dependent on DivIVA for its own positioning at the poles . To investigate MinD activities further, we have constructed and analysed a collection of minD mutants . Mutations in the conserved ATPase motifs lead to an inactive protein, possibly unable to oligomerize, but which nevertheless retains some affinity for the cell membrane . Several mutations affecting the mid- to C-terminal parts of MinD led to a protein probably unable to interact with DivIVA, but that could still stimulate division inhibition by MinC . These findings suggest that the ATPase activity of MinD is necessary for all its functions (possibly in part by controlling the oligomerization state of the protein) . The other mutations may identify a surface of MinD involved in its interactions with DivIVA and a possible mechanism for control of MinD by DivIVA.

Mol Microbiol, 2001 Dec, 42(5), 1147 - 62
A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis; Eichenberger P et al.; We present evidence for a three-protein inhibitor of polar division that locks in asymmetry after the formation of a polar septum during sporulation in Bacillus subtilis . Asymmetric division involves the formation of cytokinetic Z-rings near both poles of the developing cell . Next, a septum is formed at one of the two polar Z-rings, thereby generating a small, forespore cell and a mother cell . Gene expression under the control of the mother-cell transcription factor sigmaE is needed to block cytokinesis at the pole distal to the newly formed septum . We report that this block in polar cytokinesis is mediated partly by sigmaE-directed transcription of spoIID, spoIIM and spoIIP, sporulation genes that were known to be involved in the subsequent process of forespore engulfment . We find that a spoIID, spoIIM and spoIIP triple mutant substantially mimicked the bipolar division phenotype of a sigmaE mutant and that cells engineered to produce SpoIID, SpoIIM and SpoIIP prematurely were inhibited in septum formation at both poles . Consistent with the hypothesis that SpoIID, SpoIIM and SpoIIP function at both poles of the sporangium, a GFP--SpoIIM fusion localized to the membrane that surrounds the engulfed forespore and to the potential division site at the distal pole.

Nucleic Acids Res, 2002 Mar 15, 30(6), 1418 - 26
Mining Bacillus subtilis chromosome heterogeneities using hidden Markov models; Nicolas P et al.; We present here the use of a new statistical segmentation method on the Bacillus subtilis chromosome sequence . Maximum likelihood parameter estimation of a hidden Markov model, based on the expectation-maximization algorithm, enables one to segment the DNA sequence according to its local composition . This approach is not based on sliding windows; it enables different compositional classes to be separated without prior knowledge of their content, size and localization . We compared these compositional classes, obtained from the sequence, with the annotated DNA physical map, sequence homologies and repeat regions . The first heterogeneity revealed discriminates between the two coding strands and the non-coding regions . Other main heterogeneities arise; some are related to horizontal gene transfer, some to t-enriched composition of hydrophobic protein coding strands, and others to the codon usage fitness of highly expressed genes . Concerning potential and established gene transfers, we found 9 of the 10 known prophages, plus 14 new regions of atypical composition . Some of them are surrounded by repeats, most of their genes have unknown function or possess homology to genes involved in secondary catabolism, metal and antibiotic resistance . Surprisingly, we notice that all of these detected regions are a + t-richer than the host genome, raising the question of their remote sources.

J Biol Chem, 2002 May 24, 277(21), 18849 - 59 Epub 2002 Mar 07.
Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat; Martins LO et al.; The Bacillus subtilis endospore coat protein CotA shows laccase activity . By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase . This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3) . Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides . However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) . The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain . The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases . Optimal enzymatic activity was found at < or =pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively . The apparent K(m) values for ABTS and SGZ at 37 degrees C were of 106 +/- 11 and 26 +/- 2 microm, respectively, with corresponding k(cat) values of 16.8 +/- 0.8 and 3.7 +/- 0.1 s(-1) . Maximal enzyme activity was observed at 75 degrees C with ABTS as substrate . Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 degrees C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable.

Life Sci Space Res, 1964, 2, 357 - 71
Some biological and physical factors in dry heat sterilization: a general review; Bruch CW; There is a surprising lack of quantitative data on sterilization by dry heat so that microbiologists have little knowledge of the role played by various biological and physical factors in this sterilizing process . A recent investigation by the author has shown that the aerobic mesophilic bacterial sporeformers, such as Bacillus subtilis and Bacillus coagulans, are the most resistant among several species of sporeforming bacteria to dry heat sterilization . The type of carrier on which the spores are exposed to dry heat markedly affects their thermal resistance . An analysis of four carriers showed that spores on sand or vermiculite are more difficult to destroy than spores on paper or glass . Spores under low vacuums are more susceptible to dry heat sterilization than spores in helium, which are more susceptible than spores in air . Spores trapped in solids have thermal resistance levels two or three times greater than those found for spores exposed to dry heat in air . Preliminary results on the combination of dry heat and ionizing radiation sterilization indicate no synergistic effects, i.e., the destruction obtained with each agent is additive . Another important variable that governs the interpretation of the effectiveness of dry heat sterilization tests is the recovery medium for heat-damaged spores . The kinetics of dry heat sterilization cannot be fully interpreted from the available data . Death follows a logarithmic pattern thereby implying a monomolecular reaction . The mechanism of death is thought to be due to an oxidative process.

Microbiology, 2002 Mar, 148(Pt 3), 815 - 24
Comparison of ribitol and glycerol teichoic acid genes in Bacillus subtilis W23 and 168: identical function, similar divergent organization, but different regulation; Lazarevic V et al.; The tar genes directing the synthesis of poly(ribitol phosphate), the main teichoic acid in Bacillus subtilis strain W23, were sequenced . They are organized in two divergently transcribed operons, tarABIJKL and tarDF, as are the tag genes specifying poly(glycerol phosphate) synthesis in B . subtilis 168 . The features of the tar genes as well as the putative participation of their products in the proposed biosynthesis pathway of poly(ribitol phosphate) are presented . The tarA and tarD genes, which are most likely involved in the synthesis of the linkage unit (the entity coupling teichoic acid to peptidoglycan), are separated by 508 nt . Sequences of the outer segments of this regulatory region are similar to the two divergent promoter regions identified upstream of the tagA and tagD genes in strain 168 . However, in W23, these regions, which also included functional promoters, are separated by an additional DNA segment of about 100 nt, on which two new mRNA starts, one in each direction, were identified . The regulatory regions of teichoic acid divergons of Bacillus globigii, Bacillus licheniformis and eight strains of B . subtilis were cloned and sequenced . In four B . subtilis strains and in B . globigii, their length and sequence are similar to the regulatory region of W23 . In the others, including B . licheniformis, they are of the 168-type . Analysis of nucleotide sequences of a non-coding grey hole, present in the tag region of strain 168, revealed higher similarities to tar than to tag entities . This suggests that at least part of the tag genes specifying the synthesis of glucosylated poly(glycerol phosphate) in strain 168 was introduced by horizontal gene transfer into a strain originally synthesizing a ribitol-phosphate-containing teichoic acid.

Microbiology, 2002 Mar, 148(Pt 3), 807 - 13
Oligomerization of the Bacillus subtilis division protein DivIVA; Muchova K et al.; DivIVA appears to be a mediator of inhibition by MinCD of division at the cell poles in Bacillus subtilis . Gel permeation and ultracentrifugation techniques were used to show self-association of DivIVA into a form consisting of 10-12 monomers in vitro . Western blot analysis of non-denaturating polyacrylamide gels confirms the presence of similar oligomers in B . subtilis cell lysates . These oligomers persist in a B . subtilis strain containing the divIVA1 mutation, in which proper vegetative septum positioning is abolished . In contrast, the divIVA2 mutation, which has a similar biological impact, appears to produce a protein with different oligomerization properties . The results of the present study suggest that oligomerization of DivIVA is important, but not sufficient for its function in the cell division process.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Nov-Dec, (6), 77 - 82
{Adjuvant properties of subalin, a recombinant interferon-producing probiotic}; Beliavskaia VA et al.; The adjuvant properties of subalin, a recombinant probiotic prepared from live bacteria Bacillus subtilis producing human alpha 2-interferon were studied in the scheme of its use with vaccines against parvovirus enteritis and distemper . Subalin was shown to be capable of preventing immunosuppression caused by the injection of vaccines, accelerating the formation of the antigen-specific clone of memory cells and enhancing antigen-specific immune response . The mechanisms of the adjuvant effect of subalin were considered; this effect was shown to be due to the action of interferon excreted by bacteria of B . subtilis into the lumen of the intestine . The advantages of this method of interferon supply and the prospects of using subalin preparation as adjuvant are discussed.

Eur J Biochem, 2002 Mar, 269(5), 1456 - 63
Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli; Job V et al.; We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding alpha-keto acid, ammonia and hydrogen peroxide . By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO) . The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA) . In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g(-1) of cell (and a fermentation yield of 3.82 U x L(-1) of fermentation broth) . An N-terminal His-tagged protein (HisGO) was also successfully expressed in E . coli as a soluble protein and a fully active holoenzyme . HisGO represents approximately 3.9% of the total soluble protein content of the cell . The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 U x mg(-1) protein at 25 degrees C and with a yield of 98% . The characterization of the purified enzyme showed that GO is a homotetramer of approximately 180 kDa with the spectral properties typical of flavoproteins . GO exhibits good thermal stability, with a Tm of 46 degrees C after 30 min incubation; its stability is maximal in the 7.0-8.5 pH range . A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids.

J Bacteriol, 2002 Mar, 184(6), 1783 - 7
Induction of ResDE-dependent gene expression in Bacillus subtilis in response to nitric oxide and nitrosative stress; Nakano MM; Transcription of ResDE-controlled genes in Bacillus subtilis was induced by sodium nitroprusside and nitric oxide . This induction requires the sensor kinase ResE and the response regulator ResD . Among members of the ResDE regulon, only the flavohemoglobin gene was induced by nitrosative stress via both a ResDE-dependent mechanism and an unidentified ResDE-independent mechanism.

J Bacteriol, 2002 Mar, 184(6), 1743 - 9
Partitioning of chromosomal DNA during establishment of cellular asymmetry in Bacillus subtilis; Pogliano J et al.; The switch from symmetric to asymmetric cell division is a key feature of development in many organisms, including Bacillus subtilis sporulation . Here we demonstrate that, prior to the onset of asymmetric cell division, the B . subtilis chromosome is partitioned into two unequally sized domains, with the origin-proximal one-third of the future forespore chromosome condensed near one pole of the cell . Asymmetric chromosome partitioning is independent of polar division, as it occurs in cells depleted of FtsZ but depends on two transcription factors that govern the initiation of sporulation, sigma(H) and Spo0A-P . It is also independent of chromosome partitioning proteins Spo0J and Soj, suggesting the existence of a novel mechanism controlling chromosome structure . Thus, our results demonstrate that, during sporulation, two separable events prepare B . subtilis for asymmetric cell division: the relocation of cell division sites to the cell poles and the asymmetric partitioning of the future forespore chromosome.

J Bacteriol, 2002 Mar, 184(6), 1703 - 11
Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3; Stein T et al.; A lantibiotic gene cluster was identified in Bacillus subtilis A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the spa genes in B . subtilis ATCC 6633 . The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a lanC gene . Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of B . subtilis A1/3 culture extracts confirmed the presence of two lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da) . Disruption of the lanB-homologous gene eriB resulted in loss of production of both peptides, demonstrating that they are processed in an eriB-dependent manner . Although precursors of ericins S and A show only 75% of identity, the matured lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography . Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed . For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin . Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment . For ericin A only minor antibiotic activity was found.

Appl Environ Microbiol, 2002 Mar, 68(3), 1102 - 8
Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin; Wu SC et al.; Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications . Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production . However, attempts to produce streptavidin using B . subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency . This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin . We addressed this dilemma by engineering a B . subtilis strain (WB800BIO) which overproduces intracellular biotin . The strategy involves replacing the natural regulatory region of the B . subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B . subtilis groE promoter and gluconate operator . Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage . A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium . WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

J Biol Chem, 2002 May 10, 277(19), 17023 - 31 Epub 2002 Feb 26.
Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp; Mofid MR et al.; The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein . 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity . Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B . subtilis ACP that contains the interacting residues . This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B . brevis . Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100% . The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe) . We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe . However, communication between the hybrid PCP and the epimerization domain seems to be disabled . Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.

J Mol Biol, 2002 Feb 22, 316(3), 443 - 57
Defining the Bacillus subtilis sigma(W) regulon: a comparative analysis of promoter consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional profiling approaches; Cao M et al.; The Bacillus subtilis extracytoplasmic function (ECF) sigma factor sigma(W) controls a large regulon that is strongly induced by alkali shock . To define the physiological role of sigma(W) we have sought to identify the complete set of genes under sigma(W) control . Previously, we described a promoter consensus search procedure to identify sigma(W) controlled genes . Herein, we introduce a novel method to identify additional target promoters: run-off transcription followed by macroarray analysis (ROMA) . We compare the resulting list of targets with those identified in conventional transcriptional profiling studies and using the consensus search approach . While transcriptional profiling identifies genes that are strongly dependent on sigma(W) for in vivo expression, some sigma(W)-dependent promoters are not detected due to the masking effects of other promoter elements, overlapping recognition with other ECF sigma factors, or both . Taken together, the consensus search, ROMA, and transcriptional profiling approaches establish a minimum of 30 promoter sites (controlling approximately 60 genes) as direct targets for activation by sigma(W) . Significantly, no single approach identifies more than approximately 80% of the regulon so defined . We therefore suggest that a combination of two or more complementary approaches be employed in studies seeking to achieve maximal coverage when defining bacterial regulons . Our results indicate that sigma(W) controls genes that protect the cell against agents that impair cell wall biosynthesis but fail to reveal any connection to operons likely to function in adaptation to alkaline growth conditions . This is consistent with the observation that a sigW mutant is unaffected in its ability to survive alkali shock . We conclude that in B . subtilis sudden imposition of alkali stress activates the sigma(W) stress response, perhaps by impairing the ability of the cell wall biosynthetic machinery to function .

Nucleic Acids Res, 2002 Mar 1, 30(5), 1278 - 85
Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro; Chowdhury S et al.; The active site of a protein folding reaction is in domain V of the 23S rRNA in the bacterial ribosome and its homologs in other organisms . This domain has long been known as the peptidyl transferase center . Domain V of Bacillus subtilis is split into two segments, the more conserved large peptidyl transferase loop (RNA1) and the rest (RNA2) . These two segments together act as a protein folding modulator as well as the complete domain V RNA . A number of site-directed mutations were introduced in RNA1 and RNA2 of B.subtilis, taking clues from reports of these sites being involved in various steps of protein synthesis . For example, sites like G2505, U2506, U2584 and U2585 in Escherichia coli RNA1 region are protected by deacylated tRNA at high Mg2+ concentration and A2602 is protected by amino acyl tRNA when the P site remains occupied already . Mutations A2058G and A2059G in the RNA1 region render the ribosome Ery(r )in E.coli and Lnc(r )in tobacco chloroplast . Sites in P loop G2252 and G2253 in E.coli are protected against modification by the CCA end of the P site bound tRNA . Mutations were introduced in corresponding nucleotides in B.subtilis RNA1 and RNA2 of domain V . The mutants were tested for refolding using unfolded protein binding assays with unfolded carbonic anhydrase . In the protein folding assay, the mutants showed partial to complete loss of this activity . In the filter binding assay for the RNA-refolding protein complex, the mutants showed an extent of protein binding that agreed well with their protein folding activity.

J Biotechnol, 2002 Apr 11, 94(3), 265 - 75
Enzymatic properties of a neutral endo-1,3(4)-beta-xylanase Xyl II from Bacillus subtilis; Sa-Pereira P et al.; A Bacillus sp . CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min . It produces at least two extracellular xylanases, Xyl I and Xyl II . The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile . The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3 . The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa . Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography . The enzyme showed an optimum pH and temperature of 6.0 at 60 degrees C . Xyl II was stable at 40 degrees C for 180 min at pH 6.0 . The specificity of Xyl II for different substrates was evaluated . Xyl II presents a higher affinity towards OSX, with a K(m) of 1.56 g l(-1) and showed the ability to hydrolyse laminarin, with a K(m) of 1.02 g l(-1) . Xylotetraose is the main product of xylan degradation . The Xyl II ability for binding to cellulose and/or xylan was also studied.

J Appl Microbiol, 2001 Dec, 91(6), 1051 - 8
Analysis of the killing of spores of Bacillus subtilis by a new disinfectant, Sterilox; Loshon CA et al.; AIMS: To determine the mechanism whereby the new disinfectant Sterilox kills spores of Bacillus subtilis . METHODS AND RESULTS: Bacillus subtilis spores were readily killed by Sterilox and spore resistance to this agent was due in large part to the spore coats . Spore killing by Sterilox was not through DNA damage, released essentially no spore dipicolinic acid and Sterilox-killed spores underwent the early steps in spore germination, including dipicolinic acid release, cortex degradation and initiation of metabolism . However, these germinated spores never swelled and many had altered permeability properties . CONCLUSIONS: We suggest that Sterilox treatment kills dormant spores by oxidatively modifying the inner membrane of the spores such that this membrane becomes non-functional in the germinated spore leading to spore death . SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to and spore killing by a new disinfectant.

Biochemistry, 2002 Feb 26, 41(8), 2563 - 70
Crystal structure of the tetrameric cytidine deaminase from Bacillus subtilis at 2.0 A resolution; Johansson E et al.; Cytidine deaminases (CDA, EC 3.5.4.5) are zinc-containing enzymes in the pyrimidine salvage pathway that catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively . Two different classes have been identified in the CDA family, a homodimeric form (D-CDA) with two zinc ions per dimer and a homotetrameric form (T-CDA) with four zinc ions per tetramer . We have determined the first structure of a T-CDA from Bacillus subtilis . The active form of T-CDA is assembled of four identical subunits with one active site apiece . The subunit of D-CDA is composed of two domains each exhibiting the same fold as the T-CDA subunits, but only one of them contains zinc in the active site . The similarity results in a conserved structural core in the two CDA forms . An intriguing difference between the two CDA structures is the zinc coordinating residues found at the N-terminal of two alpha-helices: three cysteine residues in the tetrameric form and two cysteine residues and one histidine residue in the dimeric form . The role of the zinc ion is to activate a water molecule and thereby generate a hydroxide ion . How the zinc ion in T-CDA surrounded with three negatively charged residues can create a similar activity of T-CDA compared to D-CDA has been an enigma . However, the structure of T-CDA reveals that the negative charge caused by the three ligands is partly neutralized by (1) an arginine residue hydrogen-bonded to two of the cysteine residues and (2) the dipoles of two alpha-helices.

J Mol Biol, 2002 Feb 15, 316(2), 235 - 45
Dimer formation and transcription activation in the sporulation response regulator Spo0A; Lewis RJ et al.; The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis . Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain . If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds . Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain . An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation . Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain . Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not . This suggests that dimers represent the activated form of Spo0A .

Mol Microbiol, 2002 Jan, 43(1), 27 - 38
An investigation into the compartmentalization of the sporulation transcription factor sigmaE in Bacillus subtilis; Fujita M et al.; Sporulation in Bacillus subtilis involves the formation of a polar septum, which divides the sporangium into a mother cell and a forespore . The sigmaE factor, which is encoded within the spoIIG operon, is a cell-specific regulatory protein that directs gene transcription in the mother cell . SigmaE is synthesized as an inactive proprotein pro-sigmaE, which is converted to the mature factor by the putative processing enzyme SpoIIGA . Processing of pro-sigmaE does not commence until after asymmetric division when sigmaE is largely confined to the mother cell . Processing depends on the signalling protein SpoIIR, which delays proteolysis until after polar septation, but the mechanism by which sigmaE is confined to the mother cell is not understood . Previous work favoured a model in which pro-sigmaE localizes to the mother cell face of the polar septum, such that sigmaE would be selectively released into mother cell cytoplasm . Based on the use of green fluorescent protein (GFP) fusions, we now report that pro-sigmaE is distributed approximately uniformly along all membrane surfaces and is not confined to the mother- cell face of the septum . Rather, our results are consistent with a model in which preferential and persistent transcription of the spoIIG operon in the mother cell and degradation of sigmaE in the forespore contribute to the selective accumulation of sigmaE in the mother cell . Persistent transcription of spoIIG after polar septation also contributes to the proper timing of pro-sigmaE processing.

Mol Microbiol, 2002 Jan, 43(1), 15 - 26
Rok (YkuW) regulates genetic competence in Bacillus subtilis by directly repressing comK; Hoa TT et al.; The Rok (YkuW) protein acts as a negative regulator of comK, which encodes the competence transcription factor of Bacillus subtilis . In the absence of Rok, ComK is overproduced, and when excess Rok is present comK transcription is inhibited . Rok acts transcriptionally to repress comK expression but does not affect ComK stability, which is controlled by the MecA switch . Gel-shift assays show that Rok binds directly to a DNA fragment that contains the comK promoter . SinR and AbrB act negatively on rok transcription, and the inactivation of rok bypasses the positive requirements for sinR and abrB for the expression of comK . Therefore, the dependence of comK expression on SinR and AbrB may be a result of their repression of rok transcription . It has also been shown in vivo that Rok and ComK can indivi-dually repress rok transcription, and that Rok and ComK bind to the rok promoter in vitro . These interactions establish feedback loops, and the roles of these circuits are discussed.

Lett Appl Microbiol, 2002, 34(1), 42 - 5
The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea; Walker R et al.; AIMS: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L-form bacteria with Chinese cabbage . METHODS AND RESULTS: Germinating seeds of Chinese cabbage were co-cultivated with either L-forms of Bacillus subtilis or 5% (w/v) mannitol by soaking for 3 h . Seeds were then washed in sterile water, sown on a minimal medium and incubated in controlled conditions . L-form symbiosis was detected over a time course by ELISA . Conidial germination of Botrytis cinerea was significantly reduced on cotyledonous leaves of L-form-treated plants compared with controls . CONCLUSIONS: Symbiosis of B . subtilis L-form bacteria during seed germination of Chinese cabbage inhibits conidial germination in plants on subsequent exposure to Botrytis cinerea . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first account of plant symbiosis with L-form bacteria showing antagonism to a fungal plant pathogen . This has promising implications for the use of this L-form as a biocontrol agent.

J Appl Microbiol, 2002, 92(2), 362 - 75
Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol; Setlow B et al.; AIMS: To determine the mechanisms of killing of Bacillus subtilis spores by ethanol or strong acid or alkali . METHODS AND RESULTS: Killing of B . subtilis spores by ethanol or strong acid or alkali was not through DNA damage and the spore coats did not protect spores against these agents . Spores treated with ethanol or acid released their dipicolinic acid (DPA) in parallel with spore killing and the core wet density of ethanol- or acid-killed spores fell to a value close to that for untreated spores lacking DPA . The core regions of spores killed by these two agents were stained by nucleic acid stains that do not penetrate into the core of untreated spores and acid-killed spores appeared to have ruptured . Spores killed by these two agents also did not germinate in nutrient and non-nutrient germinants and were not recovered by lysozyme treatment . Spores killed by alkali did not lose their DPA, did not exhibit a decrease in their core wet density and their cores were not stained by nucleic acid stains . Alkali-killed spores released their DPA upon initiation of spore germination, but did not initiate metabolism and degraded their cortex very poorly . However, spores apparently killed by alkali were recovered by lysozyme treatment . CONCLUSIONS: The data suggest that spore killing by ethanol and strong acid involves the disruption of a spore permeability barrier, while spore killing by strong alkali is due to the inactivation of spore cortex lytic enzymes.SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide further information on the mechanisms of spore killing by various chemicals.

BMC Microbiol . 2002;2(1):1 . Epub 2002 Jan 28.
The mechanisms responsible for 2-dimensional pattern formation in bacterial macrofiber populations grown on solid surfaces: fiber joining and the creation of exclusion zones; Mendelson NH et al.; BACKGROUND: When Bacillus subtilis is cultured in a complex fluid medium under conditions where cell separation is suppressed, populations of multicellular macrofibers arise that mature into ball-like structures . The final sedentary forms are found distributed in patterns on the floor of the growth chamber although individual cells have no flagellar-driven motility . The nature of the patterns and their mode of formation are described in this communication . RESULTS: Time-lapse video films reveal that fiber-fiber contact in high density populations of macrofibers resulted in their joining either by entwining or supercoiling . Joining led to the production of aggregate structures that eventually contained all of the fibers located in an initial area . Fibers were brought into contact by convection currents and motions associated with macrofiber self-assembly such as walking, pivoting and supercoiling . Large sedentary aggregate structures cleared surrounding areas of other structures by dragging them into the aggregate using supercoiling of extended fibers to power dragging . The spatial distribution of aggregate structures in 6 mature patterns containing a total of 637 structures was compared to that expected in random theoretical populations of the same size distributed in the same surface area . Observed and expected patterns differ significantly . The distances separating all nearest neighbors from one another in observed populations were also measured . The average distance obtained from 1451 measurements involving 519 structures was 0.73 cm . These spacings were achieved without the use of flagella or other conventional bacterial motility mechanisms . A simple mathematical model based upon joining of all structures within an area defined by the minimum observed distance between structures in populations explains the observed distributions very well . CONCLUSIONS: Bacterial macrofibers are capable of colonizing a solid surface by forming large multicellular aggregate structures that are distributed in unique two-dimensional patterns . Cell growth geometry governs in an hierarchical way the formation of these patterns using forces associated with twisting and supercoiling to drive motions and the joining of structures together . Joining by entwining, supercoiling or dragging all require cell growth in a multicellular form, and all result in tightly fused aggregate structures.

J Mol Biol, 2001 Nov 30, 314(3), 359 - 64
NMR studies of the interactions of SpoIIAA with its partner proteins that regulate sporulation in Bacillus subtilis; Clarkson J et al.; SpoIIAA is a key component in the network of interactions that regulate the first sporulation-specific transcription factor, sigma(F), in Bacillus subtilis . In its unphosphorylated form SpoIIAA is either phosphorylated by or forms a non-covalent complex with SpoIIAB, whereas in its phosphorylated form it is dephosphorylated by SpoIIE . In this work we present NMR studies of the SpoIIAA(2).SpoIIAB complex and of mutant proteins that are deficient in their ability to interact with SpoIIAB or SpoIIE . The NMR studies of the SpoIIAA(2).SpoIIAB complex allowed us to define a contiguous patch that is perturbed upon complex formation . By examining the chemical shift perturbations in the mutant proteins we have identified more specific areas that contain residues critical for the SpoIIAB and SpoIIE interactions .

J Bacteriol, 2002 Mar, 184(5), 1458 - 61
Glucose-resistant sporulation in Bacillus subtilis crsA47 mutants does not depend on promoter switching at the spo0A gene; Dixon LG et al.; We have found that sporulation in Bacillus subtilis crsA47 mutants does not require the sigma(H)-dependent promoter of the spo0A gene . This implies that the glucose-resistant sporulation phenotype of this strain is not related to the switch from the vegetative-stage sigma(A)-dependent promoter to the sigma(H)-dependent promoter at the spo0A gene.

J Bacteriol, 2002 Mar, 184(5), 1423 - 9
Mutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppress cytochrome c deficiency of CcdA-defective Bacillus subtilis cells; Erlendsson LS et al.; Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane . They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds . The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur . It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane . Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis . We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA . BdbC and BdbD are thiol-disulfide oxidoreductases . Our experimental findings indicate that these B . subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.

Planta Med, 2002 Jan, 68(1), 55 - 9
Eremophilane sesquiterpenes from Cacalia ainsliaeflora; Mao M et al.; Chemical investigation of Cacalia ainsliaeflora afforded four new eremophilane sesquiterpenes (1 - 3, 5) and a known sesquiterpene (4), which were identified as 3 beta-angeloyloxy-8 alpha-hydroxy-6 beta-methoxyeremophil-7(11),9(10)-dien-8,12-olide (1), 3 beta-angeloyloxy-6 beta,8 alpha-dihydroxyeremophil-7(11),9(10)-dien-8,12-olide (2), 3 beta-angeloyloxy-8 alpha-hydroxy-6 beta-ethoxyeremophil-7(11),9(10)-dien-8,12-olide (3), 3 beta-angeloyloxy-8-oxo-eremphila-6,9-dien-12-oic acid (4), and 3,8-oxo-eremophila-6,9-dien-12-oic acid (5) respectively . These compounds were assayed against Escherichia coli, Staphylococcus aureus and Bacillus subtilis, and compounds 1, 2, 4 and 5 showed weak antibacterial activity . The structures of the compounds were established by 1D and 2D NMR experiments.

Nucleic Acids Res, 2002 Feb 15, 30(4), 1065 - 72
In vitro structure-function studies of the Bacillus subtilis tyrS mRNA antiterminator: evidence for factor-independent tRNA acceptor stem binding specificity; Gerdeman MS et al.; Expression of many aminoacyl-tRNA synthetase, amino acid biosynthesis and transport genes in Bacillus subtilis is controlled at the level of transcription termination using the T box system and requires the formation of specific secondary structures in the mRNA leader region . One structure functions as a transcriptional terminator, while an alternate form, the antiterminator, is necessary for transcription of the downstream coding regions . We have investigated the interaction of antiterminator model RNAs, based on the B.subtilis tyrS antiterminator with tRNA(Tyr) and tRNA acceptor stem models, using a gel shift assay . Binding of the antiterminator RNA to tRNA(Tyr) was dependent on complimentarity with the acceptor end of the tRNA or microhelix; affinity for the microhelix RNA was reduced relative to the tRNA . Alteration of a conserved position in the non-base pairing region of the bulge greatly reduced tRNA binding, consistent with in vivo studies . Therefore, it appears that some of the antiterminator-tRNA binding specificity is dependent on the structure of the antiterminator bulge alone and the complex it forms with tRNA in the absence of additional trans-acting factors . During the course of these studies we also discovered that the antiterminator can form a 'kissing' bulge complex, a unique RNA motif . The ease of formation of this RNA homodimer illustrates the propensity for the bulge of the antiterminator to bind RNA.

Nucleic Acids Res, 2002 Feb 15, 30(4), 966 - 74
A functional interaction between the putative primosomal protein DnaI and the main replicative DNA helicase DnaB in Bacillus; Soultanas P; In Gram negative Escherichia coli there are two well-characterised primosomal assembly processes, the PriA- and DnaA-mediated cascades . The presence of PriA and DnaA proteins in Gram positive Bacillus spp . supports the assumption that both the PriA- and DnaA-mediated primosomal assembly cascades also operate in these organisms . However, the lack of sequence homology between the rest of the primosomal proteins indicates significant differences between these two bacterial species . Central to the process of primosomal assembly is the loading of the main hexameric replicative helicase (DnaB in E.coli and DnaC in Bacillus subtilis) on the DNA . This loading is achieved by specialised proteins known as 'helicase loaders' . In E.coli DnaT and DnaC are responsible for loading DnaB onto the DNA during primosome assembly, in the PriA- and DnaA-mediated cascades, respectively . In Bacillus the identity of the helicase loader is still not established unequivocally . In this paper we provide evidence for a functional interaction between the primosomal protein DnaI from B.subtilis and the main hexameric replicative helicase DnaB from Bacillus stearothermophilus . Our results are consistent with the putative role of DnaI as the 'helicase loader' in the Gram positive Bacillus spp.

Biochemistry, 2002 Feb 19, 41(7), 2217 - 26
Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required; Brosius JL et al.; Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate . We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit {Brosius, J . L., and Colman, R . F . (2000) Biochemistry 39, 13336-13343} . Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit . Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme . Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity . Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed . Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly . For K268R, the K(m)s for all substrates are similar to WT enzyme . Binding studies using {2-3H}-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate . We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141 . Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity . These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL . Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.

Proteomics, 2002 Feb, 2(2), 206 - 11
A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography; Park SG et al.; Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168 . Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel . After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel . We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry . Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr . This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.

Curr Biol, 2002 Feb 5, 12(3), R90 - 2
Developmental biology: regulation by selective gene localization; Sonenshein AL; Recent studies have shown that, early during sporulation in Bacillus subtilis, the temporary exclusion of 70% of the chromosome from the forespore compartment is critical to the regulated activation of two major transcription factors, sigma(F) and sigma(E).

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 101 - 7
Bacillus endophyticus sp . nov., isolated from the inner tissues of cotton plants (Gossypium sp.); Reva ON et al.; Four strains of aerobic, endospore-forming bacteria were isolated from the inner tissues of healthy cotton plants (Gossypium sp., Dushanbe, Tajikistan) . The organisms had identical randomly amplified polymorphic DNA patterns that distinguished them from other bacilli that are commonly isolated from plant tissues, e.g . Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus subtilis . PCR amplification of 16S-23S rRNA spacer regions suggested that the four strains could be assigned to two highly related taxa, which correlated with differences in cell morphology . However, the cloned spacer region provided a simple and specific hybridization probe for all four strains . The virtually complete 16S rDNA sequences were prepared for representatives of the two groups (strains 2DT(T) and 12DX) and differed by only two bases, thus supporting classification of the four strains in a single taxon at the species level . Phylogenetic analyses indicated that strain 2DT(T) belonged to the genus Bacillus and was most closely related to Bacillus sporothermodurans DSM 10599T with a sequence similarity of 94.8% . It is concluded that the four strains belong to a novel species of Bacillus for which the name Bacillus endophyticus sp . nov . is proposed . The type strain is 2DT(T) (= UCM B-5715T = CIP 106778T).

J Biol Chem, 2002 Apr 19, 277(16), 14343 - 9 Epub 2002 Feb 07.
Recognition by tryptophanyl-tRNA synthetases of discriminator base on tRNATrp from three biological domains; Guo Q et al.; To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA(Trp) discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA(Trp), representing the three biological domains . When B . subtilis, A . fulgidus, and human TrpRS were used to acylate these tRNA(Trp), two distinct preference profiles regarding the discriminator base of different tRNA(Trp) substrates were found: G>A>U>C for B . subtilis TrpRS, and A>C>U>G for A . fulgidus and human TrpRS . The preference for G73 in tRNA(Trp) by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS . Cross-species reactivities between TrpRS and tRNA(Trp) from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria . NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA(Trp) elicited a conformational alteration in the G1-C72 base pair . Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA(Trp) formation . These observations identify a tRNA(Trp) structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.

Microbiology, 2002 Feb, 148(Pt 2), 507 - 18
The metIC operon involved in methionine biosynthesis in Bacillus subtilis is controlled by transcription antitermination; Auger S et al.; There are two major pathways for methionine biosynthesis in micro-organisms . Little is known about these pathways in Bacillus subtilis . The authors assigned a function to the metI (formerly yjcI) and metC (formerly yjcJ) genes of B . subtilis by complementing Escherichia coli metB and metC mutants, analysing the phenotype of B . subtilis metI and metC mutants, and carrying out enzyme activity assays . These genes encode polypeptides belonging to the cystathionine gamma-synthase family of proteins . Interestingly, the MetI protein has both cystathionine gamma-synthase and O-acetylhomoserine thiolyase activities, whereas the MetC protein is a cystathionine beta-lyase . In B . subtilis, the transsulfuration and the thiolation pathways are functional in vivo . Due to its dual activity, the MetI protein participates in both pathways . The metI and metC genes form an operon, the expression of which is subject to sulfur-dependent regulation . When the sulfur source is sulfate or cysteine the transcription of this operon is high . Conversely, when the sulfur source is methionine its transcription is low . An S-box sequence, which is located upstream of the metI gene, is involved in the regulation of the metIC operon . Northern blot experiments demonstrated the existence of two transcripts: a small transcript corresponding to the premature transcription termination at the terminator present in the S-box and a large one corresponding to transcription of the complete metIC operon . When methionine levels were limiting, the amount of the full-length transcript increased . These results substantiate a model of regulation by transcription antitermination.

Arch Biochem Biophys, 2002 Feb 15, 398(2), 170 - 8
Saccharomyces cerevisiae ferrochelatase forms a homodimer; Grzybowska E et al.; Ferrochelatase, the last enzyme of the heme biosynthetic pathway, has for years been considered to be active as a monomer . The crystal structure of Bacillus subtilis ferrochelatase confirmed its monomeric structure . However, animal ferrochelatase was found to form a functional dimer . Data presented here indicate that ferrochelatase from the yeast Saccharomyces cerevisiae is also dimeric . Following two-hybrid studies that had shown an interaction of two ferrochelatase molecules, we employed several different, complementary approaches, such as chemical crosslinking, affinity chromatography, and complementation analysis, to prove that in the yeast cells ferrochelatase forms an active dimer . We have isolated a double mutant, hem15D246V/Y248F, which is probably dimerization-defective . We propose a structural model of yeast ferrochelatase, based on the known structure of the human enzyme, which helps us to understand the differences in dimerization between the wild-type and mutant proteins.

Biometals, 2001 Sep-Dec, 14(3-4), 239 - 49
Bacterial zinc transporters and regulators; Hantke K; Zn2+ homeostasis in bacteria is achieved by export systems and uptake systems which are separately regulated by their own regulators . Three types of Zn2+ export systems that protect cells from high toxic concentrations of Zn2+ have been identified: RND multi-drug efflux transporters, P-type ATPases, and cation-diffusion facilitators . The RND type exporters for Zn2+ are only found in a few gram-negative bacteria; they allow a very efficient export across the cytoplasmic membrane and the outer membrane of the cell . P-type ATPases and cation-diffusion facilitators belong to protein families that are also found in eukaryotes . The exporters are regulated in bacteria by MerR-like repressor/activators or by ArsR-like repressors . For the high-affinity uptake of Zn2+, several binding-protein-dependent ABC transporters belonging to one class have been identified in different bacteria . Zn2+ ABC transporters are regulated by Zur repressors, which belong to the Fur protein family of iron regulators . Little is known about low-affinity Zn2+ uptake under zinc-replete conditions . One known example is the phosphate uptake system Pit, which may cotransport Zn2+ in Escherichia coli . Similarly, the citrate-metal cotransporter CitM in Bacillus subtilis may help to supply Zn2+.

Biochemistry, 2002 Feb 12, 41(6), 1886 - 92
Enzymatic reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy; Matsunaga I et al.; Cytochromes P450SP(alpha) (CYP152B1) and P450BS(beta) (CYP152A1), which are isolated from Sphingomonas paucimobilis and Bacillus subtilis, respectively, belong to the P450 superfamily, but catalyze hydroxylation reactions, in which an oxygen atom from H2O2 is efficiently introduced into fatty acids (e.g., myristic acid) . P450SP(alpha) produces the alpha-hydroxylated (alpha-OH) products at 100%, while P450BS(beta) produces alpha- and beta-hydroxylated (beta-OH) products at 33 and 67%, respectively . Using deuterium-substituted fatty acids ({2,2-d2}-myristic acid and d27-myristic acid) as a substrate, the peroxygenase reactions of the two bacterial P450s were investigated . In the P450SP(alpha) reaction, we observed an intermolecular noncompetitive kinetic isotope effect on Vmax (DV = 4.1) when {2,2-d2}-myristic acid was used, suggesting that an isotopically sensitive step involving the alpha-hydrogen of the fatty acid is present in the catalytic cycle . On the other hand, D(V/K) was masked, in sharp contrast to the features of usual monooxygenases P450 . The characteristic kinetic features can be interpreted in terms of the faster product formation than the substrate dissociation . A similar kinetic isotope effect was observed {DV = 4.9, D(V/K) approximately 1} for the P450BS(beta) reaction, when d27-myristic acid was used as a substrate, indicating that the reaction mechanism is the same for both peroxygenases . The resonance Raman spectral data of P450BS(beta) in the ferric and ferrous-CO forms in the presence and absence of myristic acid demonstrated that the catalytic pocket of the enzyme is polar, so that the location of the carboxylate of the substrate close to the sixth ligand of the heme could be allowed . On the basis of these results on the kinetic isotope effects and spectroscopy, we discuss the possible mechanisms of the alpha- and beta-hydroxylation of fatty acids catalyzed by peroxygenases P450SP(alpha) and P450BS(beta).

J Mol Biol, 2002 Feb 1, 315(5), 1075 - 85
Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis; Chami M et al.; YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters . Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer . Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy . Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni(2+)-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction . From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane . The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC . Each subunit was composed of three domains: a 5 nm height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter . The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD) . The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer . In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers .

Appl Environ Microbiol, 2002 Feb, 68(2), 456 - 63
Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure; Wemekamp-Kamphuis HH et al.; Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations . Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP) . Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4 . In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L . monocytogenes LO28 . After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated . Pressurization of L . monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively . Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C . These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.

Can J Microbiol, 2001 Dec, 47(12), 1116 - 25
Osmoregulation in Bacillus subtilis under potassium limitation: a new inducible K+-stimulated, VO4(3-)-inhibited ATPase; Sebestian J et al.; Bacillus subtilis exhibited an inducible K+-transporting ATPase activity with apparent Km and maximum velocity Vmax of 12.9 microM and 25.1 micromol x min(-1) x (g cell protein)(-1), respectively, when cultivated on a synthetic medium containing less than 400 microM K+ . Due to this enzyme, the growth rate of the bacterium in synthetic medium was not changed down to 115 microM K+, and the bacterium was able to grow down to 20 microM K+ . The limiting K+ concentration was higher in media with osmolarity increased by NaCl or sucrose . The ATPase was inhibited by micromolar concentrations of vanadate (Ki = 1.6 microM) . The ATPase activity was not stimulated by any other monovalent cation . The subunit of this ATPase, with an Mr of 52000, covalently bound the gamma phosphate group of ATP . This phosphorylated intermediate was unstable in neutral and basic pH as well as in the presence of potassium and was stable in acid pH . The enzyme did not show immunological cross-reactivity with antibody against Kdp ATPase of Escherichia coli.

J Biol Chem, 2002 Apr 19, 277(16), 13528 - 38 Epub 2002 Jan 30.
Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis; Aono S et al.; HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis . The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme . Oxygen binding and dissociation rate constants were determined to be k(on) = 32 microm(-1) s(-1) and k(off) = 23 s(-1), respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb) . The rate constant for autoxidation at 37 degrees C was 0.06 h(-1), which is also close to that of Mb . Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra . Oxygen-bound HemAT-Bs gave the nu(Fe-O(2)) band at a noticeably low frequency (560 cm(-1)), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule . Deoxy HemAT-Bs gave the nu(Fe-His) band at a higher frequency (225 cm(-1)) than those of ordinary His-coordinated deoxy heme proteins . CO-bound HemAT-Bs gave the nu(Fe-CO) and nu(C-O) bands at 494 and 1964 cm(-1), respectively, which fall on the same nu(C-O) versus nu(Fe-CO) correlation line as that of Mb . Based on these results, the structural and functional properties of HemAT-Bs are discussed.

Salud Publica Mex, 2001 Nov-Dec, 43(6), 570 - 3
{Benzalkonium chloride: unacceptable to sterilize or disinfect medical or dental instruments}; Acosta-Gio E et al.; OBJECTIVE: To compare the sporicidal activity of benzalkonium chloride (BKC) with that of glutaraldehyde . MATERIAL AND METHODS: A comparative study was conducted at the microbiology laboratory of Facultad de Odontologia, Universidad Nacional Autonoma de Mexico . Bacillus subtilis ATCC 9372 spores were exposed to these germicides (1 spore x mL) on a 0.22 mm filter . After completing the contact time the spores were washed and the filters were incubated on nutrient agar for 72 h at 37 degrees C . RESULTS: BKC did not eliminate B . subtilis spores at the concentration used, not even after increasing contact time to 15 h (900-fold the recommended time) . Two percent glutaraldehyde destroyed spores only after 10 h of contact . Urea and sodium chloride showed no sporicidal activity . CONCLUSIONS: The results confirm that BKC lacks sporicidal activity and confirm that this quaternary ammonium compound is not able to "sterilize" or "disinfect" medical and dental instruments.

FEMS Microbiol Lett, 2002 Jan 10, 206(2), 197 - 200
Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions; Jarmer H et al.; DNA microarrays were used to analyze the changes in gene expression in Bacillus subtilis strain 168 when nitrogen limiting (glutamate) and nitrogen excess (ammonium plus glutamate) growth conditions were compared . Among more than 100 genes that were significantly induced during nitrogen starvation we detected the comG, comF, comE, nin-nucA and comK transcription units together with recA . DNA was added to B . subtilis grown in minimal medium with glutamate as the sole nitrogen source and it was demonstrated that the cells were competent . Based on these observations we propose a simplification of previously designed one-step transformation procedures for B . subtilis strain 168.

J Mol Biol, 2002 Jan 25, 315(4), 551 - 60
Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme; Rox C et al.; We have detected by nucleotide analog interference mapping (NAIM) AMPalphaS and IMPalphaS modifications in Bacillus subtilis RNase P RNA that interfere with binding of the homologous protein subunit . Interference as well as some enhancement effects were clustered in two main areas, in P10.1a/L10.1 and P12 of the specificity domain (cluster 1, domain I) and in P2, P3, P15.1, J18/2 and J19/4 of the catalytic domain (cluster 2a, domain II) . Minor interferences in P1 and P19 and a strong and weak enhancement effect in P19 represent a third area located in domain II (cluster 2b) . Our results suggest that P3, P2-J18/2 and J19/4 are key elements for anchoring of the protein to the catalytic domain close to the scissile phosphodiester in enzyme-substrate complexes . Sites of interference or enhancement in clusters 1 and 2a are located at distances between 65 and 130 A from each other in the current 3D model of a full-length RNase P RNA-substrate complex . Taking into account that the RNase P protein monomer can bridge a maximum distance of about 40 A, simultaneous direct contacts to the two aforementioned potential RNA-binding areas would be incompatible with our current understanding of bacterial RNase P RNA architecture . Our findings suggest that the current 3D model has to be rearranged in order to reduce the distance between clusters 1 and 2a . Alternatively, based on the recent finding that B . subtilis RNase P forms a tetramer consisting of two protein and two RNA subunits, cluster 1 may reflect one protein contact site in domain I, and cluster 2a a separate one in domain II .

Mol Genet Genomics, 2002 Jan, 266(5), 899 - 906 Epub 2001 Nov 28.
Effect of the recU suppressors sms and subA on DNA repair and homologous recombination in Bacillus subtilis; Carrasco B et al.; In Bacillus subtilis, mutant alleles of the genes sms and subA partially suppress the recombination phenotype of recU cells . When present in an otherwise Rec(+) strain, Delta sms and Delta subA alleles render cells slightly sensitive to DNA-damaging agents, and impair chromosomal transformation (3- to 10-fold reduction), but do not affect plasmid transformation (less than 1.5-fold reduction) . The Delta sms and Delta subA alleles were introduced into rec-deficient strains representative of the epistatic groups alpha (recF strain), beta (addA addB), gamma (recH), epsilon (recB, Delta recU and recD strains) and zeta (Delta recS) . Both the Delta sms and Delta subA mutations were found to increase sensitivity to DNA-damaging agents in recF, Delta recS and addAB cells . In contrast, the Delta sms mutations decreased the sensitivity of recB, Delta recU, recD and recH cells, and the Delta subA mutation decreased the sensitivity of recB and Delta recU cells to DNA-damaging agents . Functions classified within the epistatic groups alpha, epsilon and zeta are required for intramolecular recombination, measured as plasmid transformation . The Delta sms and Delta subA mutations, which partially suppressed the recombinational repair phenotype of mutants for functions within epistatic group epsilon, enhanced plasmid transformation of recU (recB, recD) and recS cells by 10- to 20-fold . In the absence of the proteins Sms and SubA, the recombination machinery is apparently redirected towards (an) alternative pathway(s) . Furthermore, the shared ability of the Delta sms and Delta subA mutations to act as indirect suppressors of recB, recU and recD mutations supports the classification of the recBUD genes within epistatic group epsilon.

J Bacteriol, 2002 Feb, 184(4), 1219 - 24
Localization of the cortex lytic enzyme CwlJ in spores of Bacillus subtilis; Bagyan I et al.; The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis . CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins . However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant . These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.

J Bacteriol, 2002 Feb, 184(4), 1102 - 11
Characterization of the parB-like yyaA gene of Bacillus subtilis; Sievers J et al.; We have characterized the yyaA gene of Bacillus subtilis, located near the origin of chromosome replication (oriC) . Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins . Insertional inactivation of the yyaA gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation . Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA . Overexpression of yyaA caused a sporulation defect characterized by the formation of multiple septa within the cell . This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.

J Bacteriol, 2002 Feb, 184(4), 1010 - 8
ClpXP protease regulates the signal peptide cleavage of secretory preproteins in Bacillus subtilis with a mechanism distinct from that of the Ecs ABC transporter; Pummi T et al.; Identification and characterization of a suppressor mutation, sup-15, which partially restored secretion in the protein secretion-deficient Bacillus subtilis ecsA26 mutant, led us to discover a novel function of Clp protease . Inactivation of ClpP improved the processing of the precursor of AmyQ alpha-amylase exposed on the outer surface of the cytoplasmic membrane . A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex . In the absence of ClpXP, the transcription of the sipS, sipT, sipV, and lsp signal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation of ecs mutations . Specific overproduction of SipT enhanced the secretion . These findings extend the regulatory roles of ClpXP to protein secretion . ClpXP also influenced the processing of the lipoprotein PrsA . A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested . In contrast, Ecs did not affect transcription of the sip genes, pointing to a different mechanism of secretion regulation.

J Bacteriol, 2002 Feb, 184(4), 983 - 91
Identification of two myo-inositol transporter genes of Bacillus subtilis; Yoshida K et al.; Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the Bacillus subtilis genome project, we found that a mutant with inactivation of iolT (ydjK) exhibited a growth defect on myo-inositol as the sole carbon source . The putative product of iolT exhibits significant similarity with many bacterial sugar transporters in the databases . In B . subtilis, the iolABCDEFGHIJ and iolRS operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol . Among the iol genes, iolF was predicted to encode an inositol transporter . Inactivation of iolF alone did not cause such an obvious growth defect on inositol as the iolT inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single iolT inactivation . Determination of inositol uptake by the mutants revealed that iolT inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable . These results, as well as the K(m) and V(max) values for the IolT and IolF inositol transporters, indicated that iolT and iolF encode major and minor inositol transporters, respectively . Northern and primer extension analyses of iolT transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by final sigma(A) RNA polymerase and negatively regulated by IolR as well . The interaction between IolR and the iolT promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the iol divergon.

J Bacteriol, 2002 Feb, 184(4), 889 - 94
Tetracycline induces stabilization of mRNA in Bacillus subtilis; Wei Y et al.; The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance . Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction . Here we show that the other component of tet(L) induction is at the level of mRNA stabilization . Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in tet(L) mRNA stability . Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region . The results of these experiments, as well as experiments with a B . subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability . The key role of the 5' end in determining mRNA stability was confirmed in these experiments . Surprisingly, the stability of several other B . subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.

J Biol Chem, 2002 Apr 5, 277(14), 11838 - 44 Epub 2002 Jan 22.
Creating hetero-11-mers composed of wild-type and mutant subunits to study RNA binding to TRAP; Li PT et al.; TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacillus subtilis by binding to RNA targets that contain multiple GAG and UAG repeats . TRAP is composed of 11 identical subunits arranged symmetrically in a ring . The secondary structure of the protein consists entirely of antiparallel beta-sheets, beta-turns, and loops . We show here that the TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride . Removing the denaturant allows the protein to spontaneously renature into fully functional 11-mers . Based on this finding, we developed a subunit mixing method to hybridize wild-type and mutant subunits into heteromeric 11-mers by denaturation followed by subunit mixing renaturation . This method allows the study of subunit cooperativity in protein-ligand interaction such as RNA binding . Our data further support and extend the previously proposed two-step model for RNA binding to TRAP by showing that the initiation of binding requires at least one fully active subunit in the protein combined with one fully functional repeat in the RNA . The initiation complex tethers the RNA on the protein, thus allowing cooperative interaction with the remainder of the repeats.

Tuberculosis (Edinb), 2001, 81(5-6), 327 - 34
CD4+ lymphocyte responses to pulmonary infection with Mycobacterium tuberculosis in naïve and vaccinated BALB/c mice; Mason CM et al.; The Biostack experiments I and II were flown on board the Apollo 16 and 17 command modules in order to obtain information on the biological damage produced by the bombardment of heavy high-energy (HZE) particles of cosmic radiation during spaceflight . Such data are required for estimating radiation hazards in manned spaceflight . Seven biological systems in resting state (Bacillus subtilis spores, Colpoda cucullus cysts, Arabidopsis thaliana seeds, and eggs of Artemia salina, Tribolium castaneum and of Carausius morosus) were accommodated in the two Biostacks . By using a special sandwich construction of visual track detectors and layers of biological objects, identification of each hit biological object was achieved and the possible biological damage correlated with the physical features of the responsible HZE-particle . In the different systems the degree of damage depended on whether the hit cell was replaceable or not . A high sensitivity to HZE-particle bombardment was observed on Artemia salina eggs; 90% of the embryos, which were induced to develop from hit eggs, died at different developmental stages . Malformations of the abdomen or the extremities of the nauplius were frequently induced . In contrast, the growth of hit Vicia faba radiculae and the germination of hit Arabidopsis thaliana seeds and hit Bacillus subtilis spores were not influenced remarkably . But there was an increase in multicaulous plants and a reduction in the outgrowth of the bacteria} spores . In addition, information was obtained on the fluence of the HZE-particles, on their spectrum of charge and energy loss, and on the absorption by the Apollo spacecraft and the Biostack material itself . This will help to improve knowledge concerning radiation conditions inside of spacecrafts, necessary to secure a The Biostack experiments I and II were flown on board the Apollo 16 and 17 command modules in order to obtain information on the biological damage produced by the bombardment of heavy high-energy (HZE) particles of cosmic radiation during spaceflight . Such data are required for estimating radiation hazards in manned spaceflight . Seven biological systems in resting state (Bacillus subtilis spores, Colpoda cucullus cysts, Arabidopsis thaliana seeds, and eggs of Artemia salina, Tribolium castaneum and of Carausius morosus) were accommodated in the two Biostacks . By using a special sandwich construction of visual track detectors and layers of biological objects, identification of each hit biological object was achieved and the possible biological damage correlated with the physical features of the responsible HZE-particle . In the different systems the degree of damage depended on whether the hit cell was replaceable or not . A high sensitivity to HZE-particle bombardment was observed on Artemia salina eggs; 90% of the embryos, which were induced to develop from hit eggs, died at different developmental stages . Malformations of the abdomen or the extremities of the nauplius were frequently induced . In contrast, the growth of hit Vicia faba radiculae and the germination of hit Arabidopsis thaliana seeds and hit Bacillus subtilis spores were not influenced remarkably . But there was an increase in multicaulous plants and a reduction in the outgrowth of the bacteria} spores . In addition, information was obtained on the fluence of the HZE-particles, on their spectrum of charge and energy loss, and on the absorption by the Apollo spacecraft and the Biostack material itself . This will help to improve knowledge concerning radiation conditions inside of spacecrafts, necessary to secure a The Biostack experiments I and II were flown on board the Apollo 16 and 17 command modules in order to obtain information on the biological damage produced by the bombardment of heavy high-energy (HZE) particles of cosmic radiation during spaceflight . Such data are required for estimating radiation hazards in manned spaceflight . Seven biological systems in resting state (Bacillus subtilis spores, Colpoda cucullus cysts, Arabidopsis thaliana seeds, and eggs of Artemia salina, Tribolium castaneum and of Carausius morosus) were accommodated in the two Biostacks . By using a special sandwich construction of visual track detectors and layers of biological objects, identification of each hit biological object was achieved and the possible biological damage correlated with the physical features of the responsible HZE-particle . In the different systems the degree of damage depended on whether the hit cell was replaceable or not . A high sensitivity to HZE-particle bombardment was observed on Artemia salina eggs; 90% of the embryos, which were induced to develop from hit eggs, died at different developmental stages . Malformations of the abdomen or the extremities of the nauplius were frequently induced . In contrast, the growth of hit Vicia faba radiculae and the germination of hit Arabidopsis thaliana seeds and hit Bacillus subtilis spores were not influenced remarkably . But there was an increase in multicaulous plants and a reduction in the outgrowth of the bacteria} spores . In addition, information was obtained on the fluence of the HZE-particles, on their spectrum of charge and energy loss, and on the absorption by the Apollo spacecraft and the Biostack material itself . This will help to improve knowledge concerning radiation conditions inside of spacecrafts, necessary to secure a maximum possible protection to the astronauts .

Bull Group Int Rech Sci Stomatol Odontol, 1999 Oct-Dec, 41(4), 124 - 30
Morpho-structural variations of bacterial spores after treatment in steam vacuum assisted autoclave; Fonzi M et al.; This study intended to verify, through microbiological techniques and TEM investigations, the killing of bacterial spores after treatment in steam autoclave, and to propose strictly morphological considerations about the target of this sterilisation process . Autoclave is the most common device for sterilising instruments in order to prevent cross infections in dental offices . The autoclave efficiency has been improved in the last years and part of this improvement is related to both a better and more correct use of the autoclave system and to the technological innovations introduced in the last generation of devices . However, associations as ADA or CDC suggest to regularly verify the process of 'autoclaving' through biological indicators (BI) . The most commonly used BI are made of spores strips or suspensions of Bacillus Subtilis (pb 168) and Bacillus Stearothermophilus (ATCC 10149) . They visually prove, changing colours on enzymatic base, the death of micro-organism and if the physical parameters, necessary for sterilisation, have been achieved . These two strains of endospore-forming bacteria were processed and prepared following two different techniques: Karnovsky fixed and epon embedded--phosphotungstic acid fixed for direct observation . The kind and the extent of analysed modifications are extremely various: from deep lacerations, which changed the spore structure, to little clefts which let the cytoplasm go out.

J Biol Chem, 2002 Mar 29, 277(13), 11362 - 7 Epub 2002 Jan 16.
A new family of phosphotransferases with a P-loop motif; Galinier A et al.; In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the histidine-containing protein kinase/phosphatase (HprK/P) . Based either on its primary sequence or on its recently solved three-dimensional structure, no straightforward homology with other known proteins was found . However, we showed here that HprK/P exhibits a restricted homology with an unrelated phosphotransferase, the phosphoenolpyruvate carboxykinase . This includes notably two consecutive Asp residues from the phosphoenolpyruvate carboxykinase active site, whose equivalent residues were mutated in Bacillus subtilis HprK/P . Characterization of the corresponding mutants emphasizes the crucial role of these Asp residues in the HprK/P functioning . Furthermore, superimposition of HprK/P and phosphoenolpyruvate carboxykinase active sites supports the view that both enzymes bear significant resemblance in their overall mechanism of functioning showing that these two enzymes constitute a new family of phosphotransferases.

Antimicrob Agents Chemother, 2002 Feb, 46(2), 315 - 20
Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168; Tamehiro N et al.; We have found a novel phospholipid antibiotic (named bacilysocin) which accumulates within (or associates with) the cells of Bacillus subtilis 168 and determined the structure by nuclear magnetic resonance and mass spectrometry analyses . The structure of bacilysocin elucidated was 1-(12-methyltetradecanoyl)-3-phosphoglyceroglycerol . Bacilysocin demonstrated antimicrobial activity, especially against certain fungi . Production of bacilysocin commenced immediately after growth ceased and before the formation of heat-resistant spores . The production of bacilysocin was completely blocked when the ytpA gene, which encodes a protein homologous to lysophospholipase, was disrupted, but blockage of the ytpA gene did not significantly affect growth . Sporulation was also impaired, with a 10-fold reduction in heat-resistant spore titers being detected . Since the ytpA disruptant actually lacked phospholipase activity, we propose that the YtpA protein functions as an enzyme for the biosynthesis of bacilysocin.

Bioconjug Chem, 2002 Jan-Feb, 13(1), 23 - 8
Novel functional biodegradable polymer: synthesis and anticoagulant activity of poly(gamma-glutamic acid)sulfonate (gamma-PGA-sulfonate); Matsusaki M et al.; gamma-Poly(glutamic acid) (gamma-PGA), which is produced by Bacillus subtilis, was sulfonated using 2-aminoethane-1-sulfonic acid (taurine) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC) to give sulfonated gamma-PGA (gamma-PGA-sulfonate) . From (1)H NMR spectroscopy and IR spectroscopy, it was confirmed that taurine was introduced to the side chain of gamma-PGA via an amide linkage . By altering the synthetic conditions, it was possible to control the content of sulfonate in gamma-PGA-sulfonate . Anticoagulant activity was investigated in order to evaluate the biological activity of gamma-PGA-sulfonate by the Lee-White test . The clotting time was prolonged when the concentration of gamma-PGA-sulfonate on the degree of sulfonation was increased . It becomes clear that gamma-PGA-sulfonate is potentially useful for various medical applications, such as drug delivery, tissue engineering, and medical materials.

Protein Sci, 2002 Feb, 11(2), 271 - 9
Binding of cations in Bacillus subtilis phosphoribosyldiphosphate synthetase and their role in catalysis; Eriksen TA et al.; The binding sites for the two cations essential for the catalytic function of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthases have been identified from the structure of the Bacillus subtilis phosphoribosyldiphosphate synthetase (PRPPsase) with bound Cd(2+) . The structure determined from X-ray diffraction data to 2.8-A resolution reveals the same hexameric arrangement of the subunits that was observed in the complexes of the enzyme with the activator sulfate and the allosteric inhibitor ADP . Two cation binding sites were localized in each of the two domains of the subunits that compose the hexamer; each domain of the subunit has an associated cation . In addition to the bound Cd(2+), the Cd(2+)-PRPPsase structure contains a sulfate ion in the regulatory site, a sulfate ion at the ribose-5-phosphate binding site, and an AMP moiety at the ATP binding site . Comparison of the Cd(2+)-PRPPsase to the structures of the PRPPsase complexed with sulfate and mADP reveals the structural rearrangement induced by the binding of the free cation, which is essential for the initiation of the reaction . The comparison to the cPRPP complex of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli, a type I phosphoribosyltransferase, provided information about the binding of PRPP . This strongly indicates that the binding of both substrates must lead to a stabilized conformation of the loop region, which remains unresolved in the known PRPPsase complex structures.

J Bacteriol, 2002 Feb, 184(3), 718 - 27
High-salinity-induced iron limitation in Bacillus subtilis; Hoffmann T et al.; Proteome analysis of Bacillus subtilis cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively . Most of these protein spots were identified by mass spectrometry . Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin . These proteins are encoded by the dhbACEBF operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation . We found that iron limitation and high salinity derepressed dhb expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant . DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron . Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed B . subtilis cultures . Taken together, these findings strongly suggest that B . subtilis cells grown at high salinity experience iron limitation . In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress . The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of B . subtilis in natural settings, in which bioavailable iron is usually scarce.

J Biol Chem, 2002 Mar 22, 277(12), 10608 - 13 Epub 2002 Jan 10.
The anti-trp RNA-binding attenuation protein (Anti-TRAP), AT, recognizes the tryptophan-activated RNA binding domain of the TRAP regulatory protein; Valbuzzi A et al.; In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of genes involved in tryptophan metabolism in response to the accumulation of l-tryptophan . Tryptophan-activated TRAP negatively regulates expression by binding to specific mRNA sequences and either promoting transcription termination or blocking translation initiation . Conversely, the accumulation of uncharged tRNA(Trp) induces synthesis of an anti-TRAP protein (AT), which forms a complex with TRAP and inhibits its activity . In this report, we investigate the structural features of TRAP required for AT recognition . A collection of TRAP mutant proteins was examined that were known to be partially or completely defective in tryptophan binding and/or RNA binding . Analyses of AT interactions with these proteins were performed using in vitro transcription termination assays and cross-linking experiments . We observed that TRAP mutant proteins that had lost the ability to bind RNA were no longer recognized by AT . Our findings suggest that AT acts by competing with messenger RNA for the RNA binding domain of TRAP . B . subtilis AT was also shown to interact with TRAP proteins from Bacillus halodurans and Bacillus stearothermophilus, implying that the structural elements required for AT recognition are conserved in the TRAP proteins of these species . Analyses of AT interaction with B . stearothermophilus TRAP at 60 degrees C demonstrated that AT is active at this elevated temperature.

Biochem Biophys Res Commun, 2002 Jan 18, 290(2), 806 - 12
Bacillus subtilis inorganic pyrophosphatase: the C-terminal signature sequence is essential for enzyme activity and conformational integrity; Konopka MA et al.; Bacillus subtilis inorganic pyrophosphatase is the first member of a newly identified Family II of PPases . To examine the role of a signature sequence found near the C-terminus, two truncated variants and a series of site-specific mutants were produced . A truncation of 17 residues (17AATR) but also single alanine substitutions, R295A and K296A, produced inactive enzyme . Removal of 5 nonconserved terminal residues (5AATR) markedly affected enzyme stability . Replacing S294 with A, T, C, or V decreased activity, the latter two mutations showing the greatest effect . Substitutions V299I and V300I had no or minor effects, whereas V300W and V299G/V300W significantly reduced activity . The sizes of truncated proteins and the full-length PPase were indistinguishable by gel-filtration . We conclude that the C-terminus has no role in multimerization, while both its conserved and nonconserved regions are essential for full enzyme activity . The signature sequence is required for both the conformation and composition of the active site.

Genetika, 2001 Dec, 37(12), 1598 - 603
{High frequency conjugative mobilization in natural strains of Bacillus subtilis, bearing a large plasmid}; Lotareva OV et al.; Conjugative properties of the strain Bacillus subtilis that carrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described . The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis 168 strain with a frequency of approximately 10(-2) . The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium . The latter fact makes this system suitable as a model for studying conjugal mobilization in bacilli . A large plasmid cannot be transferred to recipients . An optimal temperature for conjugation of donor and recipient cells was 37 degrees C, but conjugation also proceeded at lower temperatures, up to 21 degrees C.

Microbiology, 2002 Jan, 148(Pt 1), 307 - 14
Killing of spores of Bacillus subtilis by peroxynitrite appears to be caused by membrane damage; Genest PC et al.; During an infection of a higher eukaryote, dormant spores of a Bacillus species have been previously shown to be present in cells that can generate the toxic agent peroxynitrite (PON) . Dormant spores of Bacillus subtilis were much more resistant to killing by PON than were growing cells, and spore-coat alteration or removal greatly decreased PON resistance . Spores were not killed by PON through DNA damage and lost no dipicolinic acid (DPA) during PON treatment . However, PON-killed spores lost DPA during subsequent heat treatments that caused much less DPA release from untreated spores . Although dead, the PON-killed spores germinated and initiated metabolism but never went through outgrowth; the great majority of germinated PON-killed spores also took up propidium iodide, indicating that they had suffered significant membrane damage and were dead . Together these data suggest that spore killing by PON is through some type of damage to the spore's inner membrane.

Microbiology, 2002 Jan, 148(Pt 1), 297 - 306
Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954; Anlezark GM et al.; A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens . The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined . The gene was found to reside between homologues of the B . subtilis alsD and yswB genes; however, the ywrO and yswB genes of B . amyloliquefaciens were not separated by a fourth gene, ywsA . The B . amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD) . In common with these enzymes menadione was an electron acceptor (K(m) 3 microM) and activity with this substrate was inhibited by the presence of dicoumarol (K(i) 1.0 microM) . In contrast, YwrO showed a marked preference for NADPH as a cofactor (K(m) 40 microM) and therefore could not be classified as a DTD (EC 1.6.99.2) . The flavin FMN was an acceptor with high affinity . B . amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21.5 kDa by calculation and SDS-PAGE . The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B . amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862 . In both of these properties B . amyloliquefaciens YwrO more closely resembles DTD than NTR . Its K(m) for CB 1954 was lower than that of NTR (617 microM compared to 862 microM) . Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B . amyloliquefaciens YwrO . The work has led to the identification of a previously unknown nitroreductase, B . amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).

Mol Cell, 2001 Dec, 8(6), 1197 - 206
Visualization of mismatch repair in bacterial cells; Smith BT et al.; We determined the localizations of mismatch repair proteins in living Bacillus subtilis cells . MutS-GFP colocalized with the chromosome in all cells and formed foci in a subset of cells . MutL-GFP formed foci in a subset of cells, and its localization was MutS dependent . The introduction of mismatches by growth in 2-aminopurine caused a replication-dependent increase in the number of cells with MutS and MutL foci . Approximately half of the MutS foci colocalized with DNA polymerase foci . We conclude that MutS is associated with the entire chromosome, poised to detect mismatches . After detection, it appears that mismatch repair foci assemble at mismatches as they emerge from the DNA polymerase and are then carried away from the replisome by continuing replication.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 764 - 9
Isolation of Bacillus subtilis (chungkookjang), a poly-gamma-glutamate producer with high genetic competence; Ashiuchi M et al.; A bacterium with high poly-gamma-glutamate (PGA) productivity was isolated from the traditional Korean seasoning, Chung-Kook-Jang . This bacterium could be classified as a Bacillus subtilis, but sporulation in culture was infrequent in the absence of Mn2+ . It was judged to be a variety of B . subtilis and designated B . subtilis (chungkookjang) . L-Glutamate significantly induced PGA production, and highly elongated PGAs were synthesized . The volumetric yield reached 13.5 mg ml(-1) in the presence of 2% L-glutamate . The D-glutamate content was over 50% in every PGA produced under the conditions used . During PGA production, glutamate racemase activity was found in the cells, suggesting that the enzyme is involved in the D-glutamate supply . Molecular sizes of PGAs were changed by the salt concentration in the medium; PGAs with comparatively low molecular masses were produced in culture media containing high concentrations of NaCl . B . subtilis (chungkookjang) harbors no plasmid and is the first B . subtilis strain reported with both naturally high PGA productivity and high genetic competence.

Science, 2002 Jan 4, 295(5552), 137 - 9
Role of cell-specific SpoIIIE assembly in polarity of DNA transfer; Sharp MD et al.; SpoIIIE mediates postseptational chromosome partitioning in Bacillus subtilis, but the mechanism controlling the direction of DNA transfer remains obscure . Here, we demonstrated that SpoIIIE acts as a DNA exporter: When SpoIIIE was synthesized in the larger of the two cells necessary for sporulation, the mother cell, DNA was translocated into the smaller forespore; however, when it was synthesized in the forespore, DNA was translocated into the mother cell . Furthermore, the DNA-tracking domain of SpoIIIE inhibited SpoIIIE complex assembly in the forespore . Thus, during sporulation, chromosome partitioning is controlled by the preferential assembly of SpoIIIE in one daughter cell.

Microbiol Res, 2001, 156(4), 377 - 82
Dominant fungi in the rhizosphere of established tea bushes and their interaction with the dominant bacteria under in situ conditions; Pandey A et al.; Species of Penicillium and Trichoderma were found to dominate the rhizosphere of established tea bushes in a detailed study conducted from various tea growing locations in India . Penicillium erythromellis, P . janthinellum, P . raistrickii, Trichoderma pseudokoningii and T . koningii were found to be closely associated with tea roots . While seasonal fluctuation was observed in the case of Penicillium spp., the population of Trichoderma spp . showed less variation during the year . Both species were sensitive to low temperatures . In general, fungi associated with the tea rhizosphere were found to prefer a mesophillic temperature range (15 degrees C to 35 degrees C) . The dominant species of Penicillium and Trichoderma also exhibited tolerance to lower temperatures, i.e., 5 to 10 degrees C on agar plates . Most fungi were able to grow in a wide range of pH (4 to 12) . Lowering of soil pH in the rhizosphere of tea bushes was positively correlated with the age of the bush and may have affected the development of a specific microbial community in the rhizosphere . The populations of Penicillium and Trichoderma species were inversely correlated with the populations of two most dominant rhizosphere bacteria, Bacillus subtilis and B . mycoides . Both Bacillus species have been shown to have antagonistic activity against these two fungi under in vitro conditions . The present study demonstrates the existence of a similar antagonism under in situ conditions in the rhizosphere of established tea bushes.

Orig Life Evol Biosph, 2001 Dec, 31(6), 527 - 47
Protection of bacterial spores in space, a contribution to the discussion on Panspermia; Horneck G et al.; Spores of Bacillus subtilis were exposed to space in the BIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite . The spores were exposed either in dry layers without any protecting agent, or mixed with clay, red sandstone, Martian analogue soil or meteorite powder, in dry layers as well as in so-called 'artificial meteorites', i.e . cubes filled with clay and spores in naturally occurring concentrations . After about 2 weeks in space, their survival was tested from the number of colony formers . Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases < or = 10(-6)) . The same low survival was obtained behind a thin layer of clay acting as an optical filter . The survival rate was increased by 5 orders of magnitude and more, if the spores in the dry layer were directly mixed with powder of clay, rock or meteorites, and up to 100% survival was reached in soil mixtures with spores comparable to the natural soil to spore ratio . These data confirm the deleterious effects of extraterrestrial solar UV radiation . Thin layers of clay, rock or meteorite are only successful in UV-shielding, if they are in direct contact with the spores . The data suggest that in a scenario of interplanetary transfer of life, small rock ejecta of a few cm in diameter could be sufficiently large to protect bacterial spores against the intense insolation; however, micron-sized grains, as originally requested by Panspermia, may not provide sufficient protection for spores to survive . The data are also pertinent to search for life on Mars and planetary protection considerations for future missions to Mars.

Res Microbiol, 2001 Dec, 152(10), 901 - 5
The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity; Geslin C et al.; Superoxide dismutases (SODs) are vital components that defend against oxidative stress through decomposition of superoxide radical . Escherichia coli contains two highly homologous SODs, a manganese- and an iron-containing enzyme (Mn-SOD and Fe-SOD, respectively) . In contrast, a single Mn-SOD is present in Bacillus subtilis . In E . coli, the absence of SODs was found to be associated with an increased sensitivity to cadmium, nickel and cobalt ions . Mutants lacking either sodA or sodB exhibited metal resistance to levels comparable to that of the wild-type strain . Although sod-deficient mutant cells were more resistant to zinc than their wild-type counterpart, no differences between the strains were observed in the presence of copper . In B . subtilis, the sodA mutation had no effect on cadmium and copper resistance . These results suggest that intracellular generation of superoxide by cadmium, nickel and cobalt is toxic in E . coli . They support the participation of sod genes in its protection against metal stress.

Prep Biochem Biotechnol, 2001 Nov, 31(4), 389 - 400
Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis; Srivastava P et al.; A genomic library of Bacillus lyticus was constructed in lambda GEM 11 vector and screened for the xylanase gene using Congo red plate assay . A 16-kb fragment containing the xylanase gene was obtained which was further subcloned using Mbo I partial digestion in an E . coli pUC 19 vector . A 1.3-kb sub-fragment was obtained which coded for a xylanase gene of Mr 23,650 Da . This fragment was sequenced and the homology was checked with known xylanases . The maximum homology was 97%, which was obtained with an endo xylanase gene from Bacillus species at the DNA level, while the translated sequence showed only one amino acid change from alanine to serine at position number 102 . Expression was checked in E . coli, using the native promoter, and an extracellular activity of 5.25 U/mL was obtained . Cloning of the gene was done in Bacillus subtilis using a shuttle vector pHB 201, which resulted in increasing the basal level xylanase activity from 14.02 to 22.01 U/mL.

J Mol Microbiol Biotechnol, 2002 Jan, 4(1), 37 - 67
Transport capabilities encoded within the Bacillus subtilis genome; Saier MH Jr et al.; We here describe all recognized established and putative transport proteins encoded within the genome of Bacillus subtilis . These fall into four classes of established transporter types: (1) channel proteins, (2) secondary active transporters, (3) primary active transporters, and (4) group translocators of the sugar-transporting phosphotransferase system (PTS) . Additionally, some transporters are recognized that utilize an unknown mode of action or energy coupling mechanism . The secondary carriers (which represent the majority of Bacillus transporters) are subdivided according to substrate specificity and family association . Characteristics of the families as well as the individual transport systems are presented when sufficient information is available . The recognized transporters fall into 58 families including 4 channel types, 42 secondary carrier types, 3 primary carrier types, 4 PTS-types and 5 unknown types.

Salud Publica Mex, 2001 Sep-Oct, 43(5), 455 - 8
{Use of and verification with biological indicators in sterilizers belonging to dentistry surgeons from San Luis Potosi, Mexico}; Patino-Marin N et al.; OBJECTIVE: To assess the utilization of sterilizing equipment used by dentists, and verification of sterilization using biological indicators . MATERIAL AND METHODS: A cross-sectional study was conducted in 1999-2000, among 130 (65%) dentists having sterilizing equipment, at Facultad de Estomatologia, Universidad Autonoma de San Luis Potosi and Colegio Dental Potosino . Biological indicators for sterilization containing Bacillus subtilis and Bacillus stearothermophilus were used . RESULTS: Thirty autoclaves and 100 dry-heat sterilizers were evaluated: 23 (17.7%) of them showed bacterial growth . Twenty-one (16.1%) dentists already were using biological indicators to verify their sterilizing equipment . Both sterilization methods were found to allow bacterial growth with similar frequencies (p = > 0.66) . CONCLUSIONS: Few dentists verify the quality of sterilization process through biological indicators; bacterial growth and failure of sterilization were evidenced.

Res Microbiol, 2001 Nov, 152(9), 823 - 33
Using inactivated microbial biomass as fertilizer: the fate of antibiotic resistance genes in the environment; Andersen JT et al.; The waste product produced by Novo Nordisk A/S from microbial fermentations is used as agricultural fertilizer in Denmark (NovoGro) after being treated by heat and chemicals to destroy the microorganisms . The fertilizer contains DNA fragments from the genetically modified microorganisms used in industrial production . This DNA contains genes coding for the desired industrial products as well as genes used as genetic selection markers during production strain development . The antibiotic resistance markers used as genetic selection markers are chloramphenicol (Cm), kanamycin (Km) and ampicillin (Ap) . The aim of the present study was to examine whether DNA and intact genes were present in NovoGro and whether horizontal transfer of DNA isolated from inactivated production strains occurred either in the laboratory or in the fields treated with NovoGro . DNA isolated from NovoGro was analysed by PCR and intact genes coding for a protease and chloramphenicol resistance were amplified . This isolated DNA was used for in vitro experiments including electroporation and transformation but no transfer of DNA to Escherichia coli or Bacillus subtilis was observed . The antibiotic resistance profile of the indigenous bacterial population in the fields treated with NovoGro compared with fields treated with inorganic fertilizers showed no differences . In addition, DNA isolated directly from the fields treated with NovoGro for up to 7 years was analysed by PCR and no specific production gene constructs could be detected.

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