Bacillus subtilis

J Bacteriol, 2003 Aug, 185(16), 4717 - 26
Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH; Wei Y et al.; The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell . Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination . No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified . However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape . It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape . The B . subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a . We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase . A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable . When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression . Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis . We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.

FEMS Microbiol Lett, 2003 Jul 29, 224(2), 169 - 73
A mutant Bacillus subtilis gamma-glutamyltranspeptidase specialized in hydrolysis activity; Minami H et al.; gamma-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of gamma-glutamyl compounds and the transfer of their gamma-glutamyl moieties to amino acids and peptides . The transpeptidation activity of Bacillus subtilis GGT is about 10-fold higher than its hydrolysis activity . In B . subtilis GGT, substitution of Asp-445 with Ala abolished its transpeptidation activity . The specific activity for hydrolysis of D445A GGT was 40.2% of that of the wild-type GGT . The K(m) value for L-glutamine was 15.3 mM . D445A GGT was salt tolerant like the wild-type GGT . These results indicate that D445A GGT will be highly useful as a 'glutaminase' in food industry.

Biotechnol Prog, 2003 Jul-Aug, 19(4), 1355 - 64
Potential of on-line CIMS for bioprocess monitoring; Custer TG et al.; Chemical-ionization mass spectrometry (CIMS) using flow reactors is an emerging method for on-line monitoring of trace concentrations of organic compounds in the gas phase . In this study, a flow-reactor CIMS instrument, employing the H(3)O(+) cation as the ionizing reagent, was used to simultaneously monitor several volatile metabolic products as they are released into the headspace during bacterial growth in a bioreactor . Production of acetaldehyde, ethanol, acetone, butanol, acetoin, diacetyl, and isoprene by Bacillus subtilis is reported . Ion signal intensities were related to solution-phase concentrations using empirical calibrations and, in the case of isoprene, were compared with simultaneous gas chromatography measurements . Identification of volatile and semivolatile metabolites is discussed . Flow-reactor CIMS techniques should be useful for bioprocess monitoring applications because of their ability to sensitively and simultaneously monitor many volatile metabolites on-line.

Mol Microbiol, 2003 Aug, 49(4), 1135 - 44
The BceRS two-component regulatory system induces expression of the bacitracin transporter, BceAB, in Bacillus subtilis; Ohki R et al.; BceA and bceB encode a nucleotide-binding domain (NBD) and membrane-spanning domain (MSD) subunit, respectively, of an ATP-binding cassette (ABC) transporter in Bacillus subtilis . Disruption of these genes resulted in hypersensitivity to bacitracin, a peptide antibiotic that is non-ribosomally synthesized in some strains of Bacillus . Northern hybridization analyses showed that expression of the bceAB operon is induced by bacitracin present in the growth medium . The bceRS genes encoding a two-component regulatory system are located immediately upstream of bceAB . Deletion analyses of the bceAB promoter together with DNase I footprinting experiments revealed that a sensor kinase, BceS, responds to extracellular bacitracin either directly or indirectly and transmits a signal to a cognate response regulator, BceR . The regulator binds directly to the upstream region of the bceAB promoter and upregulates the expression of bceAB genes . The bcrC gene product is additionally involved in bacitracin resistance . The expression of bcrC is dependent on the ECF sigma factors, sigmaM and sigmaX, but not on the BceRS two-component system . In view of these results, possible roles of BceA, BceB and BcrC in bacitracin resistance of B . subtilis 168 are discussed.

Mol Microbiol, 2003 Aug, 49(4), 1067 - 79
Phosphatidylethanolamine and phosphatidylglycerol are segregated into different domains in bacterial membrane . A study with pyrene-labelled phospholipids; Vanounou S et al.; To detect and characterize membrane domains that have been proposed to exist in bacteria, two kinds of pyrene-labelled phospholipids, 2-pyrene-decanoyl-phosphatidylethanolamine (PY-PE) and 2-pyrene-decanoyl-phosphatidylglycerol (PY-PG) were inserted into Escherichia coli or Bacillus subtilis membrane . The excimerization rate coefficient, calculated from the excimer-to-monomer ratio dependencies on the probe concentration, was two times higher for PY-PE than for PY-PG at 37 degrees C . This was ascribed to different local concentrations rather than to differences in mobility . The extent of mixing between the two fluorescent phospholipids, estimated by formation of their heteroexcimer, was found very low both in E . coli and B . subtilis, in contrast to model membranes . In addition, these two pyrene derivatives exhibited different temperature phase transitions and different detergent extractability, indicating that the surroundings of these phospholipids in bacterial membrane differ in organization and order . Inhibition of protein synthesis, leading to condensation of nucleoid and presumably to dissipation of membrane domains, indeed resulted in increased formation of heteroexcimers, broadening of phase transitions and equal detergent extractability of both probes . It is proposed that in bacterial membranes these phospholipids are segregated into distinct domains that differ in composition, proteo-lipid interaction and degree of order; the proteo-lipid domain being enriched by PE.

Mol Microbiol, 2003 Aug, 49(4), 895 - 903
A strand-specific model for chromosome segregation in bacteria; Rocha EP et al.; Chromosome separation and segregation must be executed within a bacterial cell in which the membrane and cytoplasm are highly structured . Here, we develop a strand-specific model based on each of the future daughter chromosomes being associated with a different set of structures or hyperstructures in an asymmetric cell . The essence of the segregation mechanism is that the genes on the same strand in the parental cell that are expressed together in a hyperstructure continue to be expressed together and segregate together in the daughter cell . The model therefore requires an asymmetric distribution of classes of genes and of binding sites and other structures on the strands of the parental chromosome . We show that the model is consistent with the asymmetric distribution of highly expressed genes and of stress response genes in Escherichia coli and Bacillus subtilis . The model offers a framework for interpreting data from genomics.

Biotechnol Lett, 2003 Jun, 25(12), 969 - 74
Regioselective synthesis of kojic acid esters by Bacillus subtilis protease; Raku T et al.; The lipophilicity of kojic acid {5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one} was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d . 1H-NMR and 13C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13-27% yield) . The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9774 - 8 Epub 2003 Jul 29.
Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli; Watanabe K et al.; Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products . Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase . We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli . The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules . AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei, which produces rifamycin, and Actinosynnema pretiosum, which produces ansamitocin . Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E . coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate . A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis . Fermentation of this strain of E . coli yielded P8/1-OG, an N-acetyl P8/1-OG analog, and AHBA . In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.

J Mol Biol, 2003 Aug 8, 331(2), 473 - 84
A core mutation affecting the folding properties of a soluble domain of the ATPase protein CopA from Bacillus subtilis; Banci L et al.; The two N-terminal domains of the P-type copper ATPase, CopAa and CopAb, from Bacillus subtilis differ in their folding capabilities in vitro . Whereas CopAb has the typical betaalphabetabetaalphabeta structure and is a rigid protein, CopAa is found to be largely unfolded . A sequence analysis of the two and of orthologue homologous proteins indicates that Ser46 in CopAa may destabilise the hydrophobic core, as also confirmed through a bioinformatic energy study . CopAb has a Val in the corresponding position . The S46V and S46A mutants are found to be folded, although the latter displays multiple conformations . S46VCopAa, in both apo and copper(I) loaded forms, has very similar structural and dynamic properties with respect to CopAb, besides a different length of strand beta2 and beta4 . It is intriguing that the oxygen of Thr16 is found close, though at longer than bonding distance, to copper in both domains, as it also occurs in a human orthologue domain . This study contributes to understanding the behaviour of proteins that do not properly fold in vitro . A possible biological significance of the peculiar folding behaviour of this domain is discussed.

Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 91 - 3
{Characterization of highly purified metalloprotease produced by Bacillus subtilis}; Khaziev AF et al.; Proteolytic enzyme produced by Bacillus subtilis is characterized as typical metalloprotease with a molecular weight of 27.9 kD; the enzyme shows its highest activity at pH 7.0-9.0, possesses substrate specificity with respect to different proteins, its temperature optimum is 52 degrees C and its specific activity exceeds that of all known commercial analogues . At 37 degrees C the enzyme is half inactivated in 72 hours.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Mar-Apr, (2), 102 - 9
{Biological effects of interferon, produced by recombinant bacteria of the probiotic preparation subalin}; Beliavskaia VA et al.; The present review deals with the analysis of biological and functional activities of recombinant bacteria Bacillus subtilis IF-alpha 2335 are producing a human interferon . The interferon-producing bacteria are constructed on a basis commercial probiotic strain B.subtilis 2335, carrying a recombinant plasmid pMBM 105 with the gene of human alpha-2 interferon . The implementation of the recombinant strain in the preparation probiotic, received a designation "Subalin", necessitates to verify a number of immunologic activities and to perform successive protective effects . Interferon, synthesized by recombinant bacteria shows the activity on macroorganism at oral and rectal application of preparation . Subalin was shown antivirus and antitumor activity and preservation by recombinat bacteria of antagonistic properties . The mechanisms of the positive effect of subalin were considered: this effect was shown to be due to the action of interferon excreted by recombinant bacteria into the mucous of different biotopes of host.

Biochemistry, 2003 Aug 5, 42(30), 9060 - 6
Engineering the primary substrate specificity of Streptomyces griseus trypsin; Page MJ et al.; Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity . Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity . The recombinant and native proteases have nearly identical enzymatic properties and structures . Four SGT mutants with alterations in the S1 substrate binding pocket (T190A, T190P, T190S, and T190V) were also expressed . The T190P mutant demonstrated the largest shift to a preference for Arg versus Lys in the P1 site . This was shown by a minor reduction in catalytic activity toward an Arg-containing substrate (k(cat) reduction of 25%) . The crystal structures of the recombinant SGT and the T190P mutant in a complex with the inhibitor benzamidine were obtained at high resolution (approximately 1.9 A) . The increase in P1 specificity, achieved with minimal effect on the catalytic efficiency, demonstrates that the T190P mutant is an ideal candidate for the design of additional substrate specificity engineered into the S2 to S4 binding pockets.

Mol Genet Genomics, 2003 Aug, 269(5), 706 - 14 Epub 2003 Jul 23.
Genome-wide survey of mRNA half-lives in Bacillus subtilis identifies extremely stable mRNAs; Hambraeus G et al.; We have used DNA microarrays to survey rates of mRNA decay on a genomic scale in early stationary-phase cultures of Bacillus subtilis . The decay rates for mRNAs corresponding to about 1500 genes could be estimated . About 80% of these mRNAs had a half-life of less than 7 min . More than 30 mRNAs, including both mono- and polycistronic transcripts, were found to be extremely stable, i.e . to have a half-life of > or =15 min . Only two such transcripts were known previously in B . subtilis . The results provide the first overview of mRNA decay rates in a gram-positive bacterium and help to identify polycistronic operons . We could find no obvious correlation between the stability of an mRNA and the function of the encoded protein . We have also not found any general features in the 5' regions of mRNAs that distinguish stable from unstable transcripts . The identified set of extremely stable mRNAs may be useful in the construction of stable recombinant genes for the overproduction of biomolecules in Bacillus species.

Med Sci Monit, 2003 Jul, 9(7), BR283 - 8
Production of high-molecular-weight ribonuclease Bsn from the recombinant strain of Bacillus subtilis; Kharitonova MA et al.; BACKGROUND: Ribonucleases (RNases) can be used in both basic and clinical sciences, e.g . in research on developmental processes or on antiviral and antitumor therapy . RNases have great potential as therapeutic entities . On the basis of new ribonucleases new medications can be created . Bacilli synthesize two types of secretory ribonucleases, the well-studied low-molecular-weight ribonucleases and high-molecular-weight ribonucleases . Only two RNases of the second type have so far been described: RNase Bsn from B . subtilis and binase II from B . intermedius . MATERIAL/METHODS: The activity of ribonucleases was determined from the amount of the acid-soluble products of RNA hydrolysis . The cultivation media were optimized for maximum RNase production in terms of the experimental factorial design B2 using BIOPT software . RESULTS: Our investigation of a novel secretory ribonuclease, the Bacillus subtilis RNase Bsn expressed in the recombinant B . subtilis strain 168, showed that it is synthesized in the growth retardation phase, when inorganic phosphate is exhausted in the medium . The biosynthesis of Bsn was found to be suppressed by inorganic phosphate in the medium and activated by small amounts of the transcriptional inhibitor actinomycin D . CONCLUSIONS: Our results show that the biosynthesis of the novel secretory ribonuclease Bsn in recombinant strain Bacillus subtilis 168 is subject to negative regulation by inorganic phosphate, and is activated by small doses of actinomycin D . The stimulating effect of this antibiotic is well pronounced during the active synthesis of ribonucleases, but insignificant when ribonuclease synthesis is inhibited by Pi.

J Nutr Sci Vitaminol (Tokyo), 2003 Feb, 49(1), 73 - 5
Investigation of 1-deoxy-D-xylulose 5-phosphate synthase and transketolase of Bacillus subtilis in relation to vitamin B6 biosynthesis; Sakai A et al.; In Escherichia coli, 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose 5-phosphate are believed to be direct precursors of vitamin B6 (B6), and 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) and transketolase could catalyze the formation of each precursor . In this report, the possible involvement Dxs and transketolase (Tkt) in B6 biosynthesis in Bacillus subtilis was investigated . The gene disruptant of tkt and conditional mutants of dxs were constructed, and their ability of B6 biosynthesis was examined . It was found that the tkt disruptants retain the ability to synthesize B6 . The conditional mutant of dxs synthesized the same amount of B6 per dry cell weight as the wild-type strain . Therefore, it is very likely that neither Dxs nor transketolase is involved in B6 biosynthesis in B . subtilis.

Biotechnol Lett, 2003 Jan, 25(2), 161 - 6
Enzymatic synthesis of hydrophilic undecylenic acid sugar esters and their biodegradability; Raku T et al.; To enhance water solubility of 10-undecylenic acid, which has anti-fungus, anti-bacterial and anti-virus activity, D-glucose, trehalose and sucrose were regioselectively esterified with vinyl 10-undecylenic acid ester in dimethyl formamide by a commercial protease, Bioprase conc., from Bacillus subtilis . 6-O-(10-Undecylenoyl) D-glucose, 6-O-(10-undecylenoyl) trehalose and 1'-O-(10-undecylenoyl) sucrose were obtained . The influence of structural variation by changing the sugar moiety was analyzed the surface tension and biodegradability.

Biotechnol Lett, 2003 Mar, 25(6), 465 - 8
Optimization of cell growth and poly(glutamic acid) production in batch fermentation by Bacillus subtilis; Richard A et al.; Poly(glutamic acid) was produced maximally by Bacillus subtilis in batch fermentations at pH 7 and using glycerol at 20 g l(-1) in a glutamic acid/citric acid medium . Poly(glutamic acid) reached 23 g l(-1) after 30 h.

Protein Expr Purif, 2003 Aug, 30(2), 203 - 9
Purification and characterization of YihA, an essential GTP-binding protein from Escherichia coli; Lehoux IE et al.; YihA has previously been characterized as an essential gene of unknown function in both Escherichia coli and Bacillus subtilis . It is conserved in bacteria and represents an attractive target for the discovery of new antibiotics . YihA encodes a putative GTP-binding protein . We have cloned and overexpressed the gene encoding E . coli YihA and initiated biochemical studies as a first step towards understanding its biological function . We showed by circular dichroism that the purified protein has a secondary structure typical of most GTP-binding proteins . It binds guanine nucleotides specifically, as demonstrated by fluorescence resonance energy transfer between 2'-(or-3')-O-(N-methylanthraniloyl) nucleotides (mant-nucleotides) and the tryptophans of YihA . The K(d) values for GDP and GTP were determined by competition with 2'-(or-3')-O-(N-methylanthraniloyl) GDP to be 3 and 27 microM, respectively . Using mutants of YihA we show that nucleotide binding occurs at the putative GTP-binding domain predicted from the primary sequence.

Arch Pharm Res, 2003 Jun, 26(6), 449 - 52
Antibacterial coumarins from Angelica gigas roots; Lee S et al.; Systematic fractionation of Angelica gigas roots led to the isolation of linear furano(pyrano)coumarins such as bergapten (1), decursinol angelate (2), decursin (3), nodakenetin (4) and nodakenin (5) . The antibacterial activities of those compounds against pathogenic bacteria were investigated . Among the compounds tested, decursinol angelate (2) and decursin (3) exhibited significant antibacterial activity against Bacillus subtilis with the minimum inhibitory concentrations (MICs) of 50 and 12.5 microg/mL, respectively.

Protein Sci, 2003 Aug, 12(8), 1694 - 705
The characterization of mutant Bacillus subtilis adenylosuccinate lyases corresponding to severe human adenylosuccinate lyase deficiencies; Palenchar JB et al.; Adenylosuccinate lyase is a homotetramer that catalyzes two discrete reactions in the de novo synthesis of purines: the cleavage of adenylosuccinate and succinylaminoimidazole carboxamide ribotide (SAICAR) . Several point mutations in the gene encoding the enzyme have been implicated in human disease . Bacillus subtilis adenylosuccinate lyase was used as a model system in which mutations were constructed corresponding to those mutations associated with severe human adenylosuccinate lyase deficiency . Site-directed mutagenesis was utilized to construct amino acid substitutions in B . subtilis adenylosuccinate lyase; Met(10), Ile(123), and Thr(367) were replaced by Leu, Trp, and Arg, respectively, and the altered enzymes were expressed in Escherichia coli . These purified enzymes containing amino acid substitutions were found to have substantial catalytic activity and exhibit relatively small changes in their kinetic parameters . The major deviations from the wild-type-like behavior were observed upon biophysical characterization . All of these enzymes with amino acid replacements are associated with marked thermal instability . I123W adenylosuccinate lyase exhibits notable changes in the circular dichroism spectra, and a native gel electrophoresis pattern indicative of some protein aggregation . T367R also exhibits alterations at the quarternary level, as reflected in native gel electrophoresis . Experimental results, combined with homology modeling, suggest that the altered enzymes are primarily structurally impaired . The enzyme instability was found to be lessened by subunit complementation with the wild-type enzyme, under mild conditions; these studies may have implications for the in vivo behavior of adenylosuccinate lyase in heterozygous patients . Residues Met(10), Ile(123), and Thr(367) appear to be located in regions of the enzyme important for maintaining the structural integrity required for a stable, functional enzyme.

Genes Cells, 2003 Aug, 8(8), 699 - 712
A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes; Morikawa K et al.; BACKGROUND: Staphylococcus aureus is a major human pathogen and causes a serious hospital infection due to the acquired multidrug resistance . Unlike the well-studied bacteria such as Escherichia coli and Bacillus subtilis, which have seven and 18 sigma factors, respectively, only two sigma factors have been known for S . aureus . We searched for possible sigma factor genes by examining the S . aureus genome with a special attention to the gene arrangement around the sigma factor genes of a close relative, B . subtilis . RESULTS: A new sigma factor gene was identified in Staphylococcus . The gene constituted a conserved gene cluster with other genes including translation- and transcription-related genes . Phylogenetic analysis and comparison of the gene sequences among species indicated that the staphylococcal sigma factor originated from a common ancestor of B . subtilis SigH . An over-expression of this sigma factor in S . aureus resulted in a drastic induction of the expression of the com operons that encode proteins required for the natural genetic competence . CONCLUSIONS: We demonstrated that the newly identified staphylococcal sigma factor participated in a regulatory network of transcription that controlled the genetic competence genes . In our phylogenetic tree, the factor was classified as a single group with a common function.

Infect Immun, 2003 Aug, 71(8), 4304 - 12
The major cold shock gene, cspA, is involved in the susceptibility of Staphylococcus aureus to an antimicrobial peptide of human cathepsin G; Katzif S et al.; A Tn551 insertional library of Staphylococcus aureus strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG) . After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136 . Southern hybridization analysis revealed that the Tn551 insert in this mutant (SK1) was carried on a 10.6-kb EcoRI chromosomal DNA fragment . Subsequent physical mapping of this Tn551 insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the cspA gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of Escherichia coli and Bacillus subtilis, respectively . This Tn551 insertion as well as a separate deletion-insertion mutation in cspA decreased the capacity of S . aureus to respond to the stress of cold shock and increased resistance to CG 117-136 . The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system.

Biochemistry, 2003 Jul 29, 42(29), 8739 - 47
Surface salt bridges modulate DNA wrapping by the type II DNA-binding protein TF1; Grove A; The histone-like protein HU is involved in compaction of the bacterial genome . Up to 37 bp of DNA may be wrapped about some HU homologues in a process that has been proposed to depend on a linked disruption of surface salt bridges that liberates cationic side chains for interaction with the DNA . Despite significant sequence conservation between HU homologues, binding sites from 9 to 37 bp have been reported . TF1, an HU homologue that is encoded by Bacillus subtilis bacteriophage SPO1, has nM affinity for 37 bp preferred sites in DNA with 5-hydroxymethyluracil (hmU) in place of thymine . On the basis of electrophoretic mobility shift assays, we show that TF1-DNA complex formation is associated with a net release of only approximately 0.5 cations . The structure of TF1 suggests that Asp13 can form a dehydrated surface salt bridge with Lys23; substitution of Asp13 with Ala increases the net release of cations to approximately 1 . These data are consistent with complex formation linked to disruption of surface salt bridges . Substitution of Glu90 with Ala, which would expose Lys87 predicted to contact DNA immediately distal to a proline-mediated DNA kink, causes an increase in affinity and an abrogation of the preference for hmU-containing DNA . We propose that hmU preference is due to finely tuned interactions at the sites of kinking that expose a differential flexibility of hmU- and T-containing DNA . Our data further suggest that the difference in binding site size for HU homologues is based on a differential ability to stabilize the DNA kinks.

Proteomics, 2003 Jul, 3(7), 1117 - 27
Using standard positions and image fusion to create proteome maps from collections of two-dimensional gel electrophoresis images; Luhn S et al.; Databases for two-dimensional protein gels pose new challenges in extracting meaningful information from large numbers of experiments . In order to create expression profiles, positions of corresponding protein spots across all gel images have to be established . In larger gel sets errors may accumulate rapidly during this spot matching process, effectively limiting the number of samples available for data mining . Here we present a novel approach for organizing spot data based on the concept of a standard position for a protein species . Standard positions are meaningful average positions that are determined using all occurrences of a protein species . They can be extended to spots that are not annotated via interpolation . The standard position of a spot can serve as a unifying index across all gels in a database, thus allowing creation and analysis of expression profiles that span the whole collection . The standard position gives a much more accurate estimation of a spot's position on a gel than can be obtained using theoretical isoelectric point and molecular weight . Positional indexing is a complement to a priori identifications (e.g . by mass spectrometry or Edman degradation) . Moreover it can be used in advance to select spots that are worth identifying because they show relevant expression profiles . Furthermore, we show how to combine all spots that occur on any of the gels into one synthetic but nevertheless realistic-looking image . This composite image is produced such that all spots have their standard positions . It can serve as a proteome reference map for an organism . As an application, we have computed a reference map from 23 gel images of Bacillus subtilis, using an enhanced prerelease version of the gel analysis software Delta2D (DECODON, Greifswald, Germany).

J Food Prot, 2003 Jul, 66(7), 1233 - 40
Characterization of UV-peroxide killing of bacterial spores; Reidmiller JS et al.; Advantage is taken in many sterilization processes, especially for food packaging materials, of the synergy between H2O2 and UV irradiation for spore killing . The nature of the synergy is currently not well defined in terms of targets and mechanisms . We found that under some experimental conditions, the synergistic killing of spores of Bacillus megaterium ATCC 19213 appeared to be mainly UV-enhanced peroxide killing, while under other conditions, it appeared to be mainly peroxide-enhanced UV killing . Lethal combinations of H2O2 and UV irradiation for spores resulted in only modest increases in auxotrophic mutations among survivors, indicative of little DNA damage, in contrast to higher mutation levels after dry-heat damage at 115 degrees C . However, the combination of UV light and peroxide did lead to major inactivation of glucose 6-phosphate dehydrogenase, an enzyme that was used to monitor the damage to bacterial protein . Synergistic UV-H2O2 killing was reduced by agents such as pyruvate, thiosulfate, and iron or copper cations, which appeared to act at least in part by reacting chemically with H2O2, and was only slightly affected by the use of UV light at a wavelength of 222 nn rather than 254 nm . Hydrogen peroxide treatment can precede UV irradiation for synergistic killing by some hours with an interim of drying for spores of Bacillus subtilis A, a spore type used commonly for the validation of aseptic processes . Synergistic killing of dried spores or those in suspensions was accelerated at higher temperatures (50 degrees C) rather than at lower temperatures (25 degrees C).

Eur J Biochem, 2003 Aug, 270(15), 3196 - 204
UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity; Gagyi C et al.; The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins . The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein . In the absence of GTP, the activity of B . subtilis enzyme is less than 10% of its maximum activity . Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly . Binding of Ant-dGTP to B . subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three . UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective . UTP inhibits UMP kinase of B . subtilis with a lower affinity than that shown towards the Escherichia coli enzyme . Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B . subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium . A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B . subtilis and E . coli enzymes.

Appl Opt, 2003 Jul 1, 42(19), 4080 - 7
Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation; Alimova A et al.; Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis (Bs), a spore-forming, and Staphylococcus aureus (Sa), a nonspore-forming bacteria subjected to conditions of starvation . Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan . Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 m, from dipicolinic acid, appeared . Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores . The intensity of the 410-nm band continued to increase over the next 3 days . The Sa fluorescence was predominantly from tryptophan and did not change over time . In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 cm and excitation peaks at 250, 270, and 400 nm . The addition of beta-hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission . The addition of Fe2+ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria . We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time.

J Bacteriol, 2003 Aug, 185(15), 4615 - 9
Analysis of the interaction between the transcription factor sigmaG and the anti-sigma factor SpoIIAB of Bacillus subtilis; Evans L et al.; The activation of sigma(G), a transcription factor, in Bacillus subtilis is coupled to the completion of engulfment during sporulation . SpoIIAB, an anti-sigma factor involved in regulation of sigma(F), is also shown to form a complex with sigma(G) in vitro . SpoIIAA, the corresponding anti-anti-sigma factor, can disrupt the SpoIIAB:sigma(G) complex, releasing free sigma(G) . The data suggest the existence of an as-yet-unknown mechanism to keep sigma(G) inactive prior to engulfment.

J Bacteriol, 2003 Aug, 185(15), 4490 - 8
Essential nature of the mreC determinant of Bacillus subtilis; Lee JC et al.; The mre genes of Escherichia coli and Bacillus subtilis are cell shape determination genes . Mutants affected in mre function are spheres instead of the normal rods . Although the mre determinants are not required for viability in E . coli, the mreB determinant is an essential gene in B . subtilis . Conflicting results have been reported as to whether the two membrane-associated proteins MreC and MreD are essential proteins . Furthermore, although the MreB protein has been studied in some detail, the roles of the MreC and MreD proteins in cell shape determination are unknown . We constructed a strain of B . subtilis in which expression of the mreC determinant is dependent upon the addition of isopropyl-beta-D-thiogalactopyranoside to the culture medium . Utilizing this conditional strain, it was shown that mreC is an essential gene in B . subtilis . Furthermore, it was shown that cells lacking sufficient quantities of MreC undergo morphological changes, namely, swelling and twisting of the cells, which is followed by cell lysis . Electron microscopy was utilized to demonstrate that a polymeric material accumulated at one side of the division septum of the cells and that the presence of this material correlated with the bending of the cell . The best explanation for the results is that the MreC protein is involved in the control of septal versus long-axis peptidoglycan synthesis, that cells lacking MreC perform aberrant septal peptidoglycan synthesis, and that lysis results from a deficiency in long-axis peptidoglycan synthesis.

J Bacteriol, 2003 Aug, 185(15), 4305 - 14
Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation; Brigulla M et al.; A variety of environmental and metabolic cues trigger the transient activation of the alternative transcription factor SigB of Bacillus subtilis, which subsequently leads to the induction of more than 150 general stress genes . This general stress regulon provides nongrowing and nonsporulated cells with a multiple, nonspecific, and preemptive stress resistance . By a proteome approach we have detected the expression of the SigB regulon during continuous growth at low temperature (15 degrees C) . Using a combination of Western blot analysis and SigB-dependent reporter gene fusions, we provide evidence for high-level and persistent induction of the sigB operon and the SigB regulon, respectively, in cells continuously exposed to low temperatures . In contrast to all SigB-activating stimuli described thus far, induction by low temperatures does not depend on the positive regulatory protein RsbV or its regulatory phosphatases RsbU and RsbP, indicating the presence of an entirely new pathway for the activation of SigB by chill stress in B . subtilis . The physiological importance of the induction of the general stress response for the adaptation of B . subtilis to low temperatures is emphasized by the observation that growth of a sigB mutant is drastically impaired at 15 degrees C . Inclusion of the compatible solute glycine betaine in the growth medium not only improved the growth of the wild-type strain but rescued the growth defect of the sigB mutant, indicating that the induction of the general stress regulon and the accumulation of glycine betaine are independent means by which B . subtilis cells cope with chill stress.

Mol Microbiol, 2003 Aug, 49(3), 581 - 90
Swarming motility in undomesticated Bacillus subtilis; Kearns DB et al.; Swarming motility was identified and characterized in an undomesticated strain of Bacillus subtilis . Rapid surface migration was preceded by a cell density-dependent lag period, which could be eliminated if actively swarming cells were used as the inoculum . The leading edge of the swarm was characterized by multicellular rafts of highly flagellated cells . Flagellum biosynthesis and surfactant production were required for swarming . Swarming was not found in any of several standard laboratory strains . Laboratory strains are characteristically unable to produce surfactant, but such a strain remained unable to swarm even when surfactant was provided by extracellular complementation . We conclude that robust swarming is a feature of undomesticated B . subtilis and that this behaviour has been lost or attenuated in laboratory strains through the accumulation of multiple genetic defects.

J Appl Microbiol, 2003, 95(2), 218 - 24
Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings; Tsomlexoglou E et al.; AIM: To detect L-form bacteria in developing Chinese cabbage seedlings . METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase) . Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium . Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings . beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems . Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues . Stable L-form bacteria were non-culturable after their association with plant material . CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B . subtilis L-form bacteria in plant material . SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.

Lett Appl Microbiol, 2003, 37(2), 169 - 73
Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread; Sorokulova IB et al.; AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread . METHODS AND RESULTS: Classical and molecular methods {16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR} were used to identify and type-isolated strains . The predominant species isolated were Bacillus subtilis and B . licheniformis . RAPD analysis demonstrated that the same sample may harbor different strains . Ten of 15 strains of B . subtilis and four of six strains of B . licheniformis were able to cause rope spoilage of the laboratory-baked bread . CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread . SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread.

Lett Appl Microbiol, 2003, 37(2), 162 - 8
Time series analysis of aerobic bacterial flora during Miso fermentation; Onda T et al.; AIMS: This article reports a microbiological study of aerobic mesophilic bacteria that are present during the fermentation process of Miso . METHODS AND RESULTS: Aerobic bacteria were enumerated and isolated from Miso during fermentation and divided into nine groups using traditional phenotypic tests . The strains were identified by biochemical analysis and 16S rRNA sequence analysis . They were identified as Bacillus subtilis, B . amyloliquefaciens, Kocuria kristinae, Staphylococcus gallinarum and S . kloosii . All strains were sensitive to the bacteriocins produced by the lactic acid bacteria isolated from Miso . CONCLUSIONS: The dominant species among the undesirable species throughout the fermentation process were B . subtilis and B . amyloliquefaciens . It is suggested that bacteriocin-producing lactic acid bacteria are effective in the growth prevention of aerobic bacteria in Miso . SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful information for controlling of bacterial flora during Miso fermentation.

Pharmazie, 2003 Jun, 58(6), 428 - 30
Iridoids from Pedicularis kansuensis forma albiflora; Yuan CS et al.; A new iridoid glycoside kansuenoside (1) and a new iridoid kansuenin (2), along with eight known compounds (3-10) were isolated from the whole plant of Pedicularis kansuensis forma albiflora Li . Their structures were elucidated by spectroscopic methods . Nine of them were assayed against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus.

Science, 2003 Jul 11, 301(5630), 211 - 3
Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA; Chen G et al.; The Bacillus subtilis AT (anti-TRAP) protein inhibits the regulatory protein TRAP (trp RNA-binding attenuation protein), thereby eliminating transcription termination in the leader region of the trp operon . Transcription of the AT operon is activated by uncharged tryptophan transfer RNA (tRNATrp) . Here we show that translation of AT also is regulated by uncharged tRNATrp . A 10-residue coding region containing three consecutive tryptophan codons is located immediately preceding the AT structural gene . Completion of translation of this coding region inhibits AT synthesis, whereas incomplete translation increases AT production . Tandem sensing of uncharged tRNATrp therefore regulates synthesis of AT, which in turn regulates TRAP's ability to inhibit trp operon expression.

Microbiology, 2003 Jul, 149(Pt 7), 1687 - 98
Phosphoenolpyruvate phosphotransferase system and N-acetylglucosamine metabolism in Bacillus sphaericus; Alice AF et al.; Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source . However, ptsHI genes encoding HPr and EI proteins belonging to a PTS were cloned, sequenced and characterized . Both HPr and EI proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins . HPr(His(6)) was purified from wild-type and a Ser46/Gln mutant of B . sphaericus, and used for in vitro phosphorylation experiments using extracts from either B . sphaericus or Bacillus subtilis as kinase source . The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained . The findings also proved indirectly the existence of an HPr kinase activity in B . sphaericus . The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orf2) . In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic . Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an EII(Nag) element of the PTS, as yet uncharacterized . In addition, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc . These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B . sphaericus . They also open new possibilities for genetic improvements in industrial applications.

FEMS Microbiol Lett, 2003 Jul 15, 224(1), 107 - 12
Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei; Braaz R et al.; The recently discovered extracellular poly{(R)-3-hydroxybutyrate} (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids . In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800 . The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B . subtilis as shown by Western blots of cell-free culture fluid proteins . No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity . Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine . Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity . In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.

J Biol Chem, 2003 Oct 10, 278(41), 39323 - 9 Epub 2003 Jul 07.
Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase; Grangeasse C et al.; Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria . However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes . Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid . The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569 . The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity . In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd . These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis . Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.

Metab Eng, 2003 Apr, 5(2), 133 - 49
Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis: regulation of the central metabolic pathways; Blencke HM et al.; Chemoheterotrophic bacteria use a few central metabolic pathways for carbon catabolism and energy production as well as for the generation of the main precursors for anabolic reactions . All sources of carbon and energy are converted to intermediates of these central pathways and then further metabolized . While the regulation of genes encoding enzymes used to introduce specific substrates into the central metabolism has already been studied to some detail, much less is known about the regulation of the central metabolic pathways . In this study, we investigated the responses of the Bacillus subtilis transcriptome to the presence of glucose and analyzed the role of the pleiotropic transcriptional regulator CcpA in these responses . We found that CcpA directly represses genes involved in the utilization of secondary carbon sources . In contrast, induction by glucose seems to be mediated by a variety of different mechanisms . In the presence of glucose, the genes encoding glycolytic enzymes are induced . Moreover, the genes responsible for the production of acetate from pyruvate with a concomitant substrate-level phosphorylation are induced by glucose . In contrast, the genes required for the complete oxidation of the sugar (Krebs cycle, respiration) are repressed if excess glucose is available for the bacteria . In the absence of glucose, the genes of the Krebs cycle as well as gluconeogenic genes are derepressed . The genes encoding enzymes of the pentose phosphate pathway are expressed both in the presence and the absence of glucose, as suggested by the central role of this pathway in generating anabolic precursors.

Nat Genet, 2003 Aug, 34(4), 377 - 8
Essentiality, not expressiveness, drives gene-strand bias in bacteria; Rocha EP et al.; Preferential positioning of bacterial genes in the leading strand was thought to result from selection to avoid high head-on collision rates between DNA and RNA polymerases . Here we show, however, that in Bacillus subtilis and Escherichia coli, essentiality (the transcript product), not expressiveness (the collision rate), selectively drives the biased gene distribution.

Nat Struct Biol, 2003 Aug, 10(8), 652 - 7
Structure of the manganese-bound manganese transport regulator of Bacillus subtilis; Glasfeld A et al.; The Bacillus subtilis manganese transport regulator, MntR, binds Mn2+ as an effector and is a repressor of transporters that import manganese . A member of the diphtheria toxin repressor (DtxR) family of metalloregulatory proteins, MntR exhibits selectivity for Mn2+ over Fe2+ . Replacement of a metal-binding residue, Asp8, with methionine (D8M) relaxes this specificity . We report here the X-ray crystal structures of wild-type MntR and the D8M mutant bound to manganese with 1.75 A and 1.61 A resolution, respectively . The 142-residue MntR homodimer has substantial structural similarity to the 226-residue DtxR but lacks the C-terminal SH3-like domain of DtxR . The metal-binding pockets of MntR and DtxR are substantially different . The cation-to-cation distance between the two manganese ions bound by MntR is 3.3 A, whereas that between the metal ions bound by DtxR is 9 A . D8M binds only a single Mn2+ per monomer, owing to alteration of the metal-binding site . The sole retained metal site adopts pseudo-hexacoordinate geometry rather than the pseudo-heptacoordinate geometry of the MntR metal sites.

Mol Genet Genomics, 2003 Aug, 269(5), 640 - 8 Epub 2003 Jul 04.
Patterns of protein carbonylation following oxidative stress in wild-type and sigB Bacillus subtilis cells; Mostertz J et al.; Oxidative stress causes damage to nucleic acids, membrane lipids and proteins . One striking effect is the metal-catalyzed, site-specific carbonylation of proteins . In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the sigmaB-dependent general stress response act together to make cells more resistant to oxidative stress . In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B . subtilis . Furthermore, we asked whether the sigmaB-dependent response to oxidative stress also confers protection against protein carbonylation . To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis . The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide . Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated . Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation . Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells . We could not detect any differences between wild-type and deltasigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress . However, artificial preloading with proteins that are normally induced by sigmaB-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.

J Mol Biol, 2003 Jul 11, 330(3), 593 - 606
Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9A resolution; Vincent F et al.; Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities . Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C . The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer . This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts . A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents . By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre . This would explain the observation that the enzyme is active on a variety of small, acetylated molecules . The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues . Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold . These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation . We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.

J Mol Biol, 2003 Jul 11, 330(3), 459 - 72
Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis; Madec E et al.; We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases . In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region . This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli . PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction . PrkC also displays kinase activity with myelin basic protein . Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc . All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity . Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive . Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity . In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism . When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region . These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism . The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways . This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.

Anal Biochem, 2003 Aug 1, 319(1), 73 - 7
A screening method for endo-beta-1,4-xylanase substrate selectivity; Moers K et al.; Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation . A screening method for rapidly determining said substrate selectivity was developed . Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant . A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX . A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate . A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities.

Appl Environ Microbiol, 2003 Jul, 69(7), 4144 - 50
A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces ethanol; Becker J et al.; Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms . Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-arabinose for growth and to ferment it to ethanol . Expanding the substrate fermentation range of S . cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes . After overexpression of a bacterial L-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-arabinose-transporting yeast galactose permease, we were able to select an L-arabinose-utilizing yeast strain by sequential transfer in L-arabinose media . Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-arabinose is a lowered activity of L-ribulokinase . Moreover, high L-arabinose uptake rates and enhanced transaldolase activities favor utilization of L-arabinose . With a doubling time of about 7.9 h in a medium with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight) . h(-1) under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.

J Bacteriol, 2003 Jul, 185(14), 4099 - 109
Functional dissection of the Bacillus subtilis pur operator site; Bera AK et al.; Bacillus subtilis PurR represses transcription of several genes involved in purine synthesis, metabolism, and transport and cofactor synthesis . PurR binds specifically to DNAs containing an inverted repeat of a 14-nucleotide "PurBox" located in the upstream control regions of genes in the PurR regulon . Further biochemical investigation of the interaction of PurR with a series of shortened upstream DNA fragments of the pur operon determined the minimum length and specificity elements of the operator . The relative affinities of the two PurBoxes differ significantly, such that upstream PurBox1 (-81 to -68 relative to the transcription start site) is designated "strong" and downstream PurBox2 (-49 to -36) is designated "weak." Two PurBoxes are required for high-affinity PurR binding, and one of these must be strong . The shortest DNA construct with high affinity for PurR is a 74-bp perfect palindrome in which weak PurBox2 and its flanking sequences are replaced by strong PurBox1 and flanking sequences . Two PurR dimers bind to this symmetric construct . Phosphoribosylpyrophosphate (PRPP), the effector molecule that reduces affinity of PurR for DNA, requires one weak PurBox in the DNA construct to inhibit PurR binding . PRPP binds, as expected, to a PRPP-motif in PurR . A tracks outside the central conserved CGAA sequence of the PurBox may facilitate DNA bending, leading to a proposal for strong and weak designations of PurBoxes in the control regions of other genes regulated by PurR.

J Bacteriol, 2003 Jul, 185(14), 4087 - 98
The purine repressor of Bacillus subtilis: a novel combination of domains adapted for transcription regulation; Sinha SC et al.; The purine repressor from Bacillus subtilis, PurR, represses transcription from a number of genes with functions in the synthesis, transport, and metabolism of purines . The 2.2-A crystal structure of PurR reveals a two-domain protein organized as a dimer . The larger C-terminal domain belongs to the PRT structural family, in accord with a sequence motif for binding the inducer phosphoribosylpyrophosphate (PRPP) . The PRT domain is fused to a smaller N-terminal domain that belongs to the winged-helix family of DNA binding proteins . A positively charged surface on the winged-helix domain likely binds specific DNA sequences in the recognition site . A second positively charged surface surrounds the PRPP site at the opposite end of the PurR dimer . Conserved amino acids in the sequences of PurR homologs in 21 gram-positive bacteria cluster on the proposed recognition surface of the winged-helix domain and around the PRPP binding site at the opposite end of the molecule, supporting a common function of DNA and PRPP binding for all of the proteins . The structure supports a binding mechanism in which extended regions of DNA interact with extensive protein surface . Unlike most PRT proteins, which are phosphoribosyltransferases (PRTases), PurR lacks catalytic activity . This is explained by a tyrosine side chain that blocks the site for a nucleophile cosubstrate in PRTases . Thus, B . subtilis has adapted an enzyme fold to serve as an effector-binding domain and has used it in a novel combination with the DNA-binding winged-helix domain as a repressor of purine genes.

Nucleic Acids Res Suppl, 2001, (1), 209 - 10
In vitro hyperprocessing of tRNAs by Bacillus subtilis ribonuclease P RNA; Hori Y et al.; We have previously reported that the catalytic RNA subunit of ribonuclease P (RNase P) of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA(i)Met), alanine tRNA (tRNA(Ala)) and histidine tRNA (tRNA(His)) within the mature tRNA sequences to produce specific fragments . We call this further cleavage hyperprocessing . These cleavages were dependent on the occurrence of altered conformations of the tRNAs . Here, we found that the RNase P RNA of Bacillus subtilis can hyperprocess these three tRNAs at the same sites as does M1 RNA . The hyperprocessing activity may probably be common feature for Bacterial RNase P RNAs.

Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1299 - 301 Epub 2003 Jun 27.
Purification, crystallization and preliminary X-ray analysis of a Sco1-like protein from Bacillus subtilis, a copper-binding protein involved in the assembly of cytochrome c oxidase; Imriskova-Sosova I et al.; The putative copper-delivery protein BsSco from Bacillus subtilis is a member of the Sco family of cytochrome c oxidase assembly proteins . BsSco is a membrane protein and the soluble domain has been cloned and expressed in Escherichia coli as a fusion with glutathione-S-transferase . The fusion protein was isolated from the cell lysate using a glutathione-affinity column and the soluble domain of BsSco was released by treatment with thrombin . Sufficient amounts of the soluble domain have been obtained for crystallization . Crystals obtained by hanging-drop vapour diffusion diffract to a resolution of 2.3 A at a synchrotron source . The space group is P6 and the unit-cell parameters are a = 67.74, b = 67.74, c = 189.58 A.

Environ Sci Technol, 2003 Jun 1, 37(11), 2376 - 82
Influence of Bacillus subtilis cell walls and EDTA on calcite dissolution rates and crystal surface features; Friis AK et al.; This study investigates the influence of EDTA and the Gram-positive cell walls of Bacillus subtilis on the dissolution rates and development of morphological features on the calcite {1014} surface . The calcite dissolution rates are compared at equivalent saturation indicies (SI) and relative to its dissolution behavior in distilled water (DW) . Results indicate that the presence of metabolically inactive B . subtilis does not affect the dissolution rates significantly . Apparent increases in dissolution rates in the presence of the dead bacterial cells can be accounted for by a decrease of the saturation state of the solution with respect to calcite resulting from bonding of dissolved Ca2+ by functional groups on the cell walls . In contrast, the addition of EDTA to the experimental solutions results in a distinct increase in dissolution rates relative to those measured in DW and the bacterial cell suspensions . These results are partly explained by the 6.5-8 orders of magnitude greater stability of the Ca-EDTA complex relative to the Ca-B . subtilis complexes as well as its free diffusion to and direct attack of the calcite surface . Atomic force microscopy images of the {1014} surface of calcite crystals exposed to our experimental solutions reveal the development of dissolution pits with different morphologies according to the nature and concentration of the ligand . Highly anisotropic dissolution pits develop in the early stages of the dissolution reaction at low B . subtilis concentrations (0.004 mM functional group sites) and in DW . In contrast, at high functional group concentrations (4.0 mM EDTA or equivalent B . subtilis functional group sites), dissolution pits are more isotropic . These results suggest that the mechanism of calcite dissolution is modified by the presence of high concentrations of organic ligands . Since all the pits that developed on the calcite surfaces display some degree of anisotropy and dissolution rates are strongly SI dependent, the rate-limiting step is most likely a surface reaction for all systems investigated in this study . Results of this study emphasize the importance of solution chemistry and speciation in determining calcite reaction rates and give a more accurate and thermodynamically sound representation of dead bacterial cell wall-mineral interactions . In studies of natural aquatic systems, the presence of organic ligands is most often ignored in speciation calculations . This study clearly demonstrates that this oversight may lead to an overestimation of the saturation state of the solutions with respect to calcite and thermodynamic inconsistencies.

FEMS Microbiol Lett, 2003 Jun 27, 223(2), 221 - 5
Differences in effects on DNA gyrase activity between two glutamate racemases of Bacillus subtilis, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC (MurI) isozyme; Ashiuchi M et al.; Bacillus subtilis possesses two isogenes encoding glutamate racemases, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC isozyme, and produces abundant amounts of the Glr enzyme . The YrpC isozyme, but not the Glr enzyme, was found to influence the activity of DNA gyrase, as did the MurI-type glutamate racemase of Escherichia coli, which is involved in peptidoglycan synthesis during cell division.

FEMS Microbiol Lett, 2003 Jun 27, 223(2), 153 - 7
The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli; Ladds JC et al.; The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment . Most response regulators are transcription factors that alter the level of transcription of specific sets of genes . Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase . For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state . For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated . This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA . Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding . It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule.

Mycol Res, 2003 Apr, 107(Pt 4), 421 - 7
Efficacy of microorganisms antagonistic to Rhizoctonia cerealis and their cell wall degrading enzymatic activities; Innocenti G et al.; The effect of Trichoderma atroviride, T . harzianum, T . longibrachiatum, Clonostachys rosea and Bacillus subtilis isolates applied to wheat seeds against Rhizoctonia cerealis disease of seedlings was investigated under controlled greenhouse conditions . Most Trichoderma isolates significantly reduced the incidence of disease compared with the infected control . Bacillus subtilis was also effective against sharp eyespot, although less active than Trichoderma spp . Interactions between the antagonistic microorganisms and the cereal pathogenic fungus in dual culture experiments on agar growth medium were also studied . Almost all tested antagonists showed competitive activity against R . cerealis: inhibition of its mycelial growth and hyphal interaction . The production of extracellular beta-N-acetylhexosaminidase, chitin 1,4-beta-chitobiosidase, glucan 1,3-beta-glucosidase and protease activity by the tested microorganisms in the presence of cell walls of R . cerealis was then determined . All isolates showed glucosaminidase and chitobiosidase activity . They also produced glucosidase activity, except B . subtilis, whereas only C . rosea, B . subtilis and one isolate of T . harzianum showed detectable levels of protease activity.

Mol Microbiol, 2003 Jul, 49(1), 157 - 65
Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box; Yoshida K et al.; Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth . In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box . A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box . Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box . The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA . The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets . It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets . The physiological role of the TnrA regulon is discussed.

Science, 2003 Jul 25, 301(5632), 510 - 3 Epub 2003 Jun 19.
Cannibalism by sporulating bacteria; Gonzalez-Pastor JE et al.; Spore formation by the bacterium Bacillus subtilis is an elaborate developmental process that is triggered by nutrient limitation . Here we report that cells that have entered the pathway to sporulate produce and export a killing factor and a signaling protein that act cooperatively to block sister cells from sporulating and to cause them to lyse . The sporulating cells feed on the nutrients thereby released, which allows them to keep growing rather than to complete morphogenesis . We propose that sporulation is a stress-response pathway of last resort and that B . subtilis delays a commitment to spore formation by cannibalizing its siblings.

J Bacteriol, 2003 Jul, 185(13), 3905 - 17
Expression of spoIIIJ in the prespore is sufficient for activation of sigma G and for sporulation in Bacillus subtilis; Serrano M et al.; During sporulation in Bacillus subtilis, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor sigma(F) . sigma(F) directs transcription of spoIIIG, encoding the late forespore-specific regulator sigma(G) . Following synthesis, sigma(G) is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor . Activation of sigma(G) occurs only after the complete engulfment of the prespore by the mother cell . Mutations in spoIIIJ arrest sporulation soon after conclusion of the engulfment process and prevent activation of sigma(G) . Here we show that sigma(G) accumulates but is mostly inactive in a spoIIIJ mutant . We also show that expression of the spoIIIGE155K allele, encoding a form of sigma(G) that is not efficiently bound by SpoIIAB in vitro, restores sigma(G)-directed gene expression to a spoIIIJ mutant . Expression of spoIIIJ occurs during vegetative growth . However, we show that expression of spoIIIJ in the prespore is sufficient for sigma(G) activation and for sporulation . Mutations in the mother cell-specific spoIIIA locus are known to arrest sporulation just after completion of the engulfment process . Previous work has also shown that sigma(G) accumulates in an inactive form in spoIIIA mutants and that the need for spoIIIA expression for sigma(G) activation can be circumvented by the spoIIIGE155K allele . However, in contrast to the case for spoIIIJ, we show that expression of spoIIIA in the prespore does not support efficient sporulation . The results suggest that the activation of sigma(G) at the end of the engulfment process involves the action of spoIIIA from the mother cell and of spoIIIJ from the prespore.

J Bacteriol, 2003 Jul, 185(13), 3813 - 20
Characterization of LytH, a differentiation-associated peptidoglycan hydrolase of Bacillus subtilis involved in endospore cortex maturation; Horsburgh GJ et al.; The cortex peptidoglycan from endospores of Bacillus subtilis is responsible for the maintenance of dormancy . LytH (YunA) has been identified as a novel sporulation-specific component with a role in cortex structure determination . The lytH gene was expressed only during sporulation, under the control of the mother cell-specific sigma factor sigma(K) . Spores of a lytH mutant have slightly reduced heat resistance and altered staining when viewed by electron microscopy . Analysis of the peptidoglycan structure of lytH mutant spores shows the loss of muramic acid residues substituted with L-alanine and a corresponding increase in muramic acid residues substituted with tetrapeptide compared to those in the parent strain . In a lytH cwlD mutant, the lack of muramic acid residues substituted with L-alanine and delta-lactam leaves 97% of residues substituted with tetrapeptide . These results suggest that lytH encodes an L-Ala-D-Glu peptidase involved in production of single L-alanine side chains from tetrapeptides in the spore cortex . The lack of di- or tripeptides in a lytH mutant reveals the enzyme is an endopeptidase.

J Bacteriol, 2003 Jul, 185(13), 3711 - 7
Electrophoretic mobility of Bacillus subtilis knockout mutants with and without flagella; Okuda S et al.; Mutants of Bacillus subtilis 168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3 . The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength . The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene . The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment . It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy . These results indicate that the flagella are a key factor influencing cell surface properties.

J Appl Microbiol, 2003, 95(1), 167 - 79
Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis; Tovar-Rojo F et al.; AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations . METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells . This spore osmoresistance is higher on richer media . Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al . 1994) . Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB . CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose . SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.

J Appl Microbiol, 2003, 95(1), 135 - 41
Modelling the combined effects of pH, temperature and sodium chloride stresses on the thermal inactivation of Bacillus subtilis spores in a buffer system; Jagannath A et al.; AIMS: The inactivation of Bacillus subtilis 168 spores subjected to the combined stress of pH, temperature and sodium chloride in a buffer system was modelled . METHODS AND RESULTS: Bacillus subtilis 168 spore suspension in 50 mmol l-1 potassium phosphate buffer was heated in an open system using a block heater . A second order polynomial equation was used to describe the relationship between pH, temperature, sodium chloride concentration and the logarithm of the decimal reduction time (D-value) of the spores . Response surface graphs were constructed to predict the inactivation within the experimental domain . The data obtained were also compared with those reported for B . subtilis in different media and foods included in a large reference-based database of thermal inactivation (ThermoKill Database, TKDB R9100), which was constructed in the laboratory . CONCLUSIONS: All the variables studied seemed to have a significant effect on the inactivation of B . subtilis 168 spores in potassium phosphate buffer . The coefficient of determination, r2, and an analysis of the residuals from the model indicated the adequacy of the model to predict the inactivation of B . subtilis spores within the range of the experimental variables studied . SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study will enable a better understanding of the inactivation of B . subtilis spores under the influence of the studied environmental variables . The model can be used by food industries to assess and monitor the shelf life of food products in the event of a chance contamination by B . subtilis spores.

J Appl Microbiol, 2003, 95(1), 54 - 67
Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide; Young SB et al.; AIMS: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them . METHODS AND RESULTS: Spores of B . subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective alpha/beta-type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway . Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins . Spores prepared at a higher temperature were also much more resistant to these agents . Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite- and chlorine dioxide-treated spores much more readily released DPA upon a subsequent normally sub-lethal heat treatment than did untreated spores . Hypochlorite-killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+-DPA, and these spores could not be recovered by lysozyme treatment . Chlorine dioxide-treated spores also did not germinate with Ca2+-DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients . However, while germinated chlorine dioxide-killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties . CONCLUSIONS: Hypochlorite and chlorine dioxide do not kill B . subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat . Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane . While chlorine dioxide-killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage . SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide.

EMBO J, 2003 Jun 16, 22(12), 2893 - 902
Molecular architecture of the ATP-dependent CodWX protease having an N-terminal serine active site; Kang MS et al.; CodWX in Bacillus subtilis is an ATP-dependent, N-terminal serine protease, consisting of CodW peptidase and CodX ATPase . Here we show that CodWX is an alkaline protease and has a distinct molecular architecture . ATP hydrolysis is required for the formation of the CodWX complex and thus for its proteolytic function . Remarkably, CodX has a 'spool-like' structure that is formed by interaction of the intermediate domains of two hexameric or heptameric rings . In the CodWX complex, CodW consisting of two stacked hexameric rings (WW) binds to either or both ends of a CodX double ring (XX), forming asymmetric (WWXX) or symmetric cylindrical particles (WWXXWW) . CodWX can also form an elongated particle, in which an additional CodX double ring is bound to the symmetric particle (WWXXWWXX) . In addition, CodWX is capable of degrading EzrA, an inhibitor of FtsZ ring formation, implicating it in the regulation of cell division . Thus, CodWX appears to constitute a new type of protease that is distinct from other ATP-dependent proteases in its structure and proteolytic mechanism.

DNA Cell Biol, 2003 Mar, 22(3), 209 - 15
First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome; Jung HY et al.; Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects . Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced . Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma . Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons . However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly . Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional . Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas . The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute . Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis . This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.

J Med Chem, 2003 Jun 19, 46(13), 2731 - 9
Synthesis of substituted 6-anilinouracils and their inhibition of DNA polymerase IIIC and Gram-positive bacterial growth; Zhi C et al.; Certain substituted 6-anilinouracils are potent and selective inhibitors of Gram+ bacterial DNA polymerase IIIC (pol IIIC) . In addition, analogues with 3-substituents in the uracil ring have potent antibacterial activity against Gram+ organisms in culture . In an attempt to find optimal anilino substituents for pol IIIC binding and optimal 3-substituents for antibacterial activity, we have prepared several series of 3-substituted-6-aminouracils and assayed their activity against pol IIIC from Bacillus subtilis and a panel of Gram+ and Gram- bacteria in culture . The 6-(3-ethyl-4-methylanilino) group and closely related substituent patterns maximized pol IIIC inhibition potency . Among a series of 3-(substituted-butyl)-6-(3-ethyl-4-methylanilino)uracils, basic amino substituents increased pol IIIC inhibition, but decreased antibacterial activity . The most potent antibacterials were simple hydroxybutyl and methoxybutyl derivatives, and hydrophobically substituted piperidinylbutyl derivatives.

Microbiol Immunol, 2003, 47(4), 279 - 83
An effective sporicidal reagent against Bacillus subtilis spores; Kida N et al.; We developed a reagent which showed significant sporicidal activity against Bacillus subtilis spores . This reagent was composed of ethylenediaminetetraacetic acid, disodium salt (EDTA-2Na), ferric chloride hexahydrate (FeCl3 x 6H2O) and ethanol (tentatively designated as the ethanol reagent) . The ethanol reagent showed pH- and temperature-dependent sporicidal activity . At pH 0.3, its activity was almost the same as that of 0.05% sodium hypochlorite at 20 C and was higher at 37 C than at 20 C . The activity of the ethanol reagent was similar both with and without 10% serum . The ethanol reagent might be applicable for disinfecting Bacillus spores.

J Dairy Res, 2003 May, 70(2), 189 - 97
Antibacterial activity of casein-derived peptides isolated from rabbit (Oryctolagus cuniculus) milk; Baranyi M et al.; Acid-precipitated rabbit 'whole casein' was digested by trypsin, chymotrypsin, pepsin, and clostripain to screen for possible peptides with antibacterial properties . The peptide fragments were separated by reversed-phase chromatography . The collected fractions were pooled and their antibacterial properties tested against Escherichia coli, Bacillus subtilis and Staphylococcus lentus . Three antibacterial peptide fragments derived from tryptic digestion of rabbit casein were isolated and identified . Their sequences were found as follows: HVEQLLR (residues 50-56 of beta-casein), ILPFIQSLFPFAER (residues 64-77 of beta-casein), and FHLGHLK (residues 19-25 of alpha(s1)-casein) . The three peptides were synthesized and found to exert antibacterial effect against gram positive bacteria only . Proteolytic digestion of rabbit casein by chymotrypsin, pepsin and clostripain yielded several peptide fragments with antibacterial activity . Since antibiotic peptides can be released from casein during the digestion of milk proteins, our results suggest a possible antibacterial function of rabbit caseins . It is conceivable that antibacterial peptides can be generated by endopeptidases of the mammalian gastrointestinal tract possibly providing protection for new-born rabbits against aggression of micro-organisms.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3071 - 7
The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille; Landthaler M et al.; Here we describe the discovery of a group I intron in the DNA polymerase gene of Bacillus thuringiensis phage Bastille . Although the intron insertion site is identical to that of the Bacillus subtilis phages SPO1 and SP82 introns, the Bastille intron differs from them substantially in primary and secondary structure . Like the SPO1 and SP82 introns, the Bastille intron encodes a nicking DNA endonuclease of the H-N-H family, I-BasI, with a cleavage site identical to that of the SPO1-encoded enzyme I-HmuI . Unlike I-HmuI, which nicks both intron-minus and intron-plus DNA, I-BasI cleaves only intron-minus alleles, which is a characteristic of typical homing endonucleases . Interestingly, the C-terminal portions of these H-N-H phage endonucleases contain a conserved sequence motif, the intron-encoded endonuclease repeat motif (IENR1) that also has been found in endonucleases of the GIY-YIG family, and which likely comprises a small DNA-binding module with a globular betabetaalphaalphabeta fold, suggestive of module shuffling between different homing endonuclease families.

FEMS Microbiol Lett, 2003 Jun 6, 223(1), 101 - 6
Analysis of a DNA-binding motif of the Bacillus subtilis HrcA repressor protein; Wiegert T et al.; The hrcA gene of Bacillus subtilis encodes a transcriptional repressor protein which negatively controls the heat shock operons dnaK and groESL . Alignment of the HrcA protein with repressor proteins from the NCBI database revealed that it exhibits a striking homology near its N-terminal part with proteins of the DeoR family . This region contains a helix-turn-helix motif and has been shown to be involved in DNA binding . To investigate whether this is also true for the HrcA protein, three critical amino acid residues were changed within or adjacent to the recognition helix . While single amino acid replacements barely influenced the binding activity, alteration of two consecutive amino acid residues within the recognition helix completely abolished the binding activity . When this mutant hrcA allele was expressed together with the wild-type allele within the same cell, it conferred a dominant-negative phenotype to the cells underlining that these amino acid residues are crucial for specific DNA binding and that HrcA binds to DNA in an oligomeric form.

FEMS Microbiol Lett, 2003 Jun 6, 223(1), 47 - 51
Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03; Cho SJ et al.; The Bacillus subtilis strain KS03 was isolated, and identified as a biological control agent that inhibits the anthracnose disease fungus Gloeosporium gloeosporioides . The antifungal compound was purified from its culture broth through butanol extraction, diethylaminoethyl (DEAE) Sepharose CL-6B chromatography, and preparative thin layer chromatography . Tandem mass spectrometric analyses (MS/MS), with matrix-assisted laser desorption ionization (MALDI) time-of-fight/time-of-flight (TOF/TOF) mass spectrometry, showed that the antifungal compound was iturin A, a cyclic lipopeptide antibiotic . The major compound, with a molecular mass of 1042 Da, was identified as iturin A(2).

J Mol Biol, 2003 Jun 20, 329(5), 973 - 82
Crystal structure of the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Bacillus subtilis; Steinbacher S et al.; Two types of isopentenyl diphosphate:dimethylallyl diphosphate isomerases (IDI) have been characterized at present . The long known IDI-1 is only dependent on divalent metals for activity, whereas IDI-2 requires a metal, FMN and NADPH . Here, we report the first structure of an IDI-2 from Bacillus subtilis at 1.9A resolution in the ligand-free form and of the FMN-bound form at 2.8A resolution . The enzyme is an octamer that forms a D4 symmetrical open, cage-like structure . The monomers of 45 kDa display a classical TIM barrel fold . FMN is bound only with very moderate affinity and is therefore completely lost during purification . However, the enzyme can be reconstituted in the crystals by soaking with FMN . Three glycine-rich sequence stretches that are characteristic for IDI-2 participate in FMN binding within the interior of the cage . Regions harboring strictly conserved residues that are implicated in substrate binding or catalysis remain largely disordered even in the presence of FMN.

J Agric Food Chem, 2003 Jun 18, 51(13), 3770 - 5
TAXI type endoxylanase inhibitors in different cereals; Goesaert H et al.; An affinity-based purification procedure with the immobilized family 11 Bacillus subtilis endoxylanase XynA allowed us to obtain high yields of highly pure endoxylanase inhibitor fractions from rye, barley, and durum wheat . In contrast, no inhibitors interacting with the B . subtilis endoxylanase affinity column are present in corn, buckwheat, rice, and oats . Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and inhibitor specificity showed that the isolated inhibitors belonged to the TAXI endoxylanase inhibitor family, thus providing a view on the diversity of this cereal inhibitor family . The isolated inhibitors are basic proteins of ca . 40 kDa, occurring in two molecular forms, with pI values of ca . 8.5 (durum wheat) and ca . 9.0 (rye, barley) . They are, in general, strong inhibitors of family 11 endoxylanases but not of family 10 endoxylanases . Because cereal endogenous endoxylanases belong to the latter family, this probably indicates that they do not influence cereal metabolic processes . For the first time, endoxylanase inhibitors, similar to TAXI I and TAXI II from wheat, were isolated from durum wheat and characterized . For each cereal, high-resolution cation exchange chromatography revealed the presence of multiple isoinhibitors, each of which occurs in two molecular forms . However, in durum wheat and barley, a single isoform is predominantly present.

Chembiochem, 2003 Jun 6, 4(6), 485 - 93
A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases; Henke E et al.; Carboxylesterases containing the sequence motif GGGX catalyze the hydrolysis of esters of chiral tertiary alcohols, albeit with only low to moderate enantioselectivity, for three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate) . In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity for this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity . For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model . In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this amino acid for an alanine residue led to a sixfold increase of enantioselectivity (E = 19) towards 2-phenyl-3-butin-2-yl acetate . However, the effect of this mutation is specific: the same mutant had the opposite enantiopreference towards the substrate linalyl acetate . Thus, depending on the substrate structure, the same mutant has either increased enantioselectivity or opposite enantiopreference compared to the wild-type enzyme.

Microbiol Mol Biol Rev, 2003 Jun, 67(2), 157 - 74, table of contents
RNA processing and degradation in Bacillus subtilis; Condon C; This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli . A comparison of the genomes from the two organisms reveals that B . subtilis has a very different selection of RNases available for RNA maturation . Of 17 characterized ribonuclease activities thus far identified in E . coli and B . subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases . Some enzymes essential for cell viability in E . coli, such as RNase E and oligoribonuclease, do not have homologs in B . subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other . The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B . subtilis mRNAs . The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay . While RNase E binds to the 5' end and catalyzes the rate-limiting cleavage of the majority of E . coli RNAs by looping to internal sites, the equivalent nuclease in B . subtilis, although not yet identified, is predicted to scan or track from the 5' end . RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B . subtilis mRNAs appears incapable of direct entry . Thus, unlike E . coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5' end can have very long half-lives even if the RNA is not protected by translation.

J Biol Chem, 2003 Aug 22, 278(34), 32219 - 26 Epub 2003 Jun 06.
The functional glycosyltransferase signature sequence of the human beta 1,3-glucuronosyltransferase is a XDD motif; Gulberti S et al.; The human beta 1,3-glucuronosyltransferase I (GlcAT-I) is the key enzyme responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide of proteoglycans (GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta 1-O-serine) . We have investigated the role of aspartate residues Asp194-Asp195-Asp196 corresponding to the glycosyltransferase DXD signature motif, in GlcAT-I function by UDP binding experiments, kinetic analyses, and site-directed mutagenesis . We presented the first evidence that Mn2+ is not only essential for GlcAT-I activity but is also required for cosubstrate binding . In agreement, kinetic studies were consistent with a metal-activated enzyme model whereby activation probably occurs via binding of a Mn2+.UDP-GlcA complex to the enzyme . Mutational analysis showed that the Asp194-Asp195-Asp196 motif is a major element of the UDP/Mn2+ binding site . Furthermore, determination of the individual role of each aspartate showed that substitution of Asp195 as well as Asp196 to alanine strongly impaired GlcAT-I activity, whereas Asp194 replacement produced only a moderate alteration of the enzyme activity . These findings along with molecular modeling and three-dimensional structure comparison of the GlcAT-I catalytic center with that of the Bacillus subtilis glycosyltransferase SpsA provided evidence that the interactions of Asp195 with the ribose moiety of UDP and of Asp196 with the metal cation Mn2+ were crucial for GlcAT-I function . Altogether, these results indicated that, similarly to the SpsA enzyme, the nucleotide binding site of GlcAT-I contains a XDD motif rather than a DXD motif.

Mol Microbiol, 2003 Jun, 48(6), 1491 - 500
Consequences of reductive evolution for gene expression in an obligate endosymbiont; Wilcox JL et al.; The smallest cellular genomes are found in obligate symbiotic and pathogenic bacteria living within eukaryotic hosts . In comparison with large genomes of free-living relatives, these reduced genomes are rearranged and have lost most regulatory elements . To test whether reduced bacterial genomes incur reduced regulatory capacities, we used full-genome microarrays to evaluate transcriptional response to environmental stress in Buchnera aphidicola, the obligate endosymbiont of aphids . The 580 genes of the B . aphidicola genome represent a subset of the 4500 genes known from the related organism, Escherichia coli . Although over 20 orthologues of E . coli heat stress (HS) genes are retained by B . aphidicola, only five were differentially expressed after near-lethal heat stress treatments, and only modest shifts were observed . Analyses of upstream regulatory regions revealed loss or degradation of most HS (sigma32) promoters . Genomic rearrangements downstream of an intact HS promoter yielded upregulation of a functionally unrelated and an inactivated gene . Reanalyses of comparable experimental array data for E . coli and Bacillus subtilis revealed that genome-wide differential expression was significantly lower in B . aphidicola . Our demonstration of a diminished stress response validates reports of temperature sensitivity in B . aphidicola and suggests that this reduced bacterial genome exhibits transcriptional inflexibility.

Cell, 2003 May 30, 113(5), 577 - 86
Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria; Mandal M et al.; Riboswitches are metabolite binding domains within certain messenger RNAs that serve as precision sensors for their corresponding targets . Allosteric rearrangement of mRNA structure is mediated by ligand binding, and this results in modulation of gene expression . We have identified a class of riboswitches that selectively recognizes guanine and becomes saturated at concentrations as low as 5 nM . In Bacillus subtilis, this mRNA motif is located on at least five separate transcriptional units that together encode 17 genes that are mostly involved in purine transport and purine nucleotide biosynthesis . Our findings provide further examples of mRNAs that sense metabolites and that control gene expression without the need for protein factors . Furthermore, it is now apparent that riboswitches contribute to the regulation of numerous fundamental metabolic pathways in certain bacteria.

Appl Microbiol Biotechnol, 2003 Nov, 63(1), 35 - 41 Epub 2003 May 29.
Development, validation, and applications of a new laboratory-scale indirect impedancemeter for rapid microbial control; Ribeiro T et al.; We introduce a new laboratory-scale impedance-meter which is specially intended for indirect technique . It consists of a software system enabling data acquisition via a connected bus which is wired to the measuring cells . These measuring cells are individual impedance-meters that can be activated independently of one another . In the current configuration, the device is slightly affected by temperature, but it can register as little as 10.9 micromol of CO(2) . With Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae cultures, the conductance responses were highly replicable and repeatable for inocula concentrations of 1-10(8) colony-forming units (CFU) ml(-1) . The main use for such devices could be the detection of contamination in foodstuffs . Several of these foodstuffs, when incubated at 37 degrees C, spontaneously release quite large amounts of CO(2) . Our impedance meter, however, was able to detect an E . coli presence in canned French beans at 2.35 x 10(-2) CFU ml(-1) and a S . cerevisiae contamination of apple puree in glass jars at 6.1 x 10(-3) CFU ml(-1) . The conductance response and the detection time (the time needed for a significant change in conductance) were correlated to the concentration of ampicillin (an antibiotic added to E . coli cultures) . The device is thus able to detect the presence of inhibitory compounds in milk or other foodstuffs . Some industrial assays are in process to complement these laboratory tests . Compared with other available techniques for CO(2) measurement (manometry, infrared, radioactive labeling), the technique put forward here appears to be the best compromise between sensitivity, technical constraints, and cost . A commercial version of the impedance meter would enable routine measurements in the quality control of foodstuffs, pharmaceuticals, cosmetics and in R&D laboratories.

Biosens Bioelectron, 2003 Aug 1, 18(8), 1023 - 9
Co-immobilized microbial biosensor for BOD estimation based on sol-gel derived composite material; Jia J et al.; A novel type of biochemical oxygen demand (BOD) biosensor was developed for water monitor, based on co-immobilizing of Trichosporon cutaneum and Bacillus subtilis in the sol-gel derived composite material which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)) . Factors that influence the performance of the resulting biosensor were examined . The biodegradable substrate spectrum could be expanded by the co-immobilized microorganisms . The biosensor prepared also exhibited good reproducibility and long-term stability . Good agreement was obtained between the results of the sensor BOD measurement and those obtained from conventional BOD(5) method for water samples.

Biochemistry, 2003 Jun 10, 42(22), 6762 - 71
Regulation and mutational analysis of the HPr kinase/phosphorylase from Bacillus subtilis; Pompeo F et al.; In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the HPr kinase/phosphorylase (HprK/P) . It has recently been shown that HprK/P could catalyze the phosphorylation of the protein HPr by using pyrophosphate (PP(i)) as a phosphate donor instead of ATP . Here we showed that, as for ATP, PP(i) binds to the enzyme with strong positive cooperativity . However, in contrast to ATP, PP(i) binding does not modify the fluorescence properties of the unique Trp residue of Bacillus subtilis HprK/P . In addition, to understand how two conserved motifs, namely, the P-loop and the specific signature of this family, participate in the three enzymatic activities of HprK/Ps (ATP-kinase, PP(i)-kinase, and phosphorylase), several site-directed mutants were generated . Whereas the three activities are mediated by the P-loop which is directly involved in the binding of ATP, PP(i), or Pi, the signature motif seems to be involved preferentially in the dephosphorylation reaction . On the basis of these results, we propose a model in which the binding of the allosteric activator FBP induces a conformational change of a central loop located above the active site of HprK/P, thereby allowing the ATP binding . However, this conformational change is not required for the binding of PP(i).

Biometals, 2003 Dec, 16(4), 497 - 505
Bacillus subtilis CPx-type ATPases: characterization of Cd, Zn, Co and Cu efflux systems; Gaballa A et al.; Metal ion homeostasis requires the balanced expression of metal ion uptake systems, when metals are limiting, and corresponding efflux or storage systems, when metals are in excess . CPx-type ATPases are a family of membrane-bound transporters that often function to export toxic metals from cells . The Bacillus subtilis genome encodes three CPx-type ATPases: zosA, yvgW and yvgX . We demonstrate that yvgW and yvgX encode CadA and CopA, respectively, and that these genes function in metal ion resistance . A cadA mutant was sensitive to Cd(II), Zn(II) and Co(II), but not copper . Transcription of cadA initiates from a single, sigmaA-type promoter and was induced by Cd(II), Zn(II), and Co(II) . The adjacent copZA operon is expressed as a bicistronic transcript from a sigmaA-type promoter and is selectively induced by copper . Mutation of either copZ, encoding a metallochaperone, or copA sensitizes the cells to copper but not to other metal ions.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1090 - 2 Epub 2003 May 23.
Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P; Bailey S et al.; The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified . The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6(1)22/P6(5)22, but the crystals diffract to only 3.4 A and are poorly reproducible . Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues . Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation . The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P2(1)2(1)2 or P2(1)2(1)2(1), with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 A, with diffraction to 2.4 A on a synchrotron source and with greatly improved reproducibility . Calculation of V(M) values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47% . A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.

Antonie Van Leeuwenhoek, 2003, 83(4), 361 - 8
phoR1, a gene encoding a new histidine protein kinase Myxococcus xanthus; Martinez-Canamero M et al.; A soil bacterium able to undergo multicellular development and a coordinated gliding in swarms, requires an accurate regulatory network of phosphorelay proteins . Inorganic phosphate is a limiting nutrient in soil and its importance in regulation is critical . As a step towards studying phosphate regulation and its influence in the developmental process in this bacterium, we screened a Myxococcus xanthus library for clones with phosphatase activity, and found four different ones . The deduced sequence of one of the cloned inserts is similar to that of the classic transmembrane histidine protein kinase of the sensor family of the two-component signal transduction systems with a high sequence similarity to the sensor kinase in the Pho regulon of Bacillus subtilis PhoR . This gene has been named phoR1 and its deduced amino acid sequence consists of 455 residues with a predicted molecular mass of 48.5 kDa . The M . xanthus PhoR1 deduced sequence contains all the characteristic histidine protein kinase motifs in the same order and with the same spacing . A hydropathy profile indicates two membrane-spanning segments located at the extreme N-terminus, according to the putative sensor role of this domain . A gene-disrupted mutant is unable to produce normal mature fruiting bodies and produces fewer spores.

J Bacteriol, 2003 Jun, 185(12), 3491 - 8
SigM, an extracytoplasmic function sigma factor of Bacillus subtilis, is activated in response to cell wall antibiotics, ethanol, heat, acid, and superoxide stress; Thackray PD et al.; The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress . It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl . The promoter region of the sigM operon contains two promoters; one (P(A)) is sigma A dependent, and the other (P(M)) is sigma M dependent . These have been placed separately at the amy locus, directing expression of a lacZ reporter gene . Only the P(M) fusion responded to salt induction . This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 micro g per ml), and by heat shock of 50 degrees C for 10 min . It was very strongly induced by acid (pH 4.3) and 80 micro M paraquat, but after a 15- to 30-min delay . There was no induction by alkali (pH 9), 5 mM H(2)O(2), the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 micro M monensin . In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol . Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 micro g of vancomycin per ml . Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another . Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin . yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction . YraA protein was extremely important to acid survival-a mutation in yraA, like a sigM mutation, resulted in the failure of B . subtilis to grow at pH 4.3 . Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.

J Insect Physiol, 2002 Jul, 48(7), 715 - 723
Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae; Zakarian RJ et al.; Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides . The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria . The total number of bacteria adhering to all the haemocytes on the slides declined . Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides . Purified apoLp-III bound to B . subtilis . ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min . ApoLp-III bound to B . subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.

J Insect Physiol, 2002 Sep, 48(9), 903 - 914
Iron contributes to the antibacterial functions of the haemolymph of Galleria mellonella; Dunphy GB et al.; Studies with Galleria mellonella larvae and the iron chelating agent EDDA showed that iron was essential for the removal of dead Xenorhabdas nematophila and Bacillus subtilis from the haemolymph . The delay in removal of the bacteria from the iron-restricted haemolymph was attributed to reduced adhesiveness of the haemocytes and prophenoloxidase activity . Iron augmentation returned these activities to control levels . Whereas dead B . subtilis had no effect on the concentration of ferrozine-detectable iron (henceforth iron) in the haemolymph, dead X . nematophila was associated with substantially lower levels of iron as the number of damaged haemocytes increased . Haemocyte lysate lowered the concentrations of iron in both FeCl(3) solutions and deproteinized larval serum independent of serum lipids . Haemocyte lysate added to tryptic soybroth lowered the level of iron and limited the growth of X . nematophila . X . nematophila limited iron availability in the plasma by releasing lipopolysaccharides; such a mechanism may be a means of impairing the antimicrobial defences of the insects.

Protein Expr Purif, 2003 Jun, 29(2), 259 - 64
DNA gyrase and DNA topoisomerase of Bacillus subtilis: expression and characterization of recombinant enzymes encoded by the gyrA, gyrB and parC, parE genes; Barnes MH et al.; Bacillus subtilis Bs gyrA and gyrB genes specifying the DNA gyrase subunits, and parC and parE genes specifying the DNA topoisomerase IV subunits, have been separately cloned and expressed in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins . Purification of the gyrA and gyrB subunits together resulted in predominantly two bands at molecular weights of 94 and 73kDa; purification of the parC and parE subunits together resulted in predominantly two bands at molecular weights of 93 and 75kDa, as predicted by their respective sequences . The ability of the subunits to complement their partner was tested in an ATP-dependent decatenation/supercoiling assay system . The results demonstrated that the DNA gyrase and the topoisomerase IV subunits produce the expected supercoiled DNA and relaxed DNA products, respectively . Additionally, inhibition of these two enzymes by fluoroquinolones has been shown to be comparable to those of the DNA gyrases and topoisomerases of other bacterial strains . In sum, the biological and enzymatic properties of these products are consistent with their authenticity as DNA gyrase and DNA topoisomerase IV enzymes from B . subtilis.

J Biomol NMR, 2003 Jun, 26(2), 167 - 79
A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy; Eletsky A et al.; The assignment of the aliphatic (13)C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) (13)C-start and (13)C-observe NMR experiments . These experiments start with (13)C excitation and end with (13)C observation while relying on the long transverse relaxation times of (13)C spins in uniformly deuterated and (13)C,(15)N-labeled large proteins . Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce (13)C T(1) relaxation times with little effect on (13)C T(2) relaxation times . Such 2D (13)C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain (1)H spins, the transfer of the side-chain spin polarization to the (1)H(N) spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains . We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic (13)C resonances, the side-chain (1)H chemical shifts can be assigned in a semiautomatic fashion using 3D (15)N-resolved and (13)C-resolved NOESY experiments measured with a randomly partially protonated protein sample . We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the (1)H,(1)H NOEs in combination with multiple residual dipolar couplings between adjacent (13)C spins determined with 2D (13)C-only experiments.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7865 - 70 Epub 2003 May 23.
Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles; Shih YL et al.; The MinCDE proteins of Escherichia coli are required for proper placement of the division septum at midcell . The site selection process requires the rapid oscillatory redistribution of the proteins from pole to pole . We report that the three Min proteins are organized into extended membrane-associated coiled structures that wind around the cell between the two poles . The pole-to-pole oscillation of the proteins reflects oscillatory changes in their distribution within the coiled structure . We also report that the E . coli MreB protein, which is required for maintaining the rod shape of the cell, also forms extended coiled structures, which are similar to the MreB structures that have previously been reported in Bacillus subtilis . The MreB and MinCDE coiled arrays do not appear identical . The results suggest that at least two functionally distinct cytoskeletal-like elements are present in E . coli and that structures of this type can undergo dynamic changes that play important roles in division site placement and possibly other aspects of the life of the cell.

In Vitro Cell Dev Biol Anim, 2002 Nov-Dec, 38(10), 572 - 81
Growth of an ovarian cell line of Galleria mellonella and its response to immune-inducing factors; Zakarian RJ et al.; Antibacterial proteins are produced in the reproductive tracts of some insect species . The advent of a pupal ovarian cell line of the lepidopteran Galleria mellonella offered an opportunity for exploring the use of ovarian tissue culture to induce antimicrobial proteins in lieu of the larvae . The ovarian cell growth rates and cell yields were maximized by adjusting Grace's medium to pH 6.5, adding 15% (v/v) qualified heat-inactivated fetal calf serum, and lowering the sucrose concentration to 9.3 g/L . Five cell forms and biochemical profiles of the collective cell types were analyzed throughout the culture growth cycle . The final modified culture medium did not affect morphogenesis, whereas it increased the culture growth rate by 50% and the final cell yield threefold . The molting and immunoprotein-inducing hormone, 20-hydroxyecdysone, increased culture growth rate and altered the levels of cell types A and D . Neither 20-hydroxyecdysone nor the larval immunizing agents, apolipophorin-III or Bacillus subtilis, in combination or alone, induced antibacterial activity . The bacterium did induce immunity in both larval and adult stages.

J Biol Inorg Chem, 2003 Apr, 8(4), 452 - 8 Epub 2003 Jan 18.
Metal binding to Bacillus subtilis ferrochelatase and interaction between metal sites; Lecerof D et al.; Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX . The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation . In this work we demonstrate that Zn(2+), which is commonly used as substrate in assays of the ferrochelatase reaction, and Cd(2+), an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft . On the other hand, Mg(2+), which has been shown to bind close to the surface at 7 A from His183, was largely absent from its site . Activity measurements demonstrate that Mg(2+) has a stimulatory effect on the enzyme, lowering K(M) for Zn(2+) from 55 to 24 micro M . Changing one of the Mg(2+) binding residues, Glu272, to serine abolishes the effect of Mg(2+) . It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin . Metal binding to the Mg(2+) site may stimulate metal release from the protein ligands and its insertion into the porphyrin.

J Biochem (Tokyo), 2003 Apr, 133(4), 475 - 83
Transcriptional, functional and cytochemical analyses of the veg gene in Bacillus subtilis; Fukushima T et al.; A Bacillus subtilis veg mutant exhibited a small reduction of absorbance, a large reduction of hexosamine release, and slow dipicolinic acid release from spores during germination with L-alanine as a germinant . But veg spores exhibited normal resistance to chloroform, 2-propanol, lysozyme, and heat . SDS-polyacrylamide gel electrophoresis of spore coat proteins revealed no difference in coat proteins between the wild type and the veg mutant . Northern and veg-lacZ fusion analyses indicated that the veg gene is transcribed in both the vegetative growth and sporulation phases, and primer extension analysis indicated an identical transcriptional start point in both phases . The upstream sequence suggests that veg is transcribed by Esigma(A) RNA polymerase . Veg-GFP fusion protein was detected for vegetative cells and spores, but the fluorescence of mother cells disappeared completely in the late sporulation phase . Decoated spores containing Veg-GFP exhibited a strong green fluorescence in the core, but much weaker fluorescence in the cortex.

J Biochem (Tokyo), 2003 Mar, 133(3), 295 - 302
Involvement of ClpX protein in the post-transcriptional regulation of a competence specific transcription factor, ComK protein, of Bacillus subtilis; Nanamiya H et al.; ComK protein of Bacillus subtilis positively regulates the transcription of several late competence genes as well as comK itself . We constructed a clpX disrupted mutant of B . subtilis and studied its effect on the regulation of ComK activation . When Pspac, which controls the comK gene in a multicopy plasmid, was induced by the addition of IPTG, comK transcripts were detected in both the clpX mutant and the wild type . However, the ComK protein could not be detected in the clpX disrupted mutant . To obtain further information, we constructed several comK-lacZ translational fusions covering different lengths of the comK gene, whose transcription is controlled by an IPTG inducible Pspac promoter . We found that both the expression of comK-lacZ directed beta-galactosidase and the accumulation of ComK-LacZ fused protein, derived from the fusion containing the entire comK open reading frame, were extremely reduced in the clpX mutant compared with the wild type, while the accumulation of comK-lacZ transcripts in the clpX mutant after the addition of IPTG was about half that in the clpX+ background . On the other hand, transcription, translation and activity of comK-lacZ were detected in both the clpX mutant and the wild type when the comK-lacZ fusion lacking the 3' region of the comK gene was induced . These results indicate that ClpX plays an important role in the regulation of ComK at the post-transcriptional level.

RNA, 2003 Jun, 9(6), 640 - 3
On the occurrence of the T-loop RNA folding motif in large RNA molecules; Krasilnikov AS et al.; The T-loop RNA folding motif may be considered as a five-nucleotide motif composed of a U-turn flanked by a noncanonical base pair . It was recently proposed that the flanking noncanonical base pair is always a UA trans Watson-Crick/Hoogsteen base pair stacked on a Watson-Crick base pair on one side . Here we show that structural analysis of several large RNA molecules, including the recently solved crystal structure of the specificity domain of Bacillus subtilis RNase P, combined with sequence analysis, indicates a broader sequence consensus for the motif . Additionally, we show that the flanking base pair does not necessarily stack on a Watson-Crick base pair and the 3' terminus of the five-nucleotide motif is often followed by a sharp turn in the phosphate backbone rather than just a bulged base or bases.

Prikl Biokhim Mikrobiol, 2003 May-Jun, 39(3), 313 - 7
{Specific toxic effects of 2,4,6-trinitrotoluene on Bacillus subtilis SK1}; Kurinenko BM et al.; Using Bacillus subtilis SK1 as an example, it was demonstrated for the first time that 2,4,6-trinitrotoluene (TNT) transformation pathways change with TNT concentration . The growth of cultured B . subtilis SK1, delayed at 20 mg/l TNT (minimum toxic concentration), was resumed following TNT transformation . Aromatic amines were predominant metabolites detected in the culture medium at early stages of TNT transformation . The culture growth was completely inhibited by 200 mg/l TNT . As this took place, nitrites accumulated in the culture medium.

J Bacteriol, 2003 Jun, 185(11), 3317 - 24
Novel developmental genes, fruCD, of Myxococcus xanthus: involvement of a cell division protein in multicellular development; Akiyama T et al.; Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation . In the present study, two novel developmental genes, fruC and fruD, of M . xanthus were identified and characterized . The FruD protein has significant amino acid sequence similarity to the DivIVA proteins of many bacteria including Bacillus subtilis . Vegetative cells of the fruD mutant exhibited a filamentous phenotype . The fruC and fruD mutants displayed similar delayed-development phenotypes . The formation of tightly aggregated mounds by fruC and fruD mutants was slower than that by the wild-type strain . Spore formation by the fruC and fruD mutants initiated after 30 h poststarvation, whereas wild-type M . xanthus initiated spore formation after 18 h . The fruCD genes were constitutively expressed as an operon during vegetative growth and development . S1 mapping revealed that transcription initiation sites of the fruCD operon were located 114 (P1) and 55 bp (P2) upstream of the fruC initiation codon . Only the P1 promoter was active during vegetative growth, while both the P1 and P2 promoters were active during development . The FruD protein was produced as a cytoplasmic protein and formed an oligomer during vegetative growth and development.

Metab Eng, 2003 Jan, 5(1), 49 - 55
Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis; Zamboni N et al.; We present redirection of electron flow to more efficient proton pumping branches within respiratory chains as a generally applicable metabolic engineering strategy, which tailors microbial metabolism to the specific requirements of high cell density processes by improving product and biomass yields . For the example of riboflavin production by Bacillus subtilis, we reduced the rate of maintenance metabolism by about 40% in a cytochrome bd oxidase knockout mutant . Since the putative Yth and the caa(3) oxidases were of minor importance, the most likely explanation for this improvement is translocation of two protons per transported electron via the remaining cytochrome aa(3) oxidase, instead of only one proton via the bd oxidase . The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and B . subtilis biomass in fed-batch cultures.

BMC Bioinformatics . 2003 May 16;4(1):18.
Genome wide identification of regulatory motifs in Bacillus subtilis; Mwangi MM et al.; BACKGROUND: To explain the vastly different phenotypes exhibited by the same organism under different conditions, it is essential that we understand how the organism's genes are coordinately regulated . While there are many excellent tools for predicting sequences encoding proteins or RNA genes, few algorithms exist to predict regulatory sequences on a genome wide scale with no prior information . RESULTS: To identify motifs involved in the control of transcription, an algorithm was developed that searches upstream of operons for improbably frequent dimers . The algorithm was applied to the B . subtilis genome, which is predicted to encode for approximately 200 DNA binding proteins . The dimers found to be over-represented could be clustered into 317 distinct groups, each thought to represent a class of motifs uniquely recognized by some transcription factor . For each cluster of dimers, a representative weight matrix was derived and scored over the regions upstream of the operons to predict the sites recognized by the cluster's factor, and a putative regulon of the operons immediately downstream of the sites was inferred . The distribution in number of operons per predicted regulon is comparable to that for well characterized transcription factors . The most highly over-represented dimers matched sigmaA, the T-box, and sigmaW sites . We have evidence to suggest that at least 52 of our clusters of dimers represent actual regulatory motifs, based on the groups' weight matrix matches to experimentally characterized sites, the functional similarity of the component operons of the groups' regulons, and the positional biases of the weight matrix matches . All predictions are assigned a significance value, and thresholds are set to avoid false positives . Where possible, we examine our false negatives, drawing examples from known regulatory motifs and regulons inferred from RNA expression data . CONCLUSIONS: We have demonstrated that in the case of B . subtilis our algorithm allows for the genome wide identification of regulatory sites . As well as recovering known sites, we predict new sites of yet uncharacterized factors . Results can be viewed at http://www.physics.rockefeller.edu/~mwangi/.

Appl Microbiol Biotechnol, 2003 May, 61(4), 329 - 35 Epub 2003 Mar 05.
Significantly enhanced stability of glucose dehydrogenase by directed evolution; Baik SH et al.; An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution . The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes . Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the p Trc99A expression vector and expressed in the host, Escherichia coli . In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B . megaterium IWG3, was also subjected to directed evolution by the family shuffling method . A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries . This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes . The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl . In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66 degrees C), while the half-life of isolated mutant DN-46 was 540 min at 66 degrees C, i.e., 415-fold more thermostable than mutant F20 . The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.

J Protein Chem, 2003 Jan, 22(1), 51 - 60
Stability studies on a lipase from Bacillus subtilis in guanidinium chloride; Acharya P et al.; Lipase from Bacillus subtilis is a "lidless" lipase that does not show interfacial activation . Due to exposure of the active site to solvent, the lipase tends to aggregate . We have investigated the solution properties and unfolding of the lipase in guanidinium chloride (GdmCl) to understand its aggregation behavior and stability . Dynamic light scattering (DLS), near- and far-UV circular dichroism, activity and intrinsic fluorescence of lipase suggest that the protein undergoes unfolding between 1 M and 2 M GdmCl . The polarity sensitive dye, 1,1',-bis-(4anilino)naphthalene-5,5"-disulfonic acid (bis-ANS), a probe for hydrophobic pockets, binds cooperatively to the native lipase . An intermediate populated in 1.75 M GdmCl that strongly binds bis-ANS was identified . Tendency of the native protein to aggregate in solution and specific binding to bis-ANS confirms that the lipase has exposed hydrophobic pockets and this surface hydrophobicity strongly influences the unfolding pathway of the lipase in GdmCl.

J Biol Chem, 2003 Jul 25, 278(30), 28173 - 80 Epub 2003 May 08.
Crystal structures of RbsD leading to the identification of cytoplasmic sugar-binding proteins with a novel folding architecture; Kim MS et al.; RbsD is the only protein whose biochemical function is unknown among the six gene products of the rbs operon involved in the active transport of ribose . FucU, a paralogue of RbsD conserved from bacteria to human, is also the only protein whose function is unknown among the seven gene products of the l-fucose regulon . Here we report the crystal structures of Bacillus subtilis RbsD, which reveals a novel decameric toroidal assembly of the protein . Nuclear magnetic resonance and other studies on RbsD reveal that the intersubunit cleft of the protein binds specific forms of d-ribose, but it does not have an enzyme activity toward the sugar . Likewise, FucU binds l-fucose but lacks an enzyme activity toward this sugar . We conclude that RbsD and FucU are cytoplasmic sugar-binding proteins, a novel class of proteins whose functional role may lie in helping influx of the sugar substrates.

Clin Diagn Lab Immunol, 2003 May, 10(3), 367 - 75
Production of Chlamydia pneumoniae proteins in Bacillus subtilis and their use in characterizing immune responses in the experimental infection model; Airaksinen U et al.; Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious . To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium . The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the alpha-amylase gene from Bacillus amyloliquefaciens . C . pneumoniae genes were readily expressed in B . subtilis under the control of the alpha-amylase promoter . The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography . The Omp2 was separated as an insoluble fraction after 8 M urea treatment . The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test . The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C . pneumoniae in mice . Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.

Dev Cell, 2003 May, 4(5), 663 - 72
FapR, a bacterial transcription factor involved in global regulation of membrane lipid biosynthesis; Schujman GE et al.; Bacterial cells exert exquisite control over the biosynthesis of their membrane lipids, but the mechanisms are obscure . We describe the identification and purification from Bacillus subtilis of a transcription factor, FapR, that controls the expression of many genes involved in fatty acid and phospholipid metabolism (the fap regulon) . Expression of this fap regulon is influenced by antibiotics that specifically inhibit the fatty acid biosynthetic pathway . We show that FapR negatively regulates fap expression and that the effects of antibiotics on fap expression are mediated by FapR . We further show that decreasing the cellular levels of malonyl-CoA, an essential molecule for fatty acid elongation, inhibits expression of the fap regulon and that this effect is FapR dependent . Our results indicate that control of FapR by the cellular pools of malonyl-CoA provides a mechanism for sensing the status of fatty acid biosynthesis and to adjust the expression of the fap regulon accordingly.

J Biol Chem, 2003 Jul 18, 278(29), 26677 - 86 Epub 2003 May 07.
Engineering of a staphylokinase-based fibrinolytic agent with antithrombotic activity and targeting capability toward thrombin-rich fibrin and plasma clots; Lian Q et al.; Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications . To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered . Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain . This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach . After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules . This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity . The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase . In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase . Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.

Spectrochim Acta A Mol Biomol Spectrosc, 2003 Jun, 59(8), 1751 - 5
Applications of electron paramagnetic resonance spectroscopy to study interactions of metalloenzymes with Cu(II) ions; Zheng XF et al.; In the past, the method of reconstitution was used to investigate the interaction between metalloenzymes (containing Zn(II)) and metal ions . In this paper, electron paramagnetic resonance (EPR) has been employed to firstly study the direct interactions between Bacillus subtilis neutral proteinase (BSNP), nuclease P1 and Cu(II) ions added in aqueous solution, respectively . These results show that a dynamic equilibrium exists between the Zn(II) in the active site of native enzymes and the added Cu(II), the added Cu(II) partly replaces the Zn(II), forming Cu(II)-enzyme derivatives . As a result, the activity of the native enzymes is influenced . The influences of pH value on this kind of interaction have also been investigated, and the results demonstrate that the change of pH value has little influence on the system of nuclease P1, but has remarkable influence on BSNP . We firstly obtained the EPR spectra for Cu(II)-enzyme derivatives . In addition, the derivative of Cu(II)-BSNP exists in the solution with two different conformations (I type g(parallel)=2.34, A(parallel) (mT)=13.4; II type g(parallel)=2.25, A(parallel) (mT)=16.1), and this two conformations exchanged each other depending on pH.

Mycopathologia, 2003, 156(2), 81 - 5
Mycoflora of tuber surface of white yam (Dioscorea rotundata Poir) and postharvest control of pathogens with Bacillus subtilis; Okigbo RN; Bacillus subtilis (Enrenberg) Cohn was investigated for its antagonistic properties against surface mycoflora of yam (Dioscorea rotundata Poir) tubers in storage . Yam tubers inoculated with a spore suspension of B . subtilis in potato dextrose broth using a knapsack sprayer showed a drastic reduction in the range and number of mycoflora, including pathogens of the tuber surface in contrast to the control tubers, during the five-month storage period in a traditional yam barn . However, B . subtilis maintained a high frequency of occurrence during the same period . Botryodiploidia theobromae Pat, Fusarium moniliforme Wollen and Reink., Penicillium sclerotigenum Yamamoto, and Rhizoctonia sp . were displaced completely on the treated tubers . The antagonism of B . subtilis was so effective that the normal tuber surface mycoflora was greatly reduced throughout the storage period of five months by a simple initial application of the antagonist.

Appl Environ Microbiol, 2003 May, 69(5), 2491 - 7
Characterization of poly-gamma-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-gamma-glutamate; Kimura K et al.; Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a gamma-linkage, called poly-gamma-glutamate (gamma-PGA) . We identified and purified a monomeric 25-kDa degradation enzyme for gamma-PGA (designated gamma-PGA hydrolase, PghP) from bacteriophage PhiNIT1 in B . subtilis host cells . The monomeric PghP internally hydrolyzed gamma-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-gamma-glutamates . Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn(2+) or Mn(2+) ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme . The corresponding pghP gene was cloned and sequenced from the phage genome . The deduced PghP sequence (208 amino acids) with a calculated M(r) of 22,939 was not significantly similar to any known enzyme . Thus, PghP is a novel gamma-glutamyl hydrolase . Whereas phage PhiNIT1 proliferated in B . subtilis cells encapsulated with gamma-PGA, phage BS5 lacking PghP did not survive well on such cells . Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce gamma-PGA . Analogous to polysaccharide capsules, gamma-PGA appears to serve as a physical barrier to phage absorption . Phages break down the gamma-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.

Curr Opin Microbiol, 2003 Apr, 6(2), 181 - 5
The role of Fe-S proteins in sensing and regulation in bacteria; Kiley PJ et al.; Fe-S clusters are key to the sensing and transcription functions of three transcription factors, FNR, IscR and SoxR . All three proteins were discovered in Escherichia coli but experimental data and bioinformatic predictions suggest that homologs of these proteins exist in other bacterial species, highlighting the widespread nature of Fe-S-dependent regulatory networks . In addition, the nearly ubiquitous citric acid cycle enzyme, aconitase, plays a role in translational regulation in E . coli and Bacillus subtilis when it loses its Fe-S cluster . Although these regulatory proteins have the common feature of containing an Fe-S cluster, they differ in the physiological signals that they respond to . Therefore, these regulatory factors provide insights into the chemical versatility of Fe-S clusters.

J Bacteriol, 2003 May, 185(10), 3228 - 31
The Bacillus subtilis acyl lipid desaturase is a delta5 desaturase; Altabe SG et al.; Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions delta5, delta7, and delta9 (M . H . Weber, W . Klein, L . Muller, U . M . Niess, and M . A . Marahiel, Mol . Microbiol . 39:1321-1329, 2001) . Since this finding would have considerable importance in the double-bond positional specificity displayed by the B . subtilis acyl lipid desaturase, we have attempted to confirm this observation . We report that the double bond of UFAs synthesized by B . subtilis is located exclusively at the delta5 position, regardless of the growth temperature and the length chain of the fatty acids.

J Bacteriol, 2003 May, 185(10), 3218 - 22
Identification of the L-aspartate transporter in Bacillus subtilis; Lorca G et al.; YveA of Bacillus subtilis, a putative transporter of the amino acid/polyamine/organocation (APC) superfamily, is shown to mediate uptake of both L-aspartate and L-glutamate as well as having sensitivity to L-aspartate hydroxamate . This 14 TMS protein is the primary aspartate uptake system in B . subtilis and serves as the prototype for a new family within the APC superfamily.

J Bacteriol, 2003 May, 185(10), 3118 - 26
CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis; Bergara F et al.; Expression of the sigma(D)-dependent flagellin gene, hag, is repressed by the CodY protein in nutrient-rich environments . Analysis of a codY mutant bearing a hag-lacZ reporter suggests that the availability of amino acids in the environment is the specific signal that triggers this repression . Further, hag-lacZ expression appears to be sensitive to intracellular GTP levels, as demonstrated by increased expression upon addition of decoyinine . This result is consistent with the postulate that the availability of amino acids in the environment effects intracellular GTP levels through the stringent response . However, the levels of hag-lacZ measured upon the addition of subsets of amino acids suggest an additional mechanism(s) . CodY is a DNA binding protein that could repress flagellin expression directly by binding to the hag promoter region, or indirectly by binding to the fla/che promoter region that governs expression of the sigma(D) transcriptional activator required for hag gene expression . Using an electrophoretic mobility shift assay, we have demonstrated that purified CodY protein binds specifically to both the hag and fla/che promoter fragments . Additionally, CodY acts as a nutritional repressor of transcription from the fla/che promoter region that contains two functional promoters . CodY binds to both the sigma(D)- and sigma(A)-dependent promoters in this region, as demonstrated by DNase I footprint analyses . Footprint analyses of the hag gene demonstrated that CodY binds downstream of its sigma(D)-dependent promoter . Taken together, these results identify new members of the CodY regulon that encode motility functions in Bacillus subtilis and are controlled by the sigma(D) alternate sigma factor.

Genes Dev, 2003 May 1, 17(9), 1166 - 74
The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division; Fujita M et al.; Gene transcription at the onset of sporulation in Bacillus subtilis is governed by Spo0A, a member of the response regulator family of transcription factors . Spo0A is traditionally viewed as the master regulator for entry into development . We now report that Spo0A continues to function after the initiation phase of sporulation and that it becomes a cell-specific transcription factor when the sporangium is divided into a mother cell and forespore . We observed that (1) Spo0A and Spo0A-directed gene transcription reached high levels in the mother cell; (2) an activated form of Spo0A impaired sporulation when produced in the forespore but not when produced in the mother cell; and (3) an inhibitor of Spo0A called Spo0A-N impaired sporulation and Spo0A-directed transcription when produced in the mother cell but not when produced in the forespore . Spo0A-N, which corresponds to the NH(2)-terminal domain of Spo0A, was shown to compete with the full-length response regulator for phosphorylation by the phosphorelay protein Spo0B . We propose that Spo0A is the earliest-acting transcription factor in the mother-cell line of gene expression and that in terms of abundance and transcriptional activity Spo0A may function predominantly as a cell-specific regulatory protein.

Biosci Biotechnol Biochem, 2003 Mar, 67(3), 613 - 6
Cloning of structural gene of Deinococcus radiodurans UV-endonuclease beta; Kitayama S et al.; To characterize its enzymic property we cloned and sequenced the gene of Deinococcus radiodurans encoding UV-endonuclease beta, an alternative enzyme to UvrABC repairing damaged DNA . Amino acid substitutions were found in UV-sensitive mutants . The putative amino acid sequence had some similarity with those of eukaryotic UV-endonucleases and with a sequence found in a protein data base of Bacillus subtilis.

Prikl Biokhim Mikrobiol, 2003 Mar-Apr, 39(2), 194 - 8
{Effect of toxic concentrations of 2,4,6-trinirtotoluene on the physical properties and morphology of bacillus subtilis SK1}; Kurinenko BM et al.; It has been demonstrated for the first time that the toxic effect of 2,4,6-trinitrotoluene on the gram-positive strain Bacillus subtilis SK1 is accompanied by a decrease in the cell size and an increase in the refraction index (i.e., density) and thermostability of the culture . These data suggest that determination of kinetic parameters of bacteria growing under the conditions of toxic stress from optical density measurements may result in erroneous conclusions.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5682 - 7 Epub 2003 Apr 28.
Genomewide transcriptional changes associated with genetic alterations and nutritional supplementation affecting tryptophan metabolism in Bacillus subtilis; Berka RM et al.; DNA microarrays comprising approximately 95% of the Bacillus subtilis annotated protein coding ORFs were deployed to generate a series of snapshots of genomewide transcriptional changes that occur when cells are grown under various conditions that are expected to increase or decrease transcription of the trp operon segment of the aromatic supraoperon . Comparisons of global expression patterns were made between cells grown in the presence of indole acrylic acid, a specific inhibitor of tRNA(Trp) charging; cells deficient in expression of the mtrB gene, which encodes the tryptophan-activated negative regulatory protein, TRAP; WT cells grown in the presence or absence of two or three of the aromatic amino acids; and cells harboring a tryptophanyl tRNA synthetase mutation conferring temperature-sensitive tryptophan-dependent growth . Our findings validate expected responses of the tryptophan biosynthetic genes and presumed regulatory interrelationships between genes in the different aromatic amino acid pathways and the histidine biosynthetic pathway . Using a combination of supervised and unsupervised statistical methods we identified approximately 100 genes whose expression profiles were closely correlated with those of the genes in the trp operon . This finding suggests that expression of these genes is influenced directly or indirectly by regulatory events that affect or are a consequence of altered tryptophan metabolism.

Mol Cell, 2003 Apr, 11(4), 1009 - 20
A two-protein strategy for the functional loading of a cellular replicative DNA helicase; Velten M et al.; The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms . We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB . We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA . Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase . We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.

Can J Microbiol, 2003 Feb, 49(2), 71 - 7
Multidrug resistant phenotype of Bacillus subtilis spontaneous mutants isolated in the presence of puromycin and lincomycin; Murata M et al.; Spontaneous mutants were isolated by growing Bacillus subtilis 168 in the presence of high concentrations of puromycin and lincomycin . These mutants showed increased resistance to several drugs other than these two drugs . The ImrAB genes, which encode a transcriptional repressor and a drug efflux protein of the major facilitator superfamily, were involved in this phenotype . Northern hybridization analysis showed that the expression of ImrAB gene increased more than 30-fold . The following two types of mutations were found to be responsible for the multidrug resistant phenotype: (i) a nucleotide replacement in the region between the promoter and initiation codon of ImrA and (ii) nucleotide replacements that resulted in amino acid replacements in the LmrA protein . The results indicate that LmrB is a multidrug resistant protein and that LmrA is a repressor, which autogenously represses the transcription of the ImrAB operon.

Planta Med, 2003 Apr, 69(4), 356 - 60
Eremophilenolides and other constituents from the roots of Ligularia sagitta; Li XQ et al.; Chemical investigation of the roots of Ligularia sagitta has resulted in the characterization of six eremophilenolides 6beta,8beta-dimethoxy-10beta-hydroxyeremophil-7(11)-en-12,8alpha-olide (1), 6beta-angeloyloxy-10beta-hydroxy-8beta-methoxyeremophil-7(11)-en-12,8alpha-olide (2), 6beta-(2'-methylbutanoyloxy)-10beta-hydroxy-8beta-methoxyeremophil-7(11)-en-12,8alpha-olide (3), 6beta-angeloyloxy-10beta-hydroxy-8alpha-methoxyeremophil-7(11)-en-12,8beta-olide (4), 6beta-(2'-methylbutanoyloxy)-10beta-hydroxy-8alpha-methoxyeremophil-7(11)-en-12,8beta-olide (5) and 8beta,10beta-dihydroxy-6beta-methoxyeremophil-7(11)-en-12,8alpha-olide (6), together with one monoterpene (3 R,4 R,6 S)-3,6-dihydroxy-1-menthene (7), two triterpenes lupeol (8) and ursolic acid (9), and beta-sitosterol (10) . The structures of five new constituents (1-5) were elucidated by spectroscopic methods including 2D-NMR experiments . The compounds 1, 5 and 7 showed antibacterial activity by being assayed against Staphylococcus aureus, Bacillus subtilis and Escherichia coli.

Biochem Biophys Res Commun, 2003 Apr 25, 304(1), 48 - 54
Activation of subtilin precursors by Bacillus subtilis extracellular serine proteases subtilisin (AprE), WprA, and Vpr; Corvey C et al.; The maturation of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 includes posttranslational modifications of the propeptide and proteolytic cleavage of the leader peptide . To identify subtilin processing activities, we used antimicrobial inactive subtilin precursors consisting of the leader peptide which was still attached to the fully matured propeptide . Two extracellular B . subtilis proteases were able to activate subtilin precursors, the commercially available serine protease prototype subtilisin (AprE) and WprA . The latter was isolated from B . subtilis WB600, a strain deficient in six extracellular proteases . Surprisingly, the aprE wprA double mutant of the ATCC 6633 strain was still able to produce active subtilin, however, with a reduced production rate . No subtilin processing was found within the culture supernatant of the WB800 strain, which is deficient in eight extracellular proteases . Vpr was identified as the third protease capable to process subtilin.

Biochemistry, 2003 Apr 29, 42(16), 4648 - 57
Solution structure and backbone dynamics of the holo form of the frenolicin acyl carrier protein; Li Q et al.; During polyketide biosynthesis, acyl carrier proteins (ACPs) perform the central role of transferring polyketide intermediates between active sites of polyketide synthase . The 4'-phosphopantetheine prosthetic group of a holo-ACP is a long and flexible arm that can reach into different active sites and provide a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage . We have determined the solution structure and characterized backbone dynamics of the holo form of the frenolicin acyl carrier protein (fren holo-ACP) by nuclear magnetic resonance (NMR) . Unambiguous assignments were made for 433 hydrogen atoms, 333 carbon atoms, and 84 nitrogen atoms, representing a total of 94.6% of the assignable atoms in this protein . From 879 meaningful NOEs and 45 angle constraints, a family of 24 structures has been calculated . The solution structure is composed of three major alpha-helices packed in a bundle with three additional short helices in intervening loops; one of the short helices slowly exchanges between two conformations . Superposition of the major helical regions on the mean structure yields average atomic rmsd values of 0.49 +/- 0.09 and 0.91 +/- 0.08 A for backbone and non-hydrogen atoms, respectively . Although the three-helix bundle fold is conserved among acyl carrier proteins involved in fatty acid synthases and polyketide synthases, a detailed comparison revealed that ACPs from polyketide biosynthetic pathways are more related to each other in tertiary fold than to their homologues from fatty acid biosynthetic pathways . Comparison of the free form of ACPs (NMR structures of fren ACP and the Bacillus subtilis ACP) with the substrate-bound form of ACP (crystal structure of butyryl-ACP from Escherichia coli) suggests that conformational exchange plays a role in substrate binding.

Infect Immun, 2003 May, 71(5), 2810 - 8
Bacterial spores as vaccine vehicles; Duc le H et al.; For the first time, bacterial spores have been evaluated as vaccine vehicles . Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) antigen were used for oral and intranasal immunization and were shown to generate mucosal and systemic responses in a murine model . TTFC-specific immunoglobulin G titers in serum (determined by enzyme-linked immunosorbent assay) reached significant levels 33 days after oral dosing, while responses against the spore coat proteins were relatively low . Tetanus antitoxin levels were sufficient to protect against an otherwise lethal challenge of tetanus toxin (20 50% lethal doses) . The robustness and long-term storage properties of bacterial spores, coupled with simplified genetic manipulation and cost-effective manufacturing, make them particularly attractive vehicles for oral and intranasal vaccination.

J Dairy Sci, 2003 Mar, 86(3), 697 - 703
Reduction of protease activity in milk by continuous flow high-intensity pulsed electric field treatments; Bendicho S et al.; High-intensity pulsed electric field (HIPEF) is a non-thermal food processing technology that is currently being investigated to inactivate microorganisms and certain enzymes, involving a limited increase of food temperature . Promising results have been obtained on the inactivation of microbial enzymes in milk when suspended in simulated milk ultrafiltrate . The aim of this study was to evaluate the effectiveness of continuous HIPEF equipment on inactivating a protease from Bacillus subtilis inoculated in milk . Samples were subjected to HIPEF treatments of up to 866 micros of squared wave pulses at field strengths from 19.7 to 35.5 kV/cm, using a treatment chamber that consisted of eight colinear chambers connected in series . Moreover, the effects of different parameters such as pulse width (4 and 7 micros), pulse repetition rates (67, 89, and 111 Hz), and milk composition (skim and whole milk) were tested . Protease activity decreased with increased treatment time or field strength and pulse repetition rate . Regarding pulse width, no differences were observed between 4 and 7 micros pulses when total treatment time was considered . On the other hand, it was observed that milk composition affected the results since higher inactivation levels were reached in skim than in whole milk . The maximum inactivation (81%) was attained in skim milk after an 866-micros treatment at 35.5 kV/cm and 111 Hz.

EMBO J, 2003 Apr 15, 22(8), 1824 - 34
Structural basis for antibiotic recognition by the TipA class of multidrug-resistance transcriptional regulators; Kahmann JD et al.; The TipAL protein, a bacterial transcriptional regulator of the MerR family, is activated by numerous cyclic thiopeptide antibiotics . Its C-terminal drug-binding domain, TipAS, defines a subfamily of broadly distributed bacterial proteins including Mta, a central regulator of multidrug resistance in Bacillus subtilis . The structure of apo TipAS, solved by solution NMR {Brookhaven Protein Data Bank entry 1NY9}, is composed of a globin-like alpha-helical fold with a deep surface cleft and an unfolded N-terminal region . Antibiotics bind within the cleft at a position that is close to the corresponding heme pocket in myo- and hemoglobin, and induce folding of the N-terminus . Thus the classical globin fold is well adapted not only for accommodating its canonical cofactors, heme and other tetrapyrroles, but also for the recognition of a variety of antibiotics where ligand binding leads to transcriptional activation and drug resistance.

J Bacteriol, 2003 May, 185(9), 2826 - 34
Growth rate-dependent regulation of medial FtsZ ring formation; Weart RB et al.; FtsZ is an essential cell division protein conserved throughout the bacteria and archaea . In response to an unknown cell cycle signal, FtsZ polymerizes into a ring that establishes the future division site . We conducted a series of experiments examining the link between growth rate, medial FtsZ ring formation, and the intracellular concentration of FtsZ in the gram-positive bacterium Bacillus subtilis . We found that, although the frequency of cells with FtsZ rings varies as much as threefold in a growth rate-dependent manner, the average intracellular concentration of FtsZ remains constant irrespective of doubling time . Additionally, expressing ftsZ solely from a constitutive promoter, thereby eliminating normal transcriptional control, did not alter the growth rate regulation of medial FtsZ ring formation . Finally, our data indicate that overexpressing FtsZ does not dramatically increase the frequency of cells with medial FtsZ rings, suggesting that the mechanisms governing ring formation are refractile to increases in FtsZ concentration . These results support a model in which the timing of FtsZ assembly is governed primarily through cell cycle-dependent changes in FtsZ polymerization kinetics and not simply via oscillations in the intracellular concentration of FtsZ . Importantly, this model can be extended to the gram-negative bacterium Escherichia coli . Our data show that, like those in B . subtilis, average FtsZ levels in E . coli are constant irrespective of doubling time.

Mikrobiologiia, 2003 Jan-Feb, 72(1), 70 - 5
{The structure of the transposable genetic element ISBsu2 in the cryptic plasmid p1516 from a soil Bacillus subtilis strain and the presence of homologues of this element in the chromosomes of various Bacillus subtilis strains}; Holsappel S et al.; A cryptic plasmid from a soil strain of Bacillus subtilis was found to contain a sequence having features of IS element . Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B . subtilis strains.

J Biol Chem, 2003 Jun 27, 278(26), 23251 - 9 Epub 2003 Apr 14.
Bacillus subtilis bacteriophage SPP1 DNA packaging motor requires terminase and portal proteins; Camacho AG et al.; Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P . G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P . G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P . The stoichiometry of G1P on the G1P.G2P complex promotes the transition from a G2P endonuclease to an ATPase . G6P does not alter the endonuclease activity of G2P . Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P . Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.

Biochim Biophys Acta, 2003 Apr 15, 1626(1-3), 51 - 6
The effects of insertional mutations in comQ, comP, srfA, spo0H, spo0A and abrB genes on bacilysin biosynthesis in Bacillus subtilis; Karatas AY et al.; In Bacillus subtilis, two extracellular signaling peptides, ComX pheromone and CSF (competence and sporulation factor), stimulate the development of genetic competence and surfaction biosynthesis in response to high cell density (quorum sensing) by regulating the activity of transcription factor ComA . We recently showed that biosynthesis of dipeptide antibiotic bacilysin is linked to ComA and PhrC(CSF) in a Spo0K(Opp)-dependent manner by constructing phrC-, comA- and oppA-disrupted mutants of B . subtilis . In the present study, another pathway of quorum-sensing global regulation, namely, ComQ/ComX was found to be essential for bacilysin biosynthesis . ComP function in this chain was dispensable, most probably because of the existence of an alternative mean of ComA activation . The disruption of srfA operon in the bacilysin producer resulted with the bacilysin-negative phenotype; thus, our study verified that the srfA operon functions directly in the production of bacilysin . The abrB mutation suppressed the bacilysin-negative phenotype of a spo0A mutant, whereas the same mutation in the wild-type strain resulted in a significant increase in the production of bacilysin . This indicated that abrB gene product negatively controls the transcription of the gene(s) involved in bacilysin formation.

J Am Chem Soc, 2003 Apr 23, 125(16), 4726 - 7
Structure of subtilosin A, an antimicrobial peptide from Bacillus subtilis with unusual posttranslational modifications linking cysteine sulfurs to alpha-carbons of phenylalanine and threonine; Kawulka K et al.; The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled {(13)C,(15)N}medium derived from Anabaena sp . grown on sodium {(13)C}bicarbonate and {(15)N}nitrate . Additional samples of 1 were also generated by separate incorporations of {U-(13)C,(15)N}phenylalanine and {U-(13)C,(15)N}threonine using otherwise unlabeled media . The results demonstrate that in addition to having a cyclized peptide backbone (N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively . Such posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is very unusual in natural peptides or proteins . Subtilosin A (1) belongs to a new class of bacteriocins.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2002, 67(3), 647 - 55
Insecticide activity of surfactins and iturins from a biopesticide Bacillus subtilis Cohn (S499 strain); Assie LK et al.; Surfactin C14, surfactin C15, and iturin C15 are lipopeptides purified from Bacillus subtilis (S499 strain) . They were incorporated to artificial diet of the fruit fly Drosophila melanogaster (Meigen) (Diptera, Drosophilidae) to assess their potential insecticide activity . Surfactins with long fatty acid chain (C14 and C15) showed insecticide effect on the fruit fly, D . melanogaster . On the contrary, iturin was not toxic to fruit fly D . melanogaster . At 100 ppm, surfactin C14 and C15 showed respectively 85.4 and 92.6% adults mortality after one-day exposure . F1 progeny fly emergence inhibition by C14 and C15 were respectively 79.8% and 91.3% . To check whether the biocide activity of lipopeptides was due to their surface-active properties, detergent Triton X100, SDS, CTAB and Tween 80 were tested . No adult mortality was recorded with the detergents but Triton X100 and SDS showed F1 progeny emergence inhibition similar to that of surfactins . We showed that there was a dose-response activity with surfactin C15.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4678 - 83 Epub 2003 Apr 07.
Essential Bacillus subtilis genes; Kobayashi K et al.; To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes . Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work . Another 79 genes were predicted to be essential . The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics . Only 4% of essential genes encode unknown functions . Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya . However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes . Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential . Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

FEMS Microbiol Lett, 2003 Apr 11, 221(1), 125 - 30
Far different levels of gene expression provided by an oriented cloning system in Bacillus subtilis and Escherichia coli; Ohashi Y et al.; A gene expression system for both Bacillus subtilis and Escherichia coli was developed . The expression vector, pHASH102, produces any combination of promoter and open reading frame to be expressed based on the T-extended cloning method . Because the pHASH series vectors are designed to shuttle between the genome and a high copy plasmid in B . subtilis, the expression profiles of copy number dependence can be examined systematically . We demonstrated that vectors with Pr, Pspac, and PS10 promoters are suitable for the overexpression of GFPuv . Moreover, aadK encoding aminoglycoside 6-adenylyltransferase (a streptomycin-resistance gene) of B . subtilis was successfully overexpressed in both B . subtilis and E . coli . These highly expressed GFPuv and aadK genes can be used as a genetic marker for both organisms.

FEMS Microbiol Lett, 2003 Apr 11, 221(1), 103 - 10
An extracellular calcium-binding domain in bacteria with a distant relationship to EF-hands; Rigden DJ et al.; Extracellular Ca(2+)-dependent nuclease YokF from Bacillus subtilis and several other surface-exposed proteins from diverse bacteria are encoded in the genomes in two paralogous forms that differ by a approximately 45 amino acid fragment, which comprises a novel conserved domain . Sequence analysis of this domain revealed a conserved DxDxDGxxCE motif, which is strikingly similar to the Ca(2+)-binding loop of the calmodulin-like EF-hand domains, suggesting an evolutionary relationship between them . Functions of many of the other proteins in which the novel domain, named Excalibur (extracellular calcium-binding region), is found, as well as a structural model of its conserved motif are consistent with the notion that the Excalibur domain binds calcium . This domain is but one more example of the diversity of structural contexts surrounding the EF-hand-like calcium-binding loop in bacteria . This loop is thus more widespread than hitherto recognized and the evolution of EF-hand-like domains is probably more complex than previously appreciated.

BMC Microbiol . 2003 Mar 06;3(1):4.
Potassium ferrate {Fe(VI)} does not mediate self-sterilization of a surrogate Mars soil; Crawford RL et al.; BACKGROUND: Martian soil is thought to be enriched with strong oxidants such as peroxides and/or iron in high oxidation states that might destroy biological materials . There is also a high flux of ultraviolet radiation at the surface of Mars . Thus, Mars may be inhospitable to life as we know it on Earth . We examined the hypothesis that if the soil of Mars contains ferrates {Fe(VI)}, the strongest of the proposed oxidizing species, and also is exposed to high fluxes of UV radiation, it will be self-sterilizing . RESULTS: Under ambient conditions (25 degrees C, oxygen and water present) K2FeO4 mixed into sand mineralized some reactive organic molecules to CO2, while less reactive compounds were not degraded . Dried endospores of Bacillus subtilis incubated in a Mars surrogate soil comprised of dry silica sand containing 20% by weight K2FeO4 and under conditions similar to those now on Mars (extreme desiccation, cold, and a CO2-dominated atmosphere) were resistant to killing by the ferrate-enriched sand . Similar results were observed with permanganate . Spores in oxidant-enriched sand exposed to high fluxes of UV light were protected from the sporocidal activity of the radiation below about 5 mm depths . CONCLUSION: Based on our data and previously published descriptions of ancient but dormant life forms on Earth, we suggest that if entities resembling bacterial endospores were produced at some point by life forms on Mars, they might still be present and viable, given appropriate germination conditions . Endospores delivered to Mars on spacecraft would possibly survive and potentially compromise life detection experiments.

J Appl Microbiol, 2003, 94(5), 836 - 41
Mechanism of the inactivation of bacterial spores by reciprocal pressurization treatment; Furukawa S et al.; AIMS: The mechanism of the inactivation of Bacillus subtilis spores by reciprocal pressurization (RP) was unclear . Therefore, the mechanism was investigated . METHODS AND RESULTS: To investigate the effects of RP and continuous pressurization (CP) treatments on the inactivation and injury of B . subtilis spores, spores were treated at 25, 35, 45 and 55 degrees C under 200, 300 and 400 MPa . RP treatment was effective in injuring and inactivating spores . Scanning electron microscopy and transmission electron microscopy observation showed that spores treated by RP treatment were more morphologically and structurally changed than the ones treated by CP treatment . There were significant differences between the release of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by RP and CP treatments . From this result, it was concluded that the core fraction was released into the spore suspension . CONCLUSIONS: The mechanism of RP treatment is believed to work as follows: hydrostatic pressure treatment initiated germination of bacterial spores, and the repeated rapid decompression caused disruption, injury and inactivation of the germinated spores . SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that the physical injury of bacterial spores was effective to inactivate the bacterial spores through the disruption of spores and leakage of their contents.

Biochemistry (Mosc), 2003 Feb, 68(2), 177 - 81
Main physicochemical features of monofunctional flavokinase from Bacillus subtilis; Solovieva IM et al.; The main properties of a monofunctional riboflavin kinase from B . subtilis have been studied for the first time; the enzyme is responsible for a key reaction in flavin biosynthesis--the ATP-dependent phosphorylation of riboflavin with production of flavin mononucleotide . The active form of the enzyme is a monomer with molecular weight of about 26 kD with a strict specificity for reduced riboflavin . To display its maximum activity, the enzyme needs ATP and Mg(2+) . During the phosphorylation of riboflavin, Mg(2+) could be partially replaced by ions of other bivalent metals, the efficiencies of which decreased in the series Mg(2+) > Mn(2+) > Zn(2+), whereas Co(2+) and Ca2+ had inhibiting effects . The flavokinase activity was maximal at pH 8.5 and 52 degrees C . ATP could be partially replaced by other triphosphates, their donor activity decreasing in the series: ATP > dATP > CTP > UTP . The Michaelis constants for riboflavin and ATP were 0.15 and 112 micro M, respectively . As compared to riboflavin, a tenfold excess of its analog 7,8-dimethyl-10-(O-methylacetoxime)-isoalloxazine decreased the enzyme activity by 30% . Other analogs of riboflavin failed to markedly affect the enzyme activity.

Yi Chuan Xue Bao, 2002 Dec, 29(12), 1111 - 7
{Cloning and sequencing of the proBA gene from the selected mutant resistant to proline analogue from Bacillus subtilis}; Miao LX et al.; NTG was used to make chemical mutation for Bacillus subtilis 93151 . An enhanced osmotolerant mutant was obtained, which could grow in minimal medium containing 14% NaCl (w/v) and was not subject to proline-mediated feedback repression . The content of the intracellular free proline from the mutant increased rapidly with the rising of NaCl concentration . A 2.3 kb DNA fragment from the mutant was amplified using PCR method . Sequence analysis indicated that three bases changed within the proB gene, compared with the wild-type strain . One of the mutations was substitution of an A for a T at nt position 781, leading to a change of a Ser to a Thr at amino acid residue 261 of the deduced protein product, while other two were silent mutations . The recombinant vector pBE2-proB could functionally complement the proline auxotrophy E . coli 1.1252 . Sequence analysis of proA showed that proA and proB overlapped by 4 nt, and there was a SD sequence at nt 14 upstream of the start codon of proA . The deduced amino acid of proA gene shared a high similarity with that of Bacillus subtilis 168 (77%).

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 369 - 73 Epub 2003 Apr 11.
Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis; Olmos-Soto J et al.; The first amino acid residue from a proinsulin gene was fused in frame with the last amino acid residue of the aprE signal peptide sequence from Bacillus subtilis, using an overlapping PCR methodology . For expression of the fused DNA the aprE regulatory region (aprERR) was used . A six-protease-deficient strain of B . subtilis with the hpr2 and degU32 mutations was constructed for overproduction of the recombinant protein . The production of proinsulin was carried out in a mineral medium which facilitated the purification of proinsulin . Samples were taken during growth and analyzed by RIA and Western blot . Proinsulin was overproduced (1 mg ml(-1)) and 90% was secreted into the culture medium 1 h after stationary phase began.

Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Mar;67(3 Pt 1):031906 . Epub 2003 Mar 13.
Hydrodynamics of bacterial colonies: a model; Lega J et al.; We propose a hydrodynamic model for the evolution of bacterial colonies growing on soft agar plates . This model consists of reaction-diffusion equations for the concentrations of nutrients, water, and bacteria, coupled to a single hydrodynamic equation for the velocity field of the bacteria-water mixture . It captures the dynamics inside the colony as well as on its boundary and allows us to identify a mechanism for collective motion towards fresh nutrients, which, in its modeling aspects, is similar to classical chemotaxis . As shown in numerical simulations, our model reproduces both usual colony shapes and typical hydrodynamic motions, such as the whirls and jets recently observed in wet colonies of Bacillus subtilis . The approach presented here could be extended to different experimental situations and provides a general framework for the use of advection-reaction-diffusion equations in modeling bacterial colonies.

Biochemistry, 2003 Apr 15, 42(14), 4094 - 100
General base catalysis in the urate oxidase reaction: evidence for a novel Thr-Lys catalytic diad; Imhoff RD et al.; Urate oxidase catalyzes the oxidation of urate without the involvement of any cofactors . The gene encoding urate oxidase from Bacillus subtilis has been cloned and expressed, and the enzyme was purified and characterized . Formation of the urate dianion is believed to be a key step in the oxidative reaction . Rapid-mixing chemical quench studies provide evidence that the dianion is indeed an intermediate; at 15 degrees C the dianion forms within the mixing time of the rapid-quench instrument, and it disappears with a rate constant of 8 s(-)(1) . Steady-state kinetic studies indicate that an ionizable group on the enzyme with a pK of 6.4 must be unprotonated for catalysis, and it is presumed that the role of this group is to abstract a proton from the substrate . Surprisingly, examination of the active site provided by the previously reported crystal structure does not reveal any obvious candidates to act as the general base . However, Thr 69 is hydrogen-bonded to the ligand at the active site, and Lys 9, which does not contact the ligand, is hydrogen-bonded to Thr 69 . The T69A mutant enzyme has a V(max) that is 3% of wild type, and the K9M mutant enzyme has a V(max) that is 0.4% of wild type . The ionization at pH 6.4 that is observed with wild-type enzyme is absent in both of these mutants . It is proposed that these residues form a catalytic diad in which K9 deprotonates T69 to allow it to abstract the proton from the N9 position of the substrate to generate the dianion.

J Biotechnol, 1999 Jun 11, 72(1-2), 185 - 95
Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis; Wu SC et al.; pUb110-based vectors are commonly used for expression and secretion studies in Bacillus subtilis . Two of these plasmids, pUB18P43 and pWB705, have been applies to produce several proteins of interest . To offer greater flexibility and compatibility in this system, the authors have also constructed a pE194-based plasmid vector (pE18) . To determine whether the pUB110-based or the pE194-based vector serves as a better expression system, three structural genes encoding cytoplasmic BirA, extracytoplasmic PrsA and extracellular staphylokinase, respectively, were used as models . Production of these products using pUB110-based vectors was consistently 2--3-fold lower than that using the pE194-based vectors . The observed difference in the protein yield did not result from either the rearrangement of the plasmid or the difference in the plasmid copy number . By using three different approaches, the lower production yield from the pUB110-based vectors was found to be due to the transcription interference from the plasmid encoded genes . These findings illustrate that the orientation of the inserted gene in pUB110-based vector can greatly affect gene expression . Two new expression vectors, pUB19P43 and pWB980, were constructed to allow better gene expression.

J Mol Biol, 2003 Apr 18, 328(1), 167 - 82
A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus; Zhang X et al.; 6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms . The enzymes involved in lumazine biosynthesis have been studied in considerable detail . However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear . Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds . The structures were refined at resolutions of 1.72 A, 1.85 A, 2.05 A and 2.2 A, respectively . The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product . Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of single-site mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis . A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed .

J Am Chem Soc, 2003 Apr 16, 125(15), 4460 - 6
Biosynthesis of riboflavin . Single turnover kinetic analysis of 6,7-dimethyl-8-ribityllumazine synthase; Schramek N et al.; 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate, affording the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine . Single turnover experiments monitored by multiwavelength photometry were performed with the recombinant lumazine synthase of Bacillus subtilis . Mixing of the enzyme with the pyrimidine type substrate is conducive to a hypsochromic shift as well as a decrease in absorbance of the heterocyclic substrate; the rate constant for that reaction is 0.02 s(-1) microM(-1) . Rapid mixing of the complex between enzyme and pyrimidine type substrate with the second substrate, 3,4-dihydroxy-2-butanone 4-phosphate, is followed by the appearance of an early optical transient characterized by an absorption maxima at 330 nm of low intensity which was tentatively assigned as a Schiff base intermediate . The subsequent elimination of phosphate affords a transient with intense absorption maxima at 455 and 282 nm, suggesting an intermediate with an extended system of conjugated double bonds . The subsequent formation of the enzyme product, 6,7-dimethyl-8-ribityllumazine, is the rate-determining step.

Life Sci Space Res, 1976, 14, 337 - 9
Effect of extreme factors on micro-organisms used for the control of the effectiveness of sterilization; Vashkov VI et al.; Survival of micro-organisms used for control of sterilization procedures was studied under conditions simulating the Martian environment (daily temperature change from +20 degrees to -20 degrees in 99.98% CO2 + 0.02% air at 0.13 x 10(-6) N m-2 pressure, with ultraviolet radiation spanning the whole range of the solar spectrum 300-350 MkW cm-2) . The test organisms were four strains of Bacillus subtilis and one strain of Bacillus anthracoides, and were inoculated on to four materials, smooth metal, porous plastic, multilayer composition material and powdered limonite . Some organisms survived on all materials, the greatest on the limonite . The resistance of the survivors to disinfectants was the same as that of the original cultures.

Life Sci Space Res, 1976, 14, 247 - 50
Influence of heavy ions on the transforming activity of DNA; Minkova MI et al.; Changes in functional activity following treatment of DNA with heavy ions were analysed by transformation assay . Biological response to exposure to charged particles, 10B, 12C, and 22Ne, was evaluated by the extent of inactivation of ability of DNA to transfer the genetic marker of tryptophane independence . The possibility of protecting the biological activity of DNA was studied using a conventional protector, cysteamine hydrochloride, or the preparation cytriphos, synthesized at the Institute of Radiobiology and Radiation Hygiene, Sofia . Dried samples of donor DNA isolated from a Bacillus subtilis prototroph strain were exposed to doses of 50 krad to 1 Mrad . For the three types of charged particles used, dose response of the inactivation process was defined by an exponential function . Comparison of response curves for the three types of radiation showed the effect to be dependent on LET, being highest for 10B and lowest for 22Ne . In all three cases, addition of a protective substance failed to produce any change in the biological effect observed.

J Biol Chem, 2003 Jun 6, 278(23), 20898 - 905 Epub 2003 Apr 03.
Evidence in support of a docking model for the release of the transcription factor sigma F from the antisigma factor SpoIIAB in Bacillus subtilis; Ho MS et al.; Cell-specific activation of the transcription factor sigmaF during the process of sporulation in Bacillus subtilis is governed by an antisigma factor SpoIIAB and an anti-antisigma factor SpoIIAA . SpoIIAB, which exists as a dimer, binds to sigmaF in a complex of stoichiometry sigmaF.SpoIIAB2 . Escape from the complex is mediated by SpoIIAA, which reacts with the complex to cause the release of free sigmaF . Previous evidence indicated that Arg-20 in SpoIIAB is a contact site for both sigmaF and SpoIIAA and that contact with sigmaF is mediated by Arg-20 on only one of the two subunits in the sigmaF.SpoIIAB2 complex . Here we report the construction of heterodimers of SpoIIAB in which one subunit is wild type and the other subunit is a mutant for Arg-20 . We show that the dissociation constant for the binding of sigmaF to the heterodimer was similar to that for the wild type, a finding consistent with the idea that sigmaF contacts Arg-20 on only one of the two subunits . Although SpoIIAA was highly effective in causing the release of sigmaF from the wild type homodimer, the anti-antisigma factor had little effect on the release of sigmaF from the heterodimer . This finding is consistent with a model in which SpoIIAA docks on the sigmaF.SpoIIAB2 complex, making contact with the subunit in which Arg-20 is not in contact with sigmaF . SpoIIAB is both an anti-sigmaF factor and a protein kinase that phosphorylates and thereby inactivates SpoIIAA . We show that SpoIIAA effectively displaces sigmaF from a complex of sigmaF with a mutant (SpoIIABR105A) that is impaired in the kinase function of SpoIIAB . This result shows that SpoIIAA-mediated displacement of sigmaF from SpoIIAB does not require concomitant phosphorylation of SpoIIAA.

Appl Environ Microbiol, 2003 Apr, 69(4), 2284 - 91
Disinfection of water containing natural organic matter by using ozone-initiated radical reactions; Cho M et al.; Ozone is widely used to disinfect drinking water and wastewater due to its strong biocidal oxidizing properties . Recently, it was reported that hydroxyl radicals ((.)OH), resulting from ozone decomposition, play a significant role in microbial inactivation when Bacillus subtilis endospores were used as the test microorganisms in pH controlled distilled water . However, it is not yet known how natural organic matter (NOM), which is ubiquitous in sources of drinking water, affects this process of disinfection by ozone-initiated radical reactions . Two types of water matrix were considered for this study . One is water containing humic acid, which is commercially available . The other is water from the Han River . This study reported that hydroxyl radicals, initiated by the ozone chain reaction, were significantly effective at B . subtilis endospore inactivation in water containing NOM, as well as in pH-controlled distilled water . The type of NOM and the pH have a considerable effect on the percentage of disinfection by hydroxyl radicals, which ranged from 20 to 50% . In addition, the theoretical T value of hydroxyl radicals for 2-log B . subtilis removal was estimated to be about 2.4 x 10(4) times smaller than that of ozone, assuming that there is no synergistic activity between ozone and hydroxyl radicals.

J Infect Chemother, 2003 Mar, 9(1), 93 - 6
The intraocular dynamics of vancomycin hydrochloride ophthalmic ointment (TN-011) in rabbits; Fukuda M et al.; The intraocular dynamics of 1% vancomycin hydrochloride (VCM) ophthalmic ointment (TN-011) in rabbits was investigated . The animals used in the study were 42 pigmented rabbits with normal eyes (normal group) and 17 pigmented rabbits with Bacillus subtilis-infected eyes (BS group) . The infection in the BS group was induced by a method previously established by the authors . Fifty milligrams of a 1% VCM ophthalmic ointment was administered into the cul-de-sacs of the animals' eyes unilaterally in both groups . Serum, bulbar conjunctiva (only from the normal group), cornea, iris-ciliary body, and vitreous were collected 15, 30, 60, 120, and 240 min after the drug administration as samples . Drug concentration was measured by the bioassay method . In the normal group, VCM in the bulbar conjunctiva reached a maximum concentration (98.98 +/- 46.48 microg/g) at 30 min, and decreased to a level below the detection limit at 240 min . The maximum concentration of VCM in the cornea (12.04 +/- 4.73 microg/g) was observed at 30 min . VCM was not detected in the aqueous humour, iris-ciliary body, vitreous, or serum in the normal group at any point . In the BS group, the maximum concentration of VCM was 25.60 +/- 11.01 microg/g in the cornea 15 min after drug administration, and 5.18 +/- 2.60 microg/ml in the aqueous humour at 30 min . VCM in the aqueous humor could be detected (0.37 +/- 0.19 microg/ml) even after 240 min . VCM was not detected in the iris-ciliary body, vitreous, or serum at any point . The intraocular dynamics of 1% VCM ophthalmic ointment (TN-011) revealed that VCM reached the estimated effective concentrations in the aqueous humour as well as extraocular tissues of Bacillus subtilis-infected rabbit eyes.

J Bacteriol, 2003 Apr, 185(8), 2465 - 74
Bacteriophage SPP1 Chu is an alkaline exonuclease in the SynExo family of viral two-component recombinases; Vellani TS et al.; Many DNA viruses concatemerize their genomes as a prerequisite to packaging into capsids . Concatemerization arises from either replication or homologous recombination . Replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth . To dissect the molecular mechanisms of viral recombination, we and others previously identified a family of viral nucleases that comprise one component of a conserved, two-component viral recombination system . The nuclease component is related to the exonuclease of phage lambda and is common to viruses with linear double-stranded DNA genomes . To test the idea that these viruses have a common strategy for recombination and genome concatemerization, we isolated the previously uncharacterized 34.1 gene from Bacillus subtilis phage SPP1, expressed it in Escherichia coli, purified the protein, and determined its enzymatic properties . Like lambda exonuclease, Chu (the product of 34.1) forms an oligomer, is a processive alkaline exonuclease that digests linear double-stranded DNA in a Mg(2+)-dependent reaction, and shows a preference for 5'-phosphorylated DNA ends . A model for viral recombination, based on the phage lambda Red recombination system, is proposed.

J Bacteriol, 2003 Apr, 185(8), 2457 - 64
Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis; Cabrera-Martinez RM et al.; The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants . However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons . Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK . Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations . However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein . Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA . These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination . We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.

FEMS Microbiol Lett, 2003 Mar 28, 220(2), 277 - 80
A Crh-specific function in carbon catabolite repression in Bacillus subtilis; Warner JB et al.; Carbon catabolite repression in Bacillus subtilis is mediated by phosphorylation of the phosphoenolpyruvate:carbohydrate phosphotransferase system intermediate HPr at a serine residue catalyzed by HPr kinase . The orthologous protein Crh functions in a similar way, but, unlike HPr, it is not functional in carbohydrate uptake . A specific function for Crh is not known . The role of HPr and Crh in repressing the citM gene encoding the Mg(2+)-citrate transporter was investigated during growth of B . subtilis on different carbon sources . In glucose minimal medium, full repression was supported by both HPr and Crh . Strains deficient in Crh or the regulatory function of HPr revealed the same repression as the wild-type strain . In contrast, in a medium containing succinate and glutamate, repression was specifically mediated via Crh . Repression was relieved in the Crh-deficient strain, but still present in the HPr mutant strain . The data are the first demonstration of a Crh-specific function in B . subtilis and suggest a role for Crh in regulation of expression during growth on substrates other than carbohydrates.

J Agric Food Chem, 2003 Apr 9, 51(8), 2200 - 5
Bacterial susceptibility to and chemical composition of essential oils from Thymus kotschyanus and Thymus persicus; Rasooli I et al.; Susceptibility of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, and Pseudomonas aeroginosa to the essential oils extracted from two varieties of Thyme, i.e., Thymus kotschyanus Boiss . and Hohen . and Thymus persicus L . at preflowering and flowering stages were studied . The disk diffusion method was used to evaluate the zone of microbial growth inhibition at various concentrations of the oils . Minimal inhibitory concentration and minimal bactericidal concentration of the oils were determined and compared with each other . The oils from the above plants were found to be strongly bactericidal with that of T . kotschyanus being more effective . T . kotschyanusand T . persicus oils analyzed by gas chromatography (GC) and GC/mass spectrometry (MS) lead to identification of 33 and 26 components, respectively . The profile of the oil components from T . persicuswas similar to that of T . kotschyanus in almost all of the compounds but at different concentrations . The major components of T . kotschyanus oil before and at the flowering stages were carvacrol (35.06, 22.75%), thymol (26.60, 16.52%), gamma-terpinene (7.81, 0.34%), gamma-terpinene (4.34, 0%), borneol (2.29, 4.52%), myrcene (0.26, 12.65%), thymolquinone (0, 11.39%), nerol (0, 6.10%), and beta-caryophyllene (0, 5.54%), respectively, and those of T . persicus at the same stages were carvacrol (38.96, 27.07%), thymol (6.48, 11.86%), P-cymene (7.51, 10.16%), gamma-terpineol (0, 9.51%), nerol (15.66, 9.41%), gamma-terpinene (6.11, 6.51%), and thymol acetate (5.29, 5.30%), respectively . The contribution of oil components to its antibacterial property is discussed . High aromatic compound content of the phenol-rich oils seems to account for strong antibacterial activity.

Mikrobiol Z, 2002 Nov-Dec, 64(6), 73 - 9
{Physiological activity of mixed cultures of Methylcoccus capsulatus UKM B-3030 with Bacillus megaterium UKM B-5723T and Bacillus subtilis VKPM B-1489 on solid surface colonization}; Kisten' AG et al.; Physiological activity of monoculture of Methylococcus capsulatus UCM B-3030 and its mixed cultures with two bacilli species distinguished by proteolytical properties--Bacillus megaterium UCM B-5723T and Bacillus subtilis BK{symbol: see text}M B-4189 in terms of long-duration cultivation is investigated . It is shown, that the most active methane consumption by methanotrophic bacteria and their mixed cultures with bacilli under solid surface colonization occurred on the second-eighth day of cultivation . The obtained results permit one to evaluate intensity of CH4 oxidation process by typical microflora of some econiches (depleted coal mines, in particular).

Curr Genet, 2003 Jun, 43(3), 186 - 90 Epub 2003 Mar 19.
The npgA/ cfwA gene encodes a putative 4'-phosphopantetheinyl transferase which is essential for penicillin biosynthesis in Aspergillus nidulans; Keszenman-Pereyra D et al.; Non-ribosomal peptide synthetases, polyketides and fatty acid synthetases have a modular organisation of multi-enzymatic activities . In all of them, the acyl or peptidyl carrier proteins have 4'-phosphopantetheine (P-pant) as an essential prosthetic group . This is added by 4'-phosphopantetheinyl transferases (PPTases) that derive the P-pant group from coenzyme A . While many PPTases of varying specificity have now been isolated from a number of bacteria, a filamentous fungal PPTase has yet to be characterised . Through database searching of the Aspergillus fumigatus genome sequence against Sfp from Bacillus subtilis, we identified a unique sequence which appears to encode a PPTase, as deduced from conserved residues considered important in PPTases . The PPTase candidate was used to search the NCBI data base and an unexpected homologue in A . nidulans was identified as npgA . Mutations in this gene (cfwA/ npgA) were identified previously as leading to defects in growth and pigmentation . To test whether the temperature-sensitive cfwA2 mutation impairs penicillin biosynthesis, which is dependent on the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, bioassays with B . calidolactis were set up at permissive and non-permissive temperatures . The cfwA2 mutant did not produce penicillin at the non-permissive temperature . Since no other PPTase homologue has been detected in the A . fumigatus genome to date, the data suggest that a single enzyme may be able to transfer the cofactor to a broad range of enzymes with acyl or peptidyl carrier protein domains.

J Mol Biol, 2003 Apr 11, 327(5), 1111 - 9
Crystal structure of the IIB(Sor) domain of the sorbose permease from Klebsiella pneumoniae solved to 1.75A resolution; Orriss GL et al.; The phosphoenolpyruvate transferase system (PTS) is the major pathway by which bacteria import hexose sugars across the plasma membrane . The PTS transfers a phosphoryl group sequentially via several components from the glycolytic intermediate phosphoenolpyruvate (PEP) to the translocated sugar . It is comprised of the two general proteins enzyme I and HPr, and a sugar-specific enzyme II complex . Sugar translocation is through the membrane domain of the enzyme II complex . The enzyme II complex can belong to one of six families based upon sequence similarity, with the sorbose transporter from Klebsiella pneumoniae a member of the mannose family.The structure of the IIB(Sor) domain was solved to 1.75A resolution by molecular replacement . It has a central core of seven parallel beta-strands surrounded by a total of six alpha-helices . Three helices cover the front face, one the back face with the remaining two capping the central beta-sheet at the top and bottom . The catalytic His15 residue is situated on the surface-exposed loop between strand 1 and helix 1 . In addition to the features previously observed in the homologous IIB(Lev) domain from Bacillus subtilis we see new features in the IIB(Sor) structure . First, the catalytic His15 side-chain is fixed in a specific conformation by forming a short hydrogen bond with Asp10, which in turn makes a salt-bridge with Arg8 . Second, as observed in other phosphoproteins, an arginine residue (Arg12) is well poised to stabilize a phosphoryl group on His15 . Third, we see an Asp/His pair reminiscent of that observed in the IIA(Man) domain from Escherichia coli . Finally, docking of IIA(Man) to IIB(Sor) shows that Arg12 in its current conformation is well positioned to assist the subsequent transfer of the phosphoryl group onto the sugar in line with previous mutagenesis studies.

J Mol Biol, 2003 Apr 11, 327(5), 945 - 72
The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis; Eichenberger P et al.; We report the identification and characterization on a genome-wide basis of genes under the control of the developmental transcription factor sigma(E) in Bacillus subtilis . The sigma(E) factor governs gene expression in the larger of the two cellular compartments (the mother cell) created by polar division during the developmental process of sporulation . Using transcriptional profiling and bioinformatics we show that 253 genes (organized in 157 operons) appear to be controlled by sigma(E) . Among these, 181 genes (organized in 121 operons) had not been previously described as members of this regulon . Promoters for many of the newly identified genes were located by transcription start site mapping . To assess the role of these genes in sporulation, we created null mutations in 98 of the newly identified genes and operons . Of the resulting mutants, 12 (in prkA, ybaN, yhbH, ykvV, ylbJ, ypjB, yqfC, yqfD, ytrH, ytrI, ytvI and yunB) exhibited defects in spore formation . In addition, subcellular localization studies were carried out using in-frame fusions of several of the genes to the coding sequence for GFP . A majority of the fusion proteins localized either to the membrane surrounding the developing spore or to specific layers of the spore coat, although some fusions showed a uniform distribution in the mother cell cytoplasm . Finally, we used comparative genomics to determine that 46 of the sigma(E)-controlled genes in B.subtilis were present in all of the Gram-positive endospore-forming bacteria whose genome has been sequenced, but absent from the genome of the closely related but not endospore-forming bacterium Listeria monocytogenes, thereby defining a core of conserved sporulation genes of probable common ancestral origin . Our findings set the stage for a comprehensive understanding of the contribution of a cell-specific transcription factor to development and morphogenesis.

J Biol Chem, 2003 May 30, 278(22), 19891 - 7 Epub 2003 Mar 26.
Characterization of YqjM, an Old Yellow Enzyme homolog from Bacillus subtilis involved in the oxidative stress response; Fitzpatrick TB et al.; In this paper, we demonstrate that a protein from Bacillus subtilis (YqjM) shares many characteristic biochemical properties with the homologous yeast Old Yellow Enzyme (OYE); the enzyme binds FMN tightly but noncovalently, preferentially uses NADPH as a source of reducing equivalents, and forms charge transfer complexes with phenolic compounds such as p-hydroxybenzaldehyde . Like yeast OYE and other members of the family, YqjM catalyzes the reduction of the double bond of an array of alpha,beta-unsaturated aldehydes and ketones including nitroester and nitroaromatic compounds . Although yeast OYE was the first member of this family to be discovered in 1933 and was the first flavoenzyme ever to be isolated, the physiological role of the family still remains obscure . The finding that alpha,beta-unsaturated compounds are substrates provoked speculation that the OYE family might be involved in reductive degradation of xenobiotics or lipid peroxidation products . Here, for the first time, we demonstrate on the protein level that whereas YqjM shows a basal level of expression in B . subtilis, the addition of the toxic xenobiotic, trinitrotoluene, leads to a rapid induction of the protein in vivo denoting a role in detoxification . Moreover, we show that YqjM is rapidly induced in response to oxidative stress as exerted by hydrogen peroxide, demonstrating a potential physiological role for this enigmatic class of proteins.

Biochim Biophys Acta, 2003 Apr 1, 1611(1-2), 91 - 7
Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin; Carrillo C et al.; Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and possesses several other interesting biological activities . This work deals with the molecular mechanism of membrane permeabilization by incorporation of surfactin . The surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC) . The effect of the addition of cholesterol, dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylethanolamine (POPE) was also checked . It was observed that surfactin was able to induce content leakage at concentrations far below the onset surfactin/lipid ratio for membrane solubilization to occur, which in our system was around 0.92 . Electron microscopy showed that vesicles were present after addition of surfactin at a ratio below this value, whereas no vesicles could be observed at ratios above it . Cholesterol and POPE attenuated the membrane-perturbing effect of surfactin, whereas the effect of DPPC was to promote surfactin-induced leakage, indicating that bilayer sensitivity to surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that surfactin destabilizes the inverted-hexagonal structure . Fourier-transform infrared spectroscopy (FTIR) was used to specifically follow the effect of surfactin on different parts of the phospholipid bilayer . The effect on the C=O stretching mode of vibration of POPC indicated a strong dehydration induced by surfactin . On the other hand, the C-H stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization . The reported effects could be useful to explain surfactin-induced 'pore' formation underlying the antibiotic and other important biological actions of this bacterial lipopeptide.

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 749 - 51 Epub 2003 Mar 25.
Crystallization and X-ray diffraction analysis of the sensor domain of the HemAT aerotactic receptor; Zhang W et al.; HemAT is a 432-amino-acid protein with two structural domains identified from Bacillus subtilis . It is responsible for sensing oxygen and delivering the signal to the downstream signal transduction cascade through a two-component system {Hou et al . (2000), Nature (London), 403, 540-544} . The consequence of such events is to change the flagellar movement and alter the swimming behavior of bacteria . To elucidate the molecular mechanism of oxygen sensing, the sensor domain of HemAT from B . subtilis was cloned, expressed and crystallized . Multiple-wavelength anomalous dispersion (MAD) data were collected from the intrinsic anomalous scatterer, iron, using synchrotron radiation . Three-wavelength iron MAD data sets were collected to 2.8 A resolution . The native data set was collected to 2.15 A resolution . Initial crystallographic analysis revealed the crystals to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.00, b = 80.12, c = 85.95 A . There is one dimer in the asymmetric unit, with 40% solvent content . Structure determination using MAD methods and model building are currently under way.

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 627 - 36 Epub 2003 Mar 25.
Characterization of a family 11 xylanase from Bacillus subtillis B230 used for paper bleaching; Oakley AJ et al.; Enzymes such as family 11 xylanases are increasingly being used for industrial applications . Here, the cloning, structure determination and temperature-stability data of a family 11 xylanase, Xyn11X, from the alkali-tolerant Bacillus subtilis subspecies B230 are reported . This enzyme, which degrades xylan polymers, is being produced on an industrial scale for use in the paper-bleaching industry . Xyn11X adopts the canonical family 11 xylanase fold . It has a greater abundance of side chain to side chain hydrogen bonds compared with all other family 11 xylanase crystal structures . Means by which the thermostability of Xyn11X might be improved are suggested.

Appl Microbiol Biotechnol, 2003 Apr, 61(2), 133 - 9 Epub 2003 Jan 29.
Synthesis of ethyl ( S)-4-chloro-3-hydroxybutanoate using fabG-homologues; Yamamoto H et al.; This paper is a report on the successful application of bioinformatics to enzyme screening . The synthesis of ethyl ( S)-4-chloro-3-hydroxybutanoate (ECHB) by asymmetric reduction of ethyl 4-chloroacetoacetate (ECAA) using fabG-homologues was studied . beta-Ketoacyl-acyl carrier protein reductases from both Escherichia coli and Bacillus subtilis, which are components of type II fatty acid synthase, could reduce ECAA to ( S)-ECHB with 94-98% ee . Furthermore, acetoacetyl-CoA reductases (ARs) from both Ralstonia eutropha and Zoogloea ramigera, whose genes are significantly similar to fabG genes and play a physiological role in the biosynthesis of poly-beta-3-hydroxybutyrate, could also catalyze the asymmetric reduction of ECAA to ( S)-ECHB with >99% ee . ( S)-ECHB was synthesized to 48.7 g/l with an optical purity of 99.8% ee, using recombinant E . coli cells coexpressing AR from R . eutropha and glucose dehydrogenase from B . subtilis for the regeneration of NADPH.

Eur J Biochem, 2003 Apr, 270(7), 1474 - 82
Kinetic mechanisms of glycine oxidase from Bacillus subtilis; Molla G et al.; The kinetic properties of glycine oxidase from Bacillus subtilis were investigated using glycine, sarcosine, and d-proline as substrate . The turnover numbers at saturating substrate and oxygen concentrations were 4.0 s(-1), 4.2 s(-1), and 3.5 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate . Glycine oxidase was converted to a two-electron reduced form upon anaerobic reduction with the individual substrates and its reductive half-reaction was demonstrated to be reversible . The rates of flavin reduction extrapolated to saturating substrate concentration, and under anaerobic conditions, were 166 s(-1), 170 s(-1), and 26 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate . The rate of reoxidation of reduced glycine oxidase with oxygen in the absence of product (extrapolated rate approximately 3 x 10(4) M(-1) x s(-1)) was too slow to account for catalysis and thus reoxidation started from the reduced enzyme:imino acid complex . The kinetic data are compatible with a ternary complex sequential mechanism in which the rate of product dissociation from the reoxidized enzyme form represents the rate-limiting step . Although glycine oxidase and D-amino acid oxidase differ in substrate specificity and amino acid sequence, the kinetic mechanism of glycine oxidase is similar to that determined for mammalian D-amino acid oxidase on neutral D-amino acids, further supporting a close similarity between these two amine oxidases.

Protein Expr Purif, 2003 Mar, 28(1), 131 - 9
High-level expression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase of Bacillus sp . strain OxB-1, in heterologous hosts; Kato Y et al.; We examined the overexpression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase (Oxd) of Bacillus sp . strain OxB-1, in Escherichia coli and Bacillus subtilis . Several plasmids were constructed to express the enzyme under the control of the lac promoter or its own promoter, together with or without nitrilase and a possible regulatory protein that is present in the wild-type genome . The enzyme was expressed using E . coli transfected with the plasmid pOxD-9OF . Expression was under the control of the lac promoter in the pUC18 vector and was much more effective when the start codon was changed from TTG to ATG . When the transfected cells were grown at 37 degrees C, the enzyme was produced mainly in inactive inclusion bodies, whereas the enzyme was largely soluble and active when the cells were grown at 30 degrees C . The production of active enzyme was markedly enhanced by increasing the volume of culture medium . This had the effect of slowing the rate of apoenzyme synthesis . A slow rate of synthesis allows for a more efficient incorporation of heme cofactor into the apoenzyme than a fast rate of synthesis . Under optimized conditions, the enzyme was produced in an active and soluble form at 15,000U/L of culture, which is about 1500-fold higher than the amount produced by the wild-type strain . Moreover, the enzyme comprised over 40% of total extractable cellular protein.

J Chromatogr A, 2003 Mar 14, 989(2), 293 - 301
Determination of pyridine and adenine nucleotide metabolites in Bacillus subtilis cell extract by sweeping borate complexation capillary electrophoresis; Markuszewski MJ et al.; With a growing interest in new areas of bioanalytical research such as metabolome analysis, the development of sensitive capillary electrophoresis (CE) methods to analyze sub-microM concentrations of analytes in biological samples is required . In this report, the application of CE with sweeping by borate complexation is used to analyze a group of seven pyridine and adenine nucleotide metabolites derived from bacteria Bacillus subtilis cell extracts . Nanomolar (nM) detectability of analytes by CE with UV photometric detection is achieved through effective focusing of large sample plug (approximately 10% of capillary length) using sweeping by borate complexation method, reflected by a limit of detections (S/N = 3) of about 2 x 10(-8) M . Changes in metabolites concentrations were observed in cell extracts when using either glucose or malate as the carbon source in the culture medium . Concentration of pyridine and adenine nucleotides in cell extracts varied widely from 78.6 (+/-7.6) microM for nicotinamide-adenine dinucleotide in malate to 0.66 (+/-0.12) microM for nicotinamide-adenine dinucleotide phosphate in glucose culture medium . Concentrations of metabolites in a single cell were also estimated at millimolar (mM) level . The method was validated in terms of linearity, sensitivity and reproducibility . The application of CE by sweeping borate complexation allows for sensitive and reproducible analyses of nucleotide metabolites in complex biological samples such as bacteria cell extracts.

Arch Soc Esp Oftalmol, 2003 Feb, 78(2), 107 - 9
{Chronic endophthalmitis in pseudophakic patients caused by bacillus subtilis}; Romero Aroca P et al.; OBJECTIVE/METHODS: We present two cases of patients with chronic endophthalmitis . The treatment included intraocular lens exchange, total capsulectomy and posterior vitrectomy through the limbus . In both cases, samples of the vitreous were taken and sent to the laboratory together with the intraocular lens and remmants of posterior capsule for culture and antibiogram . RESULTS/CONCLUSIONS: Microbiologic examination and culture were positive in two cases for Bacillus subtilis, which is not actually identified in literature as an etiologic agent for chronic endophthalmitis.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 4209 - 14 Epub 2003 Mar 19.
A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: implications for spore dormancy; Cowan AE et al.; Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased approximately 2-fold . The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species . The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.

J Bacteriol, 2003 Apr, 185(7), 2379 - 82
Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation; Suzuki T et al.; The ywtD gene, which codes for an enzyme that degrades gamma-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449 . The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme . The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli . Histidine-tagged YwtD was purified from sonicated cells of the transformant . The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio) . This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the gamma-glutamyl bond only between D- and L-glutamic acids of PGA, and it may be designated gamma-DL-glutamyl hydrolase.

J Bacteriol, 2003 Apr, 185(7), 2346 - 53
Localization of rRNA synthesis in Bacillus subtilis: characterization of loci involved in transcription focus formation; Davies KM et al.; In Bacillus subtilis, RNA polymerase becomes concentrated into regions of the nucleoid called transcription foci . With green fluorescent protein-tagged RNA polymerase, these structures are only observed at higher growth rates and have been shown to represent the sites of rRNA synthesis . There are 10 rRNA (rrn) operons distributed around nearly half of the chromosome . In this study we analyzed the rrn composition of transcription foci with fluorescently tagged loci and showed that they comprise the origin-proximal operon rrnO but not the more dispersed rrnE or rrnD . This suggests that transcription foci comprise only the seven origin-proximal operons rrnO, rrnA, rrnJ, rrnW, rrnI, rrnH, and rrnG . These results have important implications for our understanding of microbial chromosome structure.

J Bacteriol, 2003 Apr, 185(7), 2315 - 29
Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate; Ragkousi K et al.; Bacillus subtilis spores can germinate with a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA), a compound present at high levels in the spore core . Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca(2+)-DPA, three mutations were identified . One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca(2+)-DPA . The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function . Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is sigma(E) dependent . Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat . Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA . However, other than lacking CwlJ, ywdL spores have no obvious defect in their spore coat . Because of the role for YwdL in a part of the spore germination process, we propose renaming ywdL as a spore germination gene, gerQ.

J Bacteriol, 2003 Apr, 185(7), 2153 - 60
Roles of YqjH and YqjW, homologs of the Escherichia coli UmuC/DinB or Y superfamily of DNA polymerases, in stationary-phase mutagenesis and UV-induced mutagenesis of Bacillus subtilis; Sung HM et al.; YqjH and YqjW are Bacillus subtilis homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in Escherichia coli . While the functions of YqjH and YqjW in B . subtilis are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to E . coli DNA polymerase IV (DinB protein), and YqjW has 26% identity to E . coli DNA polymerase V (UmuC protein) . In this report, we demonstrate that both YqjH and the products of the yqjW operon are involved in UV-induced mutagenesis in this bacterium . Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein . Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of B . subtilis to generate revertants at the hisC952 allele via this system . These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 155 - 60
DNA microarray analysis of Bacillus subtilis sigma factors of extracytoplasmic function family; Asai K et al.; Target gene candidates of the seven extracytoplasmic function (ECF) sigma factors of Bacillus subtilis have been surveyed using DNA microarray analysis of mRNA extracted from cells grown in Luria-Bertani broth, in which an ECF sigma factor gene was placed under the control of the spac promoter on multicopy plasmid pDG148 and overexpressed . The number of target candidates for each of the sigma factors varied greatly, and a total of 278 genes were selected . Interestingly, the above target gene candidates shared only one gene out of 94 target genes of the general stress sigma B that have been reported in the literature thus far . Furthermore, lacZ-fusion experiments based on the results of DNA microarray analysis indicated that each ECF sigma factor directs transcription of its own operon, with the exception of sigZ . The DNA microarray data collected in this study are available at the KEGG Expression Database web site .

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 105 - 12
CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA; Radford DS et al.; The structure of the hypothetical copper-metallochaperone CopZ from Bacillus subtilis and its predicted partner CopA have been studied but their respective contributions to copper export, -import, -sequestration and -supply are unknown . DeltacopA was hypersensitive to copper and contained more copper atoms cell(-1) than wild-type . Expression from the copA operator-promoter increased in elevated copper (not other metals), consistent with a role in copper export . A bacterial two-hybrid assay revealed in vivo interaction between CopZ and the N-terminal domain of CopA but not that of a related transporter, YvgW, involved in cadmium-resistance . Activity of copper-requiring cytochrome caa(3) oxidase was retained in deltacopZ and deltacopA . DeltacopZ was only slightly copper-hypersensitive but deltacopZ/deltacopA was more sensitive than deltacopA, implying some action of CopZ that is independent of CopA . Significantly, deltacopZ contained fewer copper atoms cell(-1) than wild-type under these conditions . CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 49 - 55
Degradation of vanillic acid and production of guaiacol by microorganisms isolated from cork samples; Alvarez-Rodriguez ML et al.; The presence of guaiacol in cork stoppers is responsible for some cases of cork taint causing unpleasant alterations to wine . We have performed a characterization of the cork-associated microbiota by isolating 55 different microorganisms: eight yeast, 14 filamentous fungi or molds, 13 actinomycetes and 20 non-filamentous bacteria . A screening for degradation of vanillic acid and guaiacol production showed that none of the filamentous fungi could achieve any of these processes . By contrast, five of the eight yeast strains isolated were able to degrade vanillic acid, although it was not converted to guaiacol . Guaiacol production was only detected in four bacterial strains: one isolate of Bacillus subtilis and three actinomycetes, Streptomyces sp . A3, Streptomyces sp . A5 and Streptomyces sp . A13, were able to accumulate this compound in both liquid media and cultures over cork . These results suggest that guaiacol-mediated cork taint should be attributed to the degradative action of vanillic acid by bacterial strains growing on cork.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 4233 - 8 Epub 2003 Mar 17.
A regulatory protein that interferes with activator-stimulated transcription in bacteria; Nakano S et al.; Transcriptional activator proteins in bacteria often operate by interaction with the C-terminal domain of the alpha-subunit of RNA polymerase (RNAP) . Here we report the discovery of an "anti-alpha" factor Spx in Bacillus subtilis that blocks transcriptional activation by binding to the alpha-C-terminal domain, thereby interfering with the capacity of RNAP to respond to certain activator proteins . Spx disrupts complex formation between the activator proteins ResD and ComA and promoter-bound RNAP, and it does so by direct interaction with the alpha-subunit . ResD- and ComA-stimulated transcription requires the proteolytic elimination of Spx by the ATP-dependent protease ClpXP . Spx represents a class of transcriptional regulators that inhibit activator-stimulated transcription by interaction with alpha.

J Biol Chem, 2003 May 16, 278(20), 17852 - 8 Epub 2003 Mar 07.
Bacillus subtilis ResA is a thiol-disulfide oxidoreductase involved in cytochrome c synthesis; Erlendsson LS et al.; Covalent attachment of heme to apocytochromes c in bacteria occurs on the outside of the cytoplasmic membrane and requires two reduced cysteinyls at the heme binding site . A constructed ResA-deficient Bacillus subtilis strain was found to lack c-type cytochromes . Cytochrome c synthesis was restored in the mutant by: (i) in trans expression of resA; (ii) deficiency in BdbD, a thiol-disulfide oxidoreductase that catalyzes formation of an intramolecular disulfide bond in apocytochrome c after transfer of the polypeptide across the cytoplasmic membrane; or (iii) by addition of the reductant dithiothreitol to the growth medium . In vivo studies of ResA showed that it is membrane-associated with its thioredoxin-like domain on the outside of the cytoplasmic membrane . Analysis of a soluble form of the protein revealed two redox reactive cysteine residues with a midpoint potential of about -340 mV at pH 7 . We conclude that ResA, probably together with another thiol-disulfide oxidoreductase, CcdA, is required for the reduction of the cysteinyls in the heme binding site of apocytochrome c.

J Biol Chem, 2003 May 23, 278(21), 19416 - 25 Epub 2003 Mar 13.
Crystal structure of a bacterial endospore coat component . A laccase with enhanced thermostability properties; Enguita FJ et al.; Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat . The 65-kDa CotA protein is an abundant component of the outer coat layer . CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light . Here, we report the structure of CotA at 1.7-A resolution, as determined by x-ray crystallography . This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase . The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center . This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli . However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure . We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role . Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase . Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility . The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed.

J Biol Chem, 2003 May 16, 278(20), 18002 - 7 Epub 2003 Mar 12.
Purified, recombinant TagF protein from Bacillus subtilis 168 catalyzes the polymerization of glycerol phosphate onto a membrane acceptor in vitro; Schertzer JW et al.; We report the first characterization of a recombinant protein involved in the polymerization of wall teichoic acid . Previously, a study of the teichoic acid polymerase activity associated with membranes from Bacillus subtilis 168 strains bearing thermosensitive mutations in tagB, tagD, and tagF implicated TagF as the poly(glycerol phosphate) polymerase (Pooley, H . M., Abellan, F . X., and Karamata, D . (1992) J . Bacteriol . 174, 646-649) . In the work reported here, we have demonstrated an unequivocal role for tagF in the thermosensitivity of one such mutant (tagF1) by conditional complementation at the restrictive temperature with tagF under control of the xylose promoter at the amyE locus . We have overexpressed and purified recombinant B . subtilis TagF protein, and we provide direct biochemical evidence that this enzyme is responsible for polymerization of poly(glycerol phosphate) teichoic acid in B . subtilis 168 . Recombinant hexahistidine-tagged TagF protein was purified from Escherichia coli and was used to develop a novel membrane pelleting assay to monitor poly(glycerol phosphate) polymerase activity . Purified TagF was shown to incorporate radioactivity from its substrate CDP-{(14)C}glycerol into a membrane fraction in vitro . This activity showed a saturable dependence on the concentration of CDP-glycerol (K(m) of 340 microm) and the membrane acceptor (half-maximal activity at 650 microg of protein/ml of purified B . subtilis membranes) . High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, approximately 35 glycerol phosphate units in length.

Biochim Biophys Acta, 2003 Mar 21, 1646(1-2), 196 - 206
CTP:glycerol 3-phosphate cytidylyltransferase (TarD) from Staphylococcus aureus catalyzes the cytidylyl transfer via an ordered Bi-Bi reaction mechanism with micromolar K(m) values; Badurina DS et al.; CTP:glycerol 3-phosphate cytidylyltransferase catalyzes the formation of CDP-glycerol, an activated form of glycerol 3-phosphate and key precursor to wall teichoic acid biogenesis in Gram-positive bacteria . There is high sequence identity (69%) between the CTP:glycerol 3-phosphate cytidylyltransferases from Bacillus subtilis 168 (TagD) and Staphylococcus aureus (TarD) . The B . subtilis TagD protein was shown to catalyze cytidylyltransferase via a random mechanism with millimolar K(m) values for both CTP and glycerol 3-phosphate {J . Biol . Chem . 268, (1993) 16648} and exhibited negative cooperativity in the binding of substrates but not in catalysis {J . Biol . Chem . 276, (2001) 37922} . In the work described here on the S . aureus TarD protein, we have elucidated a steady state kinetic mechanism that is markedly different from that determined for B . subtilis TagD . Steady state kinetic experiments with recombinant, purified TarD employed a high-performance liquid chromatography assay developed in this work . The data were consistent with a ternary complex model . The K(m) values for CTP and glycerol 3-phosphate were 36 and 21 microM, respectively, and the k(cat) was 2.6 s(-1) . Steady state kinetic analysis of the reverse (pyrophosphorylase) reaction was also consistent with a ternary complex model . Product inhibition studies indicated an ordered Bi-Bi reaction mechanism where glycerol 3-phosphate was the leading substrate and the release of CDP-glycerol preceded that of pyrophosphate . Finally, we investigated the capacity of S . aureus tarD to substitute for tagD in B . subtilis . The tarD gene was placed under control of the xylose promoter in a B . subtilis 168 mutant defective in tagD (temperature-sensitive, tag-12) . Growth of the resulting strain at the restrictive temperature (47 degrees C) was shown to be xylose-dependent.

Microbiology, 2003 Mar, 149(Pt 3), 751 - 61
Expression of the glycolytic gapA operon in Bacillus subtilis: differential syntheses of proteins encoded by the operon; Meinken C et al.; Glycolysis is one of the central routes of carbon catabolism in Bacillus subtilis . Several glycolytic enzymes, including the key enzyme glyceraldehyde-3-phosphate dehydrogenase, are encoded in the hexacistronic gapA operon . Expression of this operon is induced by a variety of sugars and amino acids . Under non-inducing conditions, expression is repressed by the CggR repressor protein, the product of the promoter-proximal gene of the operon . Here, it is shown that the amount of glyceraldehyde-3-phosphate dehydrogenase encoded by the second gene of the operon exceeds that of the CggR repressor by about 100-fold . This differential synthesis was attributed to an mRNA processing event that takes place at the 3' end of the cggR open reading frame and to differential segmental stabilities of the resulting cleavage products . The mRNA specifying the truncated cggR gene is quickly degraded, whereas the downstream processing products encompassing gapA are quite stable . This increased stability is conferred by the presence of a stem-loop structure at the 5' end of the processed mRNAs . Mutations were introduced in the region of the cleavage site . A mutation affecting the stability of the stem-loop structure immediately downstream of the processing site had two effects . First, the steady-state transcript pattern was drastically shifted towards the primary transcripts; second, the stability of the processed mRNA containing the destabilized stem-loop structure was strongly decreased . This results in a reduction of the amount of glyceraldehyde-3-phosphate dehydrogenase in the cell . It is concluded that mRNA processing is involved in differential syntheses of the proteins encoded by the gapA operon.

Microbiology, 2003 Mar, 149(Pt 3), 739 - 50
Specificity of the interaction of RocR with the rocG-rocA intergenic region in Bacillus subtilis; Ali NO et al.; In Bacillus subtilis, expression of the rocG gene, encoding glutamate dehydrogenase, and the rocABC operon, involved in arginine catabolism, requires SigL (sigma(54))-containing RNA polymerase as well as RocR, a positive regulator of the NtrC/NifA family . The RocR protein was purified and shown to bind specifically to the intergenic region located between rocG and the rocABC operon . DNaseI footprinting experiments were used to define the RocR-binding site as an 8 bp inverted repeat, separated by one base pair, forming an imperfect palindrome which is present twice within the rocG-rocABC intergenic region, acting as both a downstream activating sequence (DAS) and an upstream activating sequence (UAS) . Point mutations in either of these two sequences significantly lowered expression of both rocG and rocABC . This bidirectional enhancer element retained partial activity even when moved 9 kb downstream of the rocA promoter . Electron microscopy experiments indicated that an intrinsically curved region is located between the UAS/DAS region and the promoter of the rocABC operon . This curvature could facilitate interaction of RocR with sigma(54)-RNA polymerase at the rocABC promoter.

Microbiology, 2003 Mar, 149(Pt 3), 569 - 77
The extracytoplasmic folding factor PrsA is required for protein secretion only in the presence of the cell wall in Bacillus subtilis; Wahlstrom E et al.; Pulse-chase labelling was used to study the role of the cell wall microenvironment in the functioning of Bacillus subtilis PrsA, an extracellular lipoprotein and member of the parvulin family of peptidylprolyl cis/trans-isomerases . It was found that in protoplasts, and thus in the absence of a cell wall matrix, the post-translocational folding, stability and secretion of the AmyQ alpha-amylase were independent of PrsA, in contrast to the strict dependency found in rods . The results indicate that PrsA is dedicated to assisting the folding and stability of exported proteins in the particular microenvironment of the cytoplasmic membrane-cell wall interface, possibly as a chaperone preventing unproductive interactions with the wall . The data also provide evidence for a crucial role of the wall in protein secretion . The presence of the wall directly or indirectly facilitates the release of AmyQ from the cell membrane and affects the rate of the signal peptide processing.

Eur J Biochem, 2003 Mar, 270(6), 1288 - 300
DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression; Homann A et al.; We have isolated and studied the cloned sigma factors SASIG1-3 from mustard (Sinapis alba) . In functional analyses using both promoter and factor mutants, the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions . Directed muta- genesis of SASIG1 identified critical residues within the conserved regions 2.4 and 4.2 necessary for binding of the -10 and -35 promoter elements, respectively . SASIG1 and 2, but not SASIG3, each have a typical region 2.5 for binding of the extended -10 promoter element . SASIG3 has a pro-sequence reminiscent of sigma K from Bacillus subtilis, suggesting that proteolytic cleavage from an inactive precursor is involved in the regulation of plastid transcription . In addition, SASIG2 was found to be more abundant in light-grown as compared to dark-grown mustard seedlings, while the converse was true for SASIG3.

Folia Microbiol (Praha), 2002, 47(6), 654 - 8
Presence and cellular distribution of soybean agglutinin-binding epitopes in two strains of Bacillus subtilis; Stoitsova SR; The cellular location of N-acetylgalactosamine in Bacillus subtilis strains 168 and 170 was examined by electron microscopy using gold-conjugated soybean agglutinin (SBA) as a marker . Post-embedding labeling of sectioned material showed SBA-reactive, galactosamine-containing polymers associated with the cell membrane or the cytoplasm of the two strains . This intracellular location was distinct from the concanavalin A-binding epitopes that were located over the cell wall . Labeling of whole cells (native, fixed in glutaraldehyde, or treated with proteinase K, or Tween 20) before negative staining revealed no galactosamine exposed on the surface of strain 168 . On the surface of strain 168 some exposed galactosamine terminal residues were detected; their accessibility to SBA increased when Tween 20 or proteinase K was applied.

Mutat Res, 2003 Feb-Mar, 523-524, 43 - 53
Antimutagenic effects of doenjang (Korean fermented soypaste) and its active compounds; Park KY et al.; Doenjang (Korean fermented soypaste) is one of the important fermented foods of Korea . Doenjang has been traditionally manufactured from meju, which is fermented rectangular shape molded from crushed cooked soybeans . The main microorganisms involved for meju fermentation are Bacillus subtilis and molds such as Rizopus sp., Mucor sp., and Aspergillus sp . We have already reported that doenjang is safe from mycotoxin, especially, aflatoxin contamination due to the manufacturing process of the doenjang . We have demonstrated that the doenjang extracts showed strong antimutagenic activities against various carcinogens/mutagens including aflatoxin B(1) . The traditionally fermented soypaste, doenjang showed higher antimutagenic activity than the raw soybeans, cooked soybeans, meju and other fermented soybeans in the Ames test . The active compounds that were identified are genistein, linoleic acid, beta-sitosterol glucoside, soyasaponin, etc . The active compounds exhibited strong antimutagenic activities in the Ames test, SOS chromotest and Drosophila wing spot test . More genistein was formed during the doenjang fermentation from genistin in the soybeans . Genistein and linoleic acid were the most effective active compounds found in doenjang .

Biochemistry, 2003 Mar 18, 42(10), 2971 - 81
Structural and mechanistic studies on ThiO, a glycine oxidase essential for thiamin biosynthesis in Bacillus subtilis; Settembre EC et al.; The thiO gene of Bacillus subtilis encodes an FAD-dependent glycine oxidase . This enzyme is a homotetramer with a monomer molecular mass of 42 kDa . In this paper, we demonstrate that ThiO is required for the biosynthesis of the thiazole moiety of thiamin pyrophosphate and describe the structure of the enzyme with N-acetylglycine bound at the active site . The closest structural relatives of ThiO are sarcosine oxidase and d-amino acid oxidase . The ThiO structure, as well as the observation that N-cyclopropylglycine is a good substrate, supports a hydride transfer mechanism for the enzyme . A mechanistic proposal for the role of ThiO in thiazole biosynthesis is also described.

Proteomics, 2003 Mar, 3(3), 299 - 306
The role of peptide deformylase in protein biosynthesis: a proteomic study; Bandow JE et al.; Recently we investigated the influence of classical and emerging antibiotics on the proteome of Bacillus subtilis including in our studies actinonin, a potent novel inhibitor of peptide deformylase . The protein synthesis pattern under actinonin treatment changed so dramatically that a direct comparison to the control pattern was impossible . Dual channel imaging revealed that actinonin treatment caused the majority of newly synthesised proteins to accumulate in spots different from the ones usually observed, indicating a more acidic isoelectric point . Two strategies were used to investigate the nature of the charge shift . In the first place, protein patterns of a conditional peptide deformylase mutant under nonrepressing and repressing conditions were compared . Secondly, several protein pairs excised from two-dimensional (2-D) gels of the peptide deformylase mutant, exponentially growing untreated wild-type and the actinonin treated wild-type were investigated with matrix-assisted laser desorption/ionization and electrospray ionization (ESI) time of flight mass spectrometry (TOF MS) for the existence of N-terminal formylation . Under nonrepressing conditions the mutant protein pattern resembled that of the wild-type . The loss of peptide deformylase activity under repressing conditions led to the same pI shift observed for actinonin treatment in the wild-type . Quadrupole TOF-MS on 11 protein pairs proved that the remaining N-terminal formyl residue was indeed responsible for the charge shift . Eight of these protein pairs were also present on 2-D gels of exponentially growing B . subtilis, where the more acidic, still formylated protein species represented the smaller parts.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3083 - 8 Epub 2003 Mar 07.
Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA; McDaniel BA et al.; Modulation of the structure of a leader RNA to control formation of an intrinsic termination signal is a common mechanism for regulation of gene expression in bacteria . Expression of the S box genes in Gram-positive organisms is induced in response to limitation for methionine . We previously postulated that methionine availability is monitored by binding of a regulatory factor to the leader RNA and suggested that methionine or S-adenosylmethionine (SAM) could serve as the metabolic signal . In this study, we show that efficient termination of the S box leader region by bacterial RNA polymerase depends on SAM but not on methionine or other related compounds . We also show that SAM directly binds to and induces a conformational change in the leader RNA . Both binding of SAM and SAM-directed transcription termination were blocked by leader mutations that cause constitutive expression in vivo . Overproduction of SAM synthetase in Bacillus subtilis resulted in delay in induction of S box gene expression in response to methionine starvation, consistent with the hypothesis that SAM is the molecular effector in vivo . These results indicate that SAM concentration is sensed directly by the nascent transcript in the absence of a trans-acting factor.

Prikl Biokhim Mikrobiol, 2003 Jan-Feb, 39(1), 43 - 6
{Evaluation of the effects of biological preparations on phytopathogenic fungi Didymella applanata and Botrytis cinerea}; Shpatova TV et al.; Fungicidal and fungistatic effects of biological preparations involving bacteria of the genera Pseudomonas and Bacillus and fungi of the genus Chaetomium on phytopathogenic fungi Didymella applanata and Botrytis cinerea were evalauated . All the biological preparations under study inhibited the growth of colonies of the fungi; however, the degree of the inhibition depended on the nature of each particular microorganism and the concentration of each particular preparation . The preparation containing Bacillus subtilis at a concentration of 0.2% effected maximum suppression of B . cinerea (the diameter of the colonies decreased sevenfold) . The preparations containing bacteria of the genus Pseudomonas and fungi of the genus Chaetomium were most efficient in suppressing D . applanata . The preparations containing B . subtilis and Chaetomium spp . showed promise as agents against simultaneous development of spur blight and Botrytis blight.

Mol Microbiol, 2003 Mar, 47(6), 1709 - 21
Regulation of the central glycolytic genes in Bacillus subtilis: binding of the repressor CggR to its single DNA target sequence is modulated by fructose-1,6-bisphosphate; Doan T et al.; Glycolysis is one of the best and widely conserved general metabolic pathways . Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA, pgk, tpi, pgm and eno . They are transcribed in a hexacistronic operon together with cggR, the first cistron, encoding the repressor of this gapA operon . Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon . CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence . Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN6TGTCN4CGGGACN6TG TC) . Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon . The presence of glycolytic carbon sources induces expression of the gapA operon . Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector . In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition) . FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes . Genomic sequence comparisons suggest that these results can apply to numerous low-G+C, Gram-positive bacterial species.

Mol Microbiol, 2003 Mar, 47(6), 1627 - 36
The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis; Stein T et al.; The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK . Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays . A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box) . Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative . Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location . Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR . A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.

Z Naturforsch {C}, 2003 Jan-Feb, 58(1-2), 70 - 5
Constituents of antibacterial extract of Caesalpinia paraguariensis Burk; Woldemichael GM et al.; The Argentinean legume Caesalpinia paraguariensis Burk . (Fabaceae) was selected for further fractionation work based on the strong antimicrobial activity of its CH2Cl2-MeOH (1:1 v/v) extract against a host of clinically significant microorganisms, including antibiotic resistant strains . 1D and 2D NMR enabled the identification of the novel benzoxecin derivative caesalpinol along with the known compounds bilobetin, stigma-5-en-3-O-beta-6'-stearoylglucopyranoside, stigma-5-en-3-beta-6'-palmitoylglucopyranoside, stigma-5-en-3-beta-glucopyranoside, oleanolic acid, 3-O-(E)-hydroxycinnamoyl oleanolic acid, betulinic acid, 3-O-(E)-hydroxycinnamoyl betulinic acid, and lupeol from the active fractions . Oleanolic acid was found active against Bacillus subtilis and both methicillin-sensitive and -resistant Staphylococcus aureus with MICs of 8 (17.5 microM), 8 and 64 (140 microM) microg/ml, respectively . The rest of the compounds, however, did not show activity.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 233 - 9
kdpE and a putative RsbQ homologue contribute to growth of Listeria monocytogenes at high osmolarity and low temperature; Brondsted L et al.; The kdp locus of Listeria monocytogenes encodes products with homology to structural proteins of a high-affinity potassium uptake system and to a two-component signal transduction system commonly involved in controlling gene expression . We have investigated the role of kdpE, encoding the transcriptional response regulator, as well as of the downstream gene, orfX, in adaptation of L . monocytogenes EGD to NaCl and low temperature . When grown in chemically defined medium the addition of NaCl to 2% decreased the growth rate of a mutant with an insertional inactivated kdpE, while mutants carrying in-frame deletions of either kdpE or orfX were unaffected by high osmolarity . Transcriptional analysis of kdpE and orfX revealed that their products are encoded by the same transcript . Thus, our data indicate that the absence of both KdpE and OrfX influences growth under osmotic pressure . Interestingly, OrfX contains a conserved domain of alpha/beta-hydrolases and resembles RsbQ that in Bacillus subtilis participates in the activation cascade of the general stress sigma factor SigB . When shifted to low temperature the deletion mutant lacking orfX resumed growth slightly faster than the wild-type . This phenotype was shared by a mutant carrying an in-frame deletion of sigB supporting the notion that OrfX could be a RsbQ homologue.

J Bacteriol, 2003 Mar, 185(6), 2005 - 8
The membrane domain of SpoIIIE is required for membrane fusion during Bacillus subtilis sporulation; Sharp MD et al.; During Bacillus subtilis sporulation, SpoIIIE is required for both postseptational chromosome segregation and membrane fusion after engulfment . Here we demonstrate that SpoIIIE must be present in the mother cell to promote membrane fusion and that the N-terminal membrane-spanning segments constitute a minimal membrane fusion domain, as well as direct septal localization.

J Bacteriol, 2003 Mar, 185(6), 1967 - 75
Global characterization of disulfide stress in Bacillus subtilis; Leichert LI et al.; We used DNA macroarray and proteome analysis to analyze the regulatory networks in Bacillus subtilis that are affected by disulfide stress . To induce disulfide stress, we used the specific thiol oxidant diamide . After addition of 1 mM diamide to an exponentially growing culture, cell growth stopped until the medium was cleared of diamide . Global analysis of the mRNA expression pattern during growth arrest revealed 350 genes that were induced by disulfide stress by greater than threefold . Strongly induced genes included known oxidative stress genes that are under the control of the global repressor PerR and heat shock genes controlled by the global repressor CtsR . Other genes that were strongly induced encode putative regulators of gene expression and proteins protecting against toxic elements and heavy metals . Many genes were substantially repressed by disulfide stress, among them most of the genes belonging to the negative stringent response . Two-dimensional gels of radioactively labeled protein extracts allowed us to visualize and quantitate the massive changes in the protein expression pattern that occurred in response to disulfide stress . The observed dramatic alteration in the protein pattern reflected the changes found in the transcriptome experiments . The response to disulfide stress seems to be a complex combination of different regulatory networks, indicating that redox-sensing cysteines play a key role in different signaling pathways sensing oxidative stress, heat stress, toxic element stress, and growth inhibition.

J Bacteriol, 2003 Mar, 185(6), 1911 - 22
Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis; Molle V et al.; Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays . The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.

J Biol Chem, 2003 May 9, 278(19), 16482 - 7 Epub 2003 Mar 03.
Dimerization and DNA binding properties of the Bacillus licheniformis 749/I BlaI repressor; Filee P et al.; In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI . Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon . In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 microm by equilibrium ultracentrifugation . Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B . licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the beta-lactamase locus (blaP-blaI-blaR1) were lower than (1.9 microm) or in the same range as (75 microm) the dissociation constant, respectively . This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer . This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence . The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K(d)(OP1) = 1.7 +/- 0.5 10(-15) m(2), K(d)(OP2) = 3.3 +/- 0.9 10(-15) m(2), and K(d)(OP3) = 10.5 +/- 2.5 10(-15) m(2) . The role of the DNA binding properties of BlaI on the beta-lactamase regulation is discussed.

Nature, 2003 Feb 13, 421(6924), 760 - 4
Crystal structure of the specificity domain of ribonuclease P; Krasilnikov AS et al.; RNase P is the only endonuclease responsible for processing the 5' end of transfer RNA by cleaving a precursor and leading to tRNA maturation . It contains an RNA component and a protein component and has been identified in all organisms . It was one of the first catalytic RNAs identified and the first that acts as a multiple-turnover enzyme in vivo . RNase P and the ribosome are so far the only two ribozymes known to be conserved in all kingdoms of life . The RNA component of bacterial RNase P can catalyse pre-tRNA cleavage in the absence of the RNase P protein in vitro and consists of two domains: a specificity domain and a catalytic domain . Here we report a 3.15-A resolution crystal structure of the 154-nucleotide specificity domain of Bacillus subtilis RNase P . The structure reveals the architecture of this domain, the interactions that maintain the overall fold of the molecule, a large non-helical but well-structured module that is conserved in all RNase P RNA, and the regions that are involved in interactions with the substrate.

J Nat Prod, 2003 Feb, 66(2), 242 - 6
Antibacterial diterpenes from Calceolaria pinifolia; Woldemichael GM et al.; Two new isopimaranes, 19-methylmalonyloxy-ent-isopimara-8(9),15-diene (5) and 19-malonyloxy-ent-isopimara-8(9),15-diene (6), were isolated using bioassay-guided fractionation of the CH(2)Cl(2)-MeOH (1:1) extract of the aerial part of Calceolaria pinifolia along with eight other diterpenes (1-4, 7-10) and two triterpenes (11, 12) . All compounds were assayed against Staphylococcus aureus (SA), methicillin-resistant S . aureus (MRSA), Bacillus subtilis (BS), and Escherichia coli (EC) . 4-Epi-dehydroabietinol (2) and ent-isopimara-9(11),15-diene-19-ol (8) were found to be active against MRSA with MIC values of 8 and 2 microgram/mL, respectively . Mechanistic studies of 8 in BS suggested rapid and nonspecific inhibition of uptake and incorporation of radiolabeled precursors into DNA, RNA, and protein consistent with membrane-damaging effects in bacteria . Compound 8 did not afford protection against an acute infection with SA in mice.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 948 - 55
Proteomic approach to understanding antibiotic action; Bandow JE et al.; We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes . We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage . Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified . Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor . These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds . Such a method should prove useful in the process of antibiotic drug discovery.

Mol Microbiol, 2003 Mar, 47(5), 1353 - 66
The conserved cytoplasmic module of the transmembrane chemoreceptor McpC mediates carbohydrate chemotaxis in Bacillus subtilis; Kristich CJ et al.; Escherichia coli cells use two distinct sensory circuits during chemotaxis towards carbohydrates . One circuit requires the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and is independent of any specific chemoreceptor, whereas the other uses a chemoreceptor-dependent sensory mechanism analogous to that used during chemotaxis towards amino acids . Work on the carbohydrate chemotaxis sensory circuit of Bacillus subtilis reported in this article indicates that the B . subtilis circuit is different from either of those used by E . coli . Our chemotactic analysis of B . subtilis strains expressing various chimeric chemoreceptors indicates that the cytoplasmic, C-terminal module of the chemoreceptor McpC acts as a sensory-input element during carbohydrate chemotaxis . Our results also indicate that PTS-mediated carbohydrate transport, but not carbohydrate metabolism, is required for production of a chemotactic signal . We propose a model in which PTS-transport-induced chemotactic signals are transmitted to the C-terminal module of McpC for control of chemotaxis towards PTS carbohydrates.

Mol Microbiol, 2003 Mar, 47(5), 1251 - 63
Characterization of a novel inhibitory feedback of the anti-anti-sigma SpoIIAA on Spo0A activation during development in Bacillus subtilis; Arabolaza AL et al.; Compartmentalized gene expression during sporulation is initiated after asymmetric division by cell-specific activation of the transcription factors sigmaF and sigmaE . Synthesis of these sigma factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system . We report here a novel regulatory function of the anti-anti-sigmaF SpoIIAA as inhibitor of Spo0A activation . This effect did not require sigmaF activity, and it was abolished by expression of the phosphorelay-independent form Spo0A-Sad67 indicating that SpoIIAA directly interfered with Spo0A approximately P generation . IPTG-directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA-P . Furthermore, expression of the non-phosphorylatable mutant SpoIIAAS58A (SpoIIAA-like), but not SpoIIAAS58D (SpoIIAA-P-like), completely blocked Spo0A-dependent gene expression . Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis . These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of sigmaF and sigmaE during development.

Pac Symp Biocomput . 2003;:17-28.
Inferring gene regulatory networks from time-ordered gene expression data of Bacillus subtilis using differential equations; de Hoon MJ et al.; We describe a new method to infer a gene regulatory network, in terms of a linear system of differential equations, from time course gene expression data . As biologically the gene regulatory network is known to be sparse, we expect most coefficients in such a linear system of differential equations to be zero . In previously proposed methods, the number of nonzero coefficients in the system was limited based on ad hoc assumptions . Instead, we propose to infer the degree of sparseness of the gene regulatory network from the data, where we use Akaike's Information Criterion to determine which coefficients are nonzero . We apply our method to MMGE time course data of Bacillus subtilis.

Phytother Res, 2003 Feb, 17(2), 129 - 34
Isolation, characterization and biological activity of betulinic acid and ursolic acid from Vitex negundo L; Chandramu C et al.; Two pentacyclic triterpenoids, betulinic acid (3beta-hydroxylup-20-(29)-en-28-oic acid) (3), and ursolic acid (2beta-hydroxyurs-12-en-28-oic acid) (4), were isolated for the first time from leaves of Vitex negundo L . along with three other compounds; an aliphatic alcohol n-hentriacontanol (1), beta-sitosterol (2) and p-hydroxybenzoic acid (5) . Their antifeedant activity against the larvae of an agricultural pest, the castor semilooper (Achoea janata), in a no-choice laboratory assay and their antibacterial activity against Bacillus subtilis and Escherichia coli, by the paper disc method, were tested . Ursolic acid (4) showed more effective antifeedant activity than the betulinic acid (3) . However, both these compounds have shown a very mild antibacterial activity . The other three compounds; n-hentriacontanol (1), beta-sitosterol (2) and p-hydroxybenzoic acid (5) have shown a little antifeedant activity against the larvae and did not show any antibacterial activity .

Water Res, 2003 Apr, 37(7), 1667 - 77
A stochastic model of an ozonation reactor; Gujer W et al.; Disinfection of some microorganisms is characterized by a lag-phase (a minimum required ozone exposure until disinfection occurs) . This phenomenon is easy to model in laboratory batch reactors but not in continuous flow mixed reactors . This paper introduces a stochastic disinfection model where individual microorganisms are followed on their paths through full-scale reactors . Combining exponentially distributed transport processes with delayed exponential disinfection kinetics for large populations of microorganisms (up to 10,000 individuals) yields predictions which can be evaluated statistically . It could be shown that deterministic models work well for systems with good disinfection performance (more than 2 log units reduction of active microorganisms), for reactors with poor performance stochastic models have to be applied . It could be demonstrated for real reactors that Bacillus subtilis spores are poor surrogates for Cryptosporidium parvum oocysts . The differences between the two microorganisms are large for reactors that deviate significantly from plug-flow behaviour .

Water Res, 2003 Apr, 37(7), 1469 - 87
Ozonation of drinking water: part II . Disinfection and by-product formation in presence of bromide, iodide or chlorine; von Gunten U; Ozone is an excellent disinfectant and can even be used to inactivate microorganisms such as protozoa which are very resistant to conventional disinfectants . Proper rate constants for the inactivation of microorganisms are only available for six species (E . coli, Bacillus subtilis spores, Rotavirus, Giardia lamblia cysts, Giardia muris cysts, Cryptosporidium parvum oocysts) . The apparent activation energy for the inactivation of bacteria is in the same order as most chemical reactions (35-50 kJ mol(-1)), whereas it is much higher for the inactivation of protozoa (80 kJ mol(-1)) . This requires significantly higher ozone exposures at low temperatures to get a similar inactivation for protozoa . Even for the inactivation of resistant microorganisms, OH radicals only play a minor role . Numerous organic and inorganic ozonation disinfection/oxidation by-products have been identified . The by-product of main concern is bromate, which is formed in bromide-containing waters . A low drinking water standard of 10 microg l(-1) has been set for bromate . Therefore, disinfection and oxidation processes have to be evaluated to fulfil these criteria . In certain cases, when bromide concentrations are above about 50 microg l(-1), it may be necessary to use control measures to lower bromate formation (lowering of pH, ammonia addition) . Iodate is the main by-product formed during ozonation of iodide-containing waters . The reactions involved are direct ozone oxidations . Iodate is considered non-problematic because it is transformed back to iodide endogenically . Chloride cannot be oxidized during ozonation processes under drinking water conditions . Chlorate is only formed if a preoxidation by chlorine and/or chlorine dioxide has occurred .

Biochemistry, 2003 Mar 4, 42(8), 2467 - 74
X-ray absorption and NMR spectroscopic studies of CopZ, a copper chaperone in Bacillus subtilis: the coordination properties of the copper ion; Banci L et al.; XAS studies have been performed, under various experimental conditions, on a copper(I)-transporting protein, CopZ, of Bacillus subtilis . The copper(I) ion, reduced with dithiothreitol, is three-coordinate with three sulfur donor atoms, two of which presumably provided by the protein and one by dithiothreitol . If a molar excess of acetate (15 mM; 5:1 respect to CopZ) or citrate (6 mM; 2:1 respect to CopZ) is present in solution, the EXAFS spectra suggest the presence of a dimeric form involving a close contact between Cu(I) ions from two molecules, where Cu is still three-coordinate . (1)H and (15)N NMR data provide further structural details . If copper reduction is accomplished with ascorbate, the data indicate that one oxygen of ascorbate enters in the first-coordination sphere of copper, together with two sulfur atoms, in a dimeric form of the protein . These results are instructive and have been discussed with respect to the molecular basis of copper trafficking.

Biotechnol Bioeng, 2003 May 5, 82(3), 299 - 305
Rheology, oxygen transfer, and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis; Richard A et al.; Poly(glutamic acid) (PGA) is a water-soluble, biodegradable biopolymer that is produced by microbial fermentation . Recent research has shown that PGA can be used in drug delivery applications for the controlled release of paclitaxel (Taxol) in cancer treatment . A fundamental understanding of the key fermentation parameters is necessary to optimize the production and molecular weight characteristics of poly(glutamic acid) by Bacillus subtilis for paclitaxel and other applications of pharmaceuticals for controlled release . Because of its high molecular weight, PGA fermentation broths exhibit non-Newtonian rheology . In this article we present experimental results on the batch fermentation kinetics of PGA production, mass transfer of oxygen, specific oxygen uptake rate, broth rheology, and molecular weight characterization of the PGA biopolymer .

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2306 - 11 Epub 2003 Feb 21.
MecA, an adaptor protein necessary for ClpC chaperone activity; Schlothauer T et al.; ClpC of Bacillus subtilis is an ATP-dependent HSP100Clp protein involved in general stress survival . A complex of ClpC with the protease ClpP and the adaptor protein MecA also controls competence development by regulated proteolysis of the transcription factor ComK . We investigated the in vitro chaperone activity of ClpC and found that the presence of MecA was crucial for the major chaperone activities of ClpC . In particular, MecA enabled ClpC to solubilize and refold aggregated proteins . Finally, in the presence of ClpP, MecA allowed the ClpC-dependent degradation of unfolded or heat-aggregated proteins . This study demonstrates that adaptor proteins like MecA through interaction with their cognate ClpC proteins can have a dual role in the protein quality-control network by rescuing, or together with ClpP, by degrading, aggregated proteins . MecA can thereby coordinate substrate targeting with ClpC activation, adding another layer to the regulation of HSP100/Clp protein activity.

Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2594 - 9
Cloning of the maltose phosphorylase gene from Bacillus sp . strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis; Inoue Y et al.; The maltose phosphorylase (MPase) gene of Bacillus sp . strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme . The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1 . This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis . The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask . About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B . subtilis.

Life Sci Space Res, 1977, 15, 147 - 50
Assignment of particle tracks to spores of Bacillus subtilis on silver chloride detectors; Schopper E et al.; In Biostack III B, flown in the Apollo-Soyuz Test Project, AgCl detectors were used to study ionizing effects of HZE particles on spores of Bacillus subtilis or eggs of Artemia salina . The tracks of these particles inside the detectors are used to extrapolate the path of the particle near the biological objects which are fixed at the detector surface . The closest distance to the geometric centre of the object, the so-called impact parameter, is determined with a mean accuracy of 0.3 micrometers for 1 micrometers spores . From knowledge of the lateral distribution of the energy transferred by primary and secondary ionization effects of the particle, the energy deposit and its localization at the objects can be determined . We describe some technical aspects of a video-electronic scanning system, Quantimet 720, which has been adjusted to the particular requirements of these experiments . The main improvements achieved are increased precision of coordinate measurements, objective focusing of the microscopic image combined with measurements of the density profile of particle tracks, and finally speeding up of the measurements by automatic data transfer.

Int J Food Microbiol, 2003 May 15, 82(3), 255 - 64
Cytokine responses of human intestinal epithelial-like Caco-2 cells to the nonpathogenic bacterium Bacillus subtilis (natto); Hosoi T et al.; Intestinal epithelial cells produce cytokines in response to pathogenic bacteria . However, cellular responses of these cells to nonpathogenic strains, such as Bacillus subtilis, are yet to be determined . In this study, we investigate whether epithelial-like human colon carcinoma Caco-2 cells produce cytokines in response to B . subtilis or B . subtilis (natto) . The latter strain is utilized for manufacturing the fermented soy food "natto" . Live cells of nonpathogenic B . subtilis JCM 1465(T), B . subtilis (natto) and E . coli JCM 1649(T), as well as pathogenic S . enteritidis JCM 1652 and P . aeruginosa JCM 5516 strains, induced secretion of interleukin-6 (IL-6) and/or IL-8, but not IL-7, IL-15 or tumor necrosis factor alpha (TNF-alpha) . Transepithelial electrical resistance (TER) of Caco-2 cell monolayers cultured with E . coli, S . enteritidis or P . aeruginosa decreased more rapidly than that of cells cultured with B . subtilis or B . subtilis (natto) . The amounts of cytokine induced by B . subtilis (natto) cells were strain-dependent . Moreover, B . subtilis (natto) cells subjected to hydrochloric acid treatment, but not autoclaving, induced a higher secretion of IL-6 and IL-8 than intact cells . Tyrosine kinase inhibitors, including AG126 and genistein, suppressed cytokine secretion . Our results suggest that the nonpathogenic B . subtilis (natto) bacterium induces cytokine responses in intestinal epithelial cells via activation of an intracellular signaling pathway, such as that of nuclear factor-kappa B (NF-kappaB).

Astrobiology, 2002 Winter, 2(4), 417 - 25
UV photochemistry of DNA in vitro and in Bacillus subtilis spores at earth-ambient and low atmospheric pressure: implications for spore survival on other planets or moons in the solar system; Nicholson WL et al.; Two major parameters influencing the survival of Bacillus subtilis spores in space and on bodies within the Solar System are UV radiation and vacuum, both of which induce inactivating damage to DNA . To date, however, spore survival and DNA photochemistry have been explored only at the extremes of Earth-normal atmospheric pressure (101.3 kPa) and at simulated space vacuum (10(-3)-10(-6) Pa) . In this study, wild-type spores, mutant spores lacking alpha/beta-type small, acid-soluble spore proteins (SASP), naked DNA, and complexes between SASP SspC and DNA were exposed simultaneously to UV (254 nm) at intermediate pressure (1-2 Pa), and the UV photoproducts cis,syn-thymine-thymine cyclobutane dimer (c,sTT), trans,syn-thymine-thymine cyclobutane dimer (t,sTT), and "spore photoproduct" (SP) were quantified . At 101.3 kPa, UV-treated wild-type spores accumulated only SP, but spores treated with UV radiation at 1-2 Pa exhibited a spectrum of DNA damage similar to that of spores treated at 10(-6) Pa, with accumulation of SP, c,sTT, and t,sTT . The presence or absence of alpha/beta-type SASP in spores was partly responsible for the shift observed between levels of SP and c,sTT, but not t,sTT . The changes observed in spore DNA photochemistry at 1-2 Pa in vivo were not reproduced by irradiation of naked DNA or SspC:DNA complexes in vitro, suggesting that factors other than SASP are involved in spore DNA photochemistry at low pressure.

J Bacteriol, 2003 Mar, 185(5), 1672 - 80
Complex regulation of the Bacillus subtilis aconitase gene; Kim HJ et al.; The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth . CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium . A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations . However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation . An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium . All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region . Interaction of CcpC and CodY with the citB promoter region was partially competitive.

J Bacteriol, 2003 Mar, 185(5), 1590 - 8
Novel spoIIE mutation that causes uncompartmentalized sigmaF activation in Bacillus subtilis; Hilbert DW et al.; During sporulation, Bacillus subtilis undergoes an asymmetric division that results in two cells with different fates, the larger mother cell and the smaller forespore . The protein phosphatase SpoIIE, which is required for activation of the forespore-specific transcription factor sigma(F), is also required for optimal efficiency and timing of asymmetric division . We performed a genetic screen for spoIIE mutants that were impaired in sporulation but not sigma(F) activity and isolated a strain with the mutation spoIIEV697A . The mutant exhibited a 10- to 40-fold reduction in sporulation and a sixfold reduction in asymmetric division compared to the parent . Transcription of the sigma(F)-dependent spoIIQ promoter was increased more than 10-fold and was no longer confined to the forespore . The excessive sigma(F) activity persisted even when asymmetric division was prevented . Disruption of spoIIGB did not restore asymmetric division to the spoIIEV697A mutant, indicating that the deficiency is not a consequence of predivisional activation of the mother cell-specific transcription factor sigma(E) . Deletion of the gene encoding sigma(F) (spoIIAC) restored asymmetric division; however, a mutation that dramatically reduced the number of promoters responsive to sigma(F), spoIIAC561 (spoIIACV233 M), failed to do so . This result suggests that the block is due to expression of one of the small subset of sigma(F)-dependent genes expressed in this background or to unregulated interaction of sigmaF with some other factor . Our results indicate that regulation of SpoIIE plays a critical role in coupling asymmetric division to sigma(F) activation in order to ensure proper spatial and temporal expression of forespore-specific genes.

Biochemistry, 2003 Feb 25, 42(7), 1939 - 49
Understanding copper trafficking in bacteria: interaction between the copper transport protein CopZ and the N-terminal domain of the copper ATPase CopA from Bacillus subtilis; Banci L et al.; In this paper the interaction of cytoplasmic CopZ and the N-terminal domain of the CopA ATPase from Bacillus subtilis has been studied by NMR through (15)N-(1)H HSQC experiments in order to understand the role of the two proteins in the whole copper trafficking mechanism of the bacteria . It appears that the two proteins interact in a fashion similar to that of the yeast homologue proteins {Arnesano, F., Banci, L., Bertini, I., Cantini, F., Ciofi-Baffoni, S., Huffman, D . L., and O'Halloran, T . V . (2001) J . Biol . Chem . 276, 41365-41376}, although the surface potentials are reversed . A structural model for the interaction is proposed . (15)N mobility studies on the free proteins and on their complex are also reported . From these data, it appears that copper is largely transferred from CopZ to CopA, thus suggesting their possible involvement in a detoxification process . Comparing functional data of homologous proteins of other bacteria, it can be concluded that this class of proteins is involved in copper homeostasis but the specific roles are species dependent.

Biochemistry, 2003 Feb 25, 42(7), 1831 - 41
Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role; Palenchar JB et al.; Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme . Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu . The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate . Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes . We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity . An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes . The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes . The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme . We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.

J Biol Chem, 2003 Apr 25, 278(17), 15304 - 12 Epub 2003 Feb 13.
Structural analysis of Bacillus subtilis SPP1 phage helicase loader protein G39P; Bailey S et al.; The Bacillus subtilis SPP1 phage-encoded protein G39P is a loader and inhibitor of the phage G40P replicative helicase involved in the initiation of DNA replication . We have carried out a full x-ray crystallographic and preliminary NMR analysis of G39P and functional studies of the protein, including assays for helicase binding by a number of truncated mutant forms, in an effort to improve our understanding of how it both interacts with the helicase and with the phage replisome organizer, G38P . Our structural analyses reveal that G39P has a completely unexpected bipartite structure comprising a folded N-terminal domain and an essentially unfolded C-terminal domain . Although G39P has been shown to bind its G40P target with a 6:6 stoichiometry, our crystal structure and other biophysical characterization data reveal that the protein probably exists predominantly as a monomer in solution . The G39P protein is proteolytically sensitive, and our binding assays show that the C-terminal domain is essential for helicase interaction and that removal of just the 14 C-terminal residues abolishes interaction with the helicase in vitro . We propose a number of possible scenarios in which the flexibility of the C-terminal domain of G39P and its proteolytic sensitivity may have important roles for the function of G39P in vivo that are consistent with other data on SPP1 phage DNA replication.

J Appl Microbiol, 2003, 94(3), 396 - 402
Degradation of proteins during the fermentation of African locust bean (Parkia biglobosa) by strains of Bacillus subtilis and Bacillus pumilus for production of Soumbala; Ouoba LI et al.; AIMS: To examine isolates of Bacillus subtilis and B . pumilus predominant in Soumbala for their ability to degrade African locust bean proteins (ALBP) . METHODS AND RESULTS: Agar diffusion test in casein and ALBP agar was used for screening of isolates . The profiles of water-soluble proteins and free amino acids (FAA) during the fermentation of ALBP by the Bacillus isolates were studied by SDS-PAGE and cation exchange chromatography . The profile of soluble proteins changed with the fermentation time and varied depending on the isolate . The quantity of total FAA and essential FAA such as lysine was increased sharply between 24 and 48 h of fermentation and differed among the isolates . Simultaneously, a pH increase was observed . Cysteine, methionine, leucine, isoleucine, tyrosine and phenylalaline appeared during fermentation . CONCLUSION: The Bacillus isolates studied degraded ALBP leading to a profile of soluble proteins and FAA specific for each isolate . SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of Soumbala.

J Appl Microbiol, 2003, 94(3), 375 - 81
Expression of a Bacillus thuringiensisdelta-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants (Lycopersicon esculentum, Mills); Theoduloz C et al.; AIMS: To introduce a cry gene into microorganisms that naturally colonize the phylloplane of tomato plants to improve the persistence of the Cry proteins for controlling a South American tomato moth (Tuta absoluta, Meyrick, 1917) . METHODS AND RESULTS: A cry1Ab gene isolated from a native Bacillus thuringiensis strain (LM-466), showing a relevant activity against T . absoluta larvae, was cloned into the shuttle vector pHT315 (Arantes and Lereclus 1991) . The construct was introduced by electroporation into native Bacillus subtilis and Bacillus licheniformis strains, both natural inhabitants of the tomato phylloplane . Western analysis and toxicity assays against the target larvae proved that the successful expression of the gene was accomplished in host bacteria . Recombinant toxin displayed a similar LC50 value in comparison to native donor strain LM-466 . Both transformed Bacillus survived for at least 45 days on the tomato leaf surface . CONCLUSIONS: Plant-associated microorganisms that naturally colonize the phylloplane could be useful as recombinant microbial delivery systems of toxin genes of B . thuringiensis . SIGNIFICANCE AND IMPACT OF THE STUDY: Modified microorganisms capable of surviving on leaf surfaces for several weeks with insecticidal activity should allow for a reduction in pesticide application.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 299 - 304
The Bacillus subtilis transition state regulator AbrB binds to the -35 promoter region of comK; Hamoen LW et al.; Genetic competence is a differentiation process initiated by Bacillus subtilis as a result of nutritional deprivation, and is controlled by a complex signal transduction cascade . The promoter of comK, encoding the competence transcription factor, is regulated by at least four different transcription factors: Rok, CodY, DegU and ComK itself . Genetic data have shown that comK expression is influenced by the transition state regulator AbrB as well . In this paper we show that AbrB binds specifically to the comK promoter and covers the RNA polymerase binding site, making it the fifth transcription factor regulating the activity of the comK promoter.

Biochem J, 2003 May 15, 372(Pt 1), 113 - 9
Phosphorylation induces subtle structural changes in SpoIIAA, a key regulator of sporulation; Clarkson J et al.; The phosphorylation state of SpoIIAA is a key factor in the regulation of sporulation in Bacillus subtilis . Previous crystallographic studies had led to the conclusion that phosphorylation alters the binding affinity of SpoIIAA for its partner proteins solely through the additional charge and bulk of the phosphoryl group: small structural changes observed elsewhere in the protein were considered to be random fluctuations rather than the result of phosphorylation . The results presented in the present paper show that NMR studies detect the same subtle structural changes in solution as those seen in the crystal, strongly implying that they are the direct result of phosphorylation . These subtle structural changes are similar to those that occur in a non-phosphorylated mutant that is defective in binding to one of its partner proteins . We propose that the structural changes which occur in SpoIIAA on phosphorylation act in concert with the phosphoryl group to alter its binding properties.

Chemistry, 2003 Feb 17, 9(4), 984 - 91
Preorganization and reorganization as related factors in enzyme catalysis: the chorismate mutase case; Marti S et al.; In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques . The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement . To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site . Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character . With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure . This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier . Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants . This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants) . In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.

Bioinformatics, 2003 Feb 12, 19(3), 336 - 44
Genetic Network Analyzer: qualitative simulation of genetic regulatory networks; de Jong H et al.; MOTIVATION: The study of genetic regulatory networks has received a major impetus from the recent development of experimental techniques allowing the measurement of patterns of gene expression in a massively parallel way . This experimental progress calls for the development of appropriate computer tools for the modeling and simulation of gene regulation processes . RESULTS: We present Genetic Network Analyzer (GNA), a computer tool for the modeling and simulation of genetic regulatory networks . The tool is based on a qualitative simulation method that employs coarse-grained models of regulatory networks . The use of GNA is illustrated by a case study of the network of genes and interactions regulating the initiation of sporulation in Bacillus subtilis . AVAILABILITY: GNA and the model of the sporulation network are available at http://www-helix.inrialpes.fr/gna.

Plasmid, 2003 Jan, 49(1), 53 - 62
Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector; Titok MA et al.; We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus . Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb . Small plasmids were shown to carry a rolling circle replicon of the pC194 type . Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination . A B . subtilis/Escherichia coli shuttle vector based on this replicon was constructed . It has a low copy number (6 units per chromosome) and is stably inherited in B . subtilis . It might thus be a useful tool for DNA cloning . These data extend previous observations, indicating that most of the small plasmids of B . subtilis replicate as rolling circles and belong to the pC194 family . On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 31 - 8
Identification and disruption of btlA, a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes; Begley M et al.; A transposon Tn917 mutant of Listeria monocytogenes L028 was isolated on the basis of reduced growth on agar adjusted to pH 5.5 . The disrupted gene, designated btlA (bile tolerance locus), encodes a putative secondary transporter of the major facilitator superfamily, which has significant homology to yxiO in Bacillus subtilis (lmo1417 in L . monocytogenes EGDe) . The mutant demonstrated decreased growth rates relative to the wild-type when grown in sub-lethal levels of various stressors (acid, salt, ethanol, bile, SDS, ampicillin and phosphomycin) . The mutant was also more sensitive to lethal levels of bile . A pORI19 insertion mutant demonstrated similar phenotypes . Murine virulence studies indicated that disruption of btlA does not influence virulence potential . BtlA therefore represents a membrane protein essential for the maintenance of homeostasis under stress conditions, but is not involved in pathogenicity.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 23 - 30
The DNA secondary structure of the Bacillus subtilis genome; Tosato V et al.; The entire genomic DNA sequence of the Gram-positive bacterium Bacillus subtilis reported in the SubtiList database has been subjected in this work to a complete bioinformatic analysis of the potential formation of secondary DNA structures such as hairpins and bending . The most significant of these structures have been mapped with respect to their genomic location and compared to those structures already known to have a physiological role, such as the rho-independent transcription terminators . The distribution of these structures along the bacterial chromosome shows two major features: (i) . the concentration of the most curved DNA in the intergenic regions rather than within the ORFs, and (ii) . a decreasing gradient of large hairpins from the origin towards the terC end of chromosomal DNA replication . Given the increasing biological relevance of secondary DNA structures, these findings should facilitate further studies on the evolution, dynamics and expression of the genetic information stored in bacterial genomes.

Syst Appl Microbiol, 2002 Dec, 25(4), 478 - 82
Transformation of Bacillus subtilis in chocolate milk: evidence for low frequency of establishment of cells transformed under non-selective conditions; Wittke A et al.; Transformation of naturally competent Bacillus subtilis with plasmid was carried out in chocolate milk without antibiotics . Transformed cells were enumerated during the entire growth phase in chocolate milk . When DNA was added to aliquots of a batch culture after different times of incubation, transformation events were detected at all different growth stages . When DNA was added to a batch culture together with the inoculum, transformed cells were detected at the onset of exponential growth . However, apparently no or only limited growth of these transformed cells was observed . To clarify, whether the limitation of growth was due to suppression by non-transformed cells, different proportions of B . subtilis cells either carrying or not carrying the plasmid were mixed and inoculated into chocolate milk without antibiotic . Our results indicate that suppression appears to be of minor importance . Instead, plasmid-bearing cells appear to suffer from a prolonged lag-phase . However, the failure to exhibit significant growth of cells which had taken up the plasmid in chocolate milk appears to be due to failure of these cells to establish themselves as permanently transformed under non-selective conditions.

Syst Appl Microbiol, 2002 Dec, 25(4), 471 - 7
Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food; Kharazmi M et al.; A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168 . Marker rescue was detected in vitro using different types of donor DNA containing intact nptII . The efficiency of marker rescue using chromosomal DNA of E . coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene . Low efficiencies of ca . 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E . coli Sure or DNA from a transgenic potato . B . subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca . 10(-6) and 10(-8), respectively, using chromosomal DNA of E . coli Sure as donor DNA . Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E . coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B . subtilis was detectable neither in vitro nor in situ.

J Mol Biol, 2003 Feb 21, 326(3), 783 - 93
Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-dimethyl-8-ribityllumazine synthase; Fischer M et al.; 6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin . In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit . The ribH gene specifying the lumazine synthase subunit can be expressed in high yield . All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis . Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties . Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude . Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude . Surprisingly, the activation enthalpy deltaH of the lumazine synthase reaction exceeds that of the uncatalysed reaction . On the other hand, the free energy of activation deltaG of the uncatalysed reaction is characterised by a large entropic term (TdeltaS) of -37.8 kJmol(-1), whereas the entropy of activation (TdeltaS) of the enzyme-catalysed reaction is -6.7 kJmol(-1) . This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role.

Mol Microbiol, 2003 Feb, 47(4), 1113 - 22
Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis; Kawai Y et al.; A knock-out mutant of the dinR gene that encodes the SOS regulon repressor in Bacillus subtilis was constructed . The yneA, yneB and ynzC genes transcribed divergently from the dinR gene were strongly induced in mutant cells . Northern hybridization analyses revealed that these genes collectively form an operon and belong to the SOS regulon . The simultaneous deletion of dinR and yneA suppressed the filamentous phenotype of the dinR mutant . Furthermore, although yneA is suppressed in the wild-type cell in the absence of SOS induction, artificial expression of the YneA protein using an IPTG-inducible promoter resulted in cell elongation . Disruption of yneA significantly reduced cell elongation after the induction of the SOS response by mitomycin C in dinR+ cells . These results indicate that the YneA protein is responsible for cell division suppression during the SOS response in B . subtilis . Localization of the FtsZ protein to the cell division site was reduced in dinR-disrupted or yneA-expressing cells, further suggesting that the YneA protein suppresses cell division through the suppression of FtsZ ring formation . Interestingly, the B . subtilis YneA protein is structurally and phylogenetically unrelated to its functional counterpart in Escherichia coli, SulA.

Mol Microbiol, 2003 Feb, 47(4), 1061 - 73
Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria; Chastanet A et al.; Multiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria . Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis . We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus . Sequence analysis of the S . aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B . subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA . The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors . The S . aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B . subtilis as a heterologous host . Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S . aureus CtsR and HrcA proteins . DeltactsR, DeltahrcA and DeltactsRDeltahrcA mutants of S . aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress . This novel regulatory mode appears to be specific to Staphylococci.

Eur J Biochem, 2003 Feb, 270(4), 675 - 86
Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major; Iijima N et al.; We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major . Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides . Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence . The alpha-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy . The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens . The three peptides were also hemolytic . Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills . Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.

Biol Pharm Bull, 2003 Feb, 26(2), 262 - 5
Molecular cloning of the gene for edeine B1 amidinohydrolase in addition to the agmatinase activity in Bacillus subtilis; Shimotohno KW et al.; A gene with a high-nucleotide sequence homology to the edeine B1 amidinohydrolase gene of Bacillus brevis was identified in the database of the Bacillus subtilis genome . The gene was isolated, expressed in Escherichia coli, and the gene product was analyzed with regard to the characteristics of its enzyme activity . A 32-kDa protein encoded by the ywhG gene showed a 69.8% amino acid sequence-homology to the edeine B1 amidinohydrolase of B . brevis . Among various guanidino-compounds, edeine B1 and agmatine were both efficiently hydrolyzed by the protein encoded by the ywhG gene, although edeine B1 was a more potent substrate than agmatine in this assay system . These data indicate that the protein encoded by the ywhG gene is an agmatinase that is essential for polyamine biosynthesis in B . subtilis.

Microbiology, 2003 Jan, 149(Pt 1), 19 - 28
A plasmid-borne Rap-Phr system of Bacillus subtilis can mediate cell-density controlled production of extracellular proteases; Koetje EJ et al.; Bacillus subtilis uses two-component signal transduction systems to sense intra- and extracellular stimuli to adapt to fluctuating environmental situations . Regulator aspartate phosphatases (Raps) have important roles in these processes, as they can dephosphorylate certain response-regulators, and are themselves subject to cell-density-controlled inhibition by secreted Phr (phosphate regulator) peptides . Eleven chromosomal genes encode this family of phosphatases, but in addition, certain strains contain endogenous plasmids with genes for homologous Rap-Phr systems . Plasmid pTA1060 encodes Rap60 and its antagonistic signalling molecule Phr60 . Strikingly, expression of Rap60 in B . subtilis 168 strongly repressed the production of proteolytic enzymes . In fact, the transcription of the aprE gene, encoding a major extracellular protease, was shown to be decreased upon Rap60 expression, whereas this effect could be antagonized by the extracellular addition of synthetic Phr60 pentapeptide . Finally, transcription studies suggest that Rap60 dephosphorylates a component of the phosphorelay and is coupled to aprE transcription by the transition-state regulator AbrB . In conclusion, these data show that endogenous plasmids contain functional Rap-Phr systems and for the first time, that Rap-Phr systems can mediate cell-density controlled production of secreted proteases . This quorum-sensing mechanism might enable B . subtilis to suppress protease production under conditions of low cell densities when nutrients are still available in sufficient amounts.

Microbiology, 2003 Jan, 149(Pt 1), 9 - 17
Controlling competence in Bacillus subtilis: shared use of regulators; Hamoen LW et al.; Bacteria have developed a wide arsenal of survival strategies to cope with the specific problems posed by their environment . These processes are carefully regulated and complex signal transduction cascades ensure proper activation of the adequate adaptive response . An intriguing observation is that generally the regulation pathways of the different adaptive processes are highly intertwined . In this review, this phenomenon is illustrated by the regulation of genetic competence development in Bacillus subtilis . The different regulation pathways which make up the gene regulation network that controls the development of competence are described, and their connections to other adaptive processes in B . subtilis are discussed.

Genetika, 2002 Dec, 38(12), 1719 - 22
{The ISBsu2 mobile element is present in a plasmid of a soil strain and in the chromosomes of several other strains of Bacillus subtilis}; Poluektova EU et al.; Chromosomes of several Bacillus subtilis strains were shown to contain homologs of the ISBsu2 mobile genetic element, which was earlier revealed in a cryptic plasmid of a soil strain of B . subtilis.

Appl Radiat Isot, 2003 Feb, 58(2), 161 - 8
Transfer of Eu (III) associated with polymaleic acid to Bacillus subtilis; Markai S et al.; The aim of this study is to contribute to the understanding of the distribution of Eu(III) between dissolved organic matter and microorganisms, and to investigate the effect of competitive ions such as Ca(+2) on adsorption properties . Polymaleic acid (PMA), is used as synthetic organic matter, having similar properties as natural fulvic acid, and Bacillus subtilis is chosen as microorganism . A double labeling method was used: {14C}MPA and 152Eu to quantify the behavior of the various components . Preliminary experiments showed that the adsorption of polymaleic acid onto Bacillus subtilis was negligible at pH=5 in 0.15mol/l of NaCl . In the absence of Ca(+2), the transfer of Eu(III) from PMA to B . subtilis could be described by a simple empirical model based on data obtained from sorption isotherms on the reference systems Eu(III)/PMA and Eu(III)/B . subtilis . In the presence of Ca(+2), the transfer was increased . The hypothesis that Ca(+2) ions acted as a bridging agent between PMA and the bacteria was proposed.

J Biotechnol, 2003 Mar 6, 101(2), 173 - 80
Growth at low temperature suppresses readthrough of the UGA stop codon during the expression of Bacillus subtilis flgM gene in Escherichia coli; Gonzalez B et al.; The efficient production of recombinant proteins in Escherichia coli requires a proper termination of translation to ensure the synthesis of only the desired product . During the recombinant production of Bacillus subtilis flgM in E . coli, we detected an additional polypeptide of molecular mass higher than the expected, corresponding to a product of a translational readthrough of the UGA stop codon . In this paper we show that the readthrough was abolished when the synthesis of the recombinant protein was carried out at 25 degrees C . The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed .

Genome Res, 2003 Feb, 13(2), 224 - 37
Bacillus subtilis during feast and famine: visualization of the overall regulation of protein synthesis during glucose starvation by proteome analysis; Bernhardt J et al.; Dual channel imaging and warping of two-dimensional (2D) protein gels were used to visualize global changes of the gene expression patterns in growing Bacillus subtilis cells during entry into the stationary phase as triggered by glucose exhaustion . The 2D gels only depict single moments during the cells' growth cycle, but a sequential series of overlays obtained at specific points of the growth curve facilitates visualization of the developmental processes at the proteomics scale . During glucose starvation a substantial reprogramming of the protein synthesis pattern was found, with 150 proteins synthesized de novo and cessation of the synthesis of almost 400 proteins . Proteins induced following glucose starvation belong to two main regulation groups: general stress/starvation responses induced by different stresses or starvation stimuli (sigma(B)-dependent general stress regulon, stringent response, sporulation), and glucose-starvation-specific responses (drop in glycolysis, utilization of alternative carbon sources, gluconeogenesis) . Using the dual channel approach, it was not only possible to identify those regulons or stimulons, but also to follow the fate of each single protein by the three-color code: red, newly induced but not yet accumulated; yellow, synthesized and accumulated; and green, still present, but no longer being synthesized . These green proteins, which represent a substantial part of the protein pool in the nongrowing cell, are not accessible by using DNA arrays . The combination of 2D gel electrophoresis and MALDI TOF mass spectrometry with the dual channel imaging technique provides a new and comprehensive view of the physiology of growing or starving bacterial cell populations, here for the case of the glucose-starvation response.

J Bacteriol, 2003 Feb, 185(4), 1423 - 31
Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis; McPherson DC et al.; Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis . Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains . Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential . The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain . A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type . The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable . The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis . This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin . In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo . Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.

J Bacteriol, 2003 Feb, 185(4), 1391 - 8
Subcellular localization of a small sporulation protein in Bacillus subtilis; van Ooij C et al.; SpoVM is an unusually small (26-residue-long) protein that is produced in the mother cell chamber of the sporangium during the process of sporulation in Bacillus subtilis . We investigated the subcellular localization of SpoVM, which is believed to be an amphipathic alpha-helix, by using a fusion of the sporulation protein to the green fluorescence protein (GFP) . We found that SpoVM-GFP is recruited to the polar septum shortly after the sporangium undergoes asymmetric division and that the fusion protein localizes to the mother cell membrane that surrounds the forespore during the subsequent process of engulfment . We identified a patch of three residues near the N terminus of the proposed alpha-helix that is needed both for proper subcellular localization and for SpoVM function . We also identified a patch of residues on the opposite face of the helix and residues near both ends of the protein that are needed for SpoVM function but not for subcellular localization . Subcellular localization of SpoVM-GFP was found to require an unknown gene(s) under the control of the mother cell transcription factor sigmaE . We propose that the N-terminal patch binds to an unknown anchoring protein that is produced under the control of sigmaE and that other residues important in SpoVM function to recruit an unknown sporulation protein(s) to the mother cell membrane that surrounds the forespore . Our results provide evidence that SpoVM function depends on proper subcellular localization.

J Bacteriol, 2003 Feb, 185(4), 1326 - 37
Effects of the chromosome partitioning protein Spo0J (ParB) on oriC positioning and replication initiation in Bacillus subtilis; Lee PS et al.; Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species . We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations . In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters . In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell . We found, by using a Spo0J-green fluorescent protein {GFP} fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites . When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters . spo0J also affected chromosome positioning during sporulation . A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore . In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry . These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.

J Bacteriol, 2003 Feb, 185(4), 1181 - 9
CDP-2,3-Di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) in the methanogenic archaeon Methanothermobacter thermautotrophicus; Morii H et al.; CDP-2,3-di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of Methanothermobacter thermautotrophicus cells was characterized . The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and L-serine . The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation . The enzyme showed maximal activity in the presence of 10 mM Mn2+ and 1% Triton X-100 . Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase . The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate . The activity of D-serine with the enzyme was 30% of that observed for L-serine . A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment . A gene (MT1027) in M . thermautotrophicus genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to Bacillus subtilis pssA but not to Escherichia coli pssA . The substrate specificity of phosphatidylserine synthase from B . subtilis was quite similar to that observed for the M . thermautotrophicus archaetidylserine synthase, while the E . coli enzyme had a strong preference for CDP-1,2-diacyl-sn-glycerol . It was concluded that M . thermautotrophicus archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (B . subtilis type) on the basis of not only homology but also substrate specificity and some enzymatic properties . The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed.

Indian J Exp Biol, 2001 Jun, 39(6), 578 - 83
A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713; Mane RR et al.; An alkaline protease was isolated from culture filtrate of B . subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration . With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C . The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1) . The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr . During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+ . Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C . About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C . DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained . However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+ . The results indicated that this is a serine protease.

Mikrobiol Z, 2002 Sep-Oct, 64(5), 10 - 7
{Lectin activity of antitumor substances synthesized by Bacillus subtilis B-7025}; Podgorskii VS et al.; Nutrient medium, which is optimal for synthesis of biologically active substances and allows obtaining stable outflow of Bacillus subtilis B-7025 to the culture medium, has been selected under the conditions of the bacillus periodic cultivation . It has been established that these substances are extracellular lectins with high activity and carbohydrate specificity to fructose-1.6-di phosphate, N-acetylneuraminic acid and glucose derivatives: D-glucuronic acid and D-glucosamine.

Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 232 - 5
{The optimization of guanosine fermentation based on process parameter correlation analysis}; Cai X et al.; The characteristic of Bacillus subtilis fermentation process of guanosine on 50 L fermentor was analyzed . Based on determination of on-line and off-line parameter, using correlation analysis, the technology study of physiologic regulation was combined with the metabolic flux distribution of synthesis process . The metabolic flux shift from HMP to EMP and TCA cycle during fermentation was found . The reason of the flux shift was preliminary analyzed, based on which the procedure was optimized to increase the yield of guanosine to 30 g/L.

Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 163 - 8
{Cloning and expression of a osmoregulatory gene pro B from halotolerant Bacillus subtilis}; Zhang X et al.; A 1.3 kb fragment is cloned from halotolerated Bacillus subtilis 93151 with PCR amplification method, and its positively-directionally inserted fragment can complemented with proB-E . coli by function test . Halotolerated ability of E . coli DH5 alpha having this recombination plasmid rises from 2% to 4% in minimal medium . The nucleotide sequence of this fragment is obtained by primer walking method . Nucleotide sequence of this fragment 167-1269 bp translates a protein which has 370 amino acid by sequence analysis through DNAsis program . There are non-typical-10 sequences, typical-35 sequences and a Ribosome binding site of this fragment in its upstream sequence, and there is flanking sequence, which has best efficiency of beginning translating . Homologues comparision, of nucleotide and amino acid sequences of this fragment and those of gene in gene bank shows that homogenous of Nucleotide sequences and amino acid sequences of this fragement and Bacillus subtilis 168 are respectively 81%, 90%, which prove that this gene is certainly a pro B gene . This protein translated by this fragment has several absoluter conservative domain which have been correlating closely with forming active center of enzyme and tri-dimension structure of active center, compared amino acid sequences of this fragment and pro B genes of thirty kinds of different microorganism.

Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 145 - 52
{Cloning and expression in Escherichia coli of oligo-1,6-glucosidase gene from Bacillus subtilis HB002}; Jiang S et al.; The gene coding for oligo-1,6-glucosidase of Bacillus subtilis HB002 was cloned by the shotgun-cloning method and sequenced by the chain-termination method of Sanger et al . It consists of an open reading frame of 1683 bp . The amino acid sequence of oligo-1,6-glucosidase deduced from its nuecleotide sequence predicts a protein of 561 amino acid residues with a Mr of 65.985 kD, which is 81% and 67% identical to those of oligo-1,6-glucosidase from Bacillus sp . and Bacillus coagulans, respectively, 89% and 79% similar to those of oligo-1,6-glucosidase from Bacillus sp . and Bacillus coagulans, respectively . The oligo-1,6-glucosidase gene of Bacillus subtilis HB002 was cloned into Escherichia coli expression plasmid pBV220, the result of SDS-PAGE showed that the oligo-1,6-glucosidase gene had been expressed in Escherichia coli DH5 alpha, the expressed oligo-1, 6-glucosidase has enzymatic activity.

Can J Microbiol, 2002 Nov, 48(11), 986 - 94
The pathway of dephosphorylation of myo-inositol hexakisphosphate by phytate-degrading enzymes of different Bacillus spp; Greiner R et al.; The pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45 was established using a combination of high-performance ion chromatography analysis and kinetic studies . The data demonstrate that all the Bacillus phytate-degrading enzymes under investigation dephosphorylate myo-inositol hexakisphosphate by sequential removal of phosphate groups via two independent routes; the routes proceed via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3 or D-Ins(2,5,6)P3 and via D-Ins(1,2,4,5,6)P5 to D-Ins(1,2,5,6)P4 to finally D-Ins(1,2,6)P3 . The resulting myo-inositol trisphosphate D-Ins(1,2,6)P3 was degraded via D-Ins(2,6)P2 to finally Ins(2)P after prolonged incubation times in combination with increased enzyme concentration.

Wei Sheng Wu Xue Bao, 1999 Oct, 39(5), 441 - 7
{Purification and characterization of antifungal peptide LP-1}; Liu Y et al.; An antifungal peptide LP-1 from Bacillus subtilis TG26 strain was purified by acid precipitation, acetone precipitation and Hi-pore reversed phase column chromatography . The molecular weight of LP-1 is 1057.3 D as determined by MALDI-TOF mass spectrometry, and its pI is 4.75 by PAG-IEF . It was also found to be thermostable . Its antifungal spectrum showed that LP-1 has strong inhibitory activity against many plant pathogenic fungi, such as Pythium aphanidermatum, Gibberella zeae, Alternaria longipe, Fusarium oxysporum f . lycopersici, etc. . The abnormal hyphal growth of Trichoderma viride caused by LP-1 such as swollen tips, twisted, short growth and cytoplasm condensation was also observed . Both ninhydrin reaction and peptide sequencing suggested that LP-1 is a cyclic peptide.

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