Bacillus subtilis

Chem Biol, 2000 Aug, 7(8), 583 - 91
Tuning chemotactic responses with synthetic multivalent ligands; Gestwicki JE et al.; BACKGROUND: Multivalent ligands have been used previously to investigate the role of ligand valency and receptor clustering in eliciting biological responses . Studies of multivalent ligand function, however, typically have employed divalent ligands or ligands of undefined valency . How cells respond to multivalent ligands of distinct valencies, which can cluster a signaling receptor to different extents, has never been examined . The chemoreceptors, which mediate chemotactic responses in bacteria, are localized, and clustering has been proposed to play a role in their function . Using multivalent ligands directed at the chemoreceptors, we hypothesized that we could exploit ligand valency to control receptor occupation and clustering and, ultimately, the cellular response . RESULTS: To investigate the effects of ligand valency on the bacterial chemotactic response, we generated a series of linear multivalent arrays with distinct valencies by ring-opening metathesis polymerization . We report that these synthetic ligands elicit bacterial chemotaxis in both Escherichia coli and Bacillus subtilis . The chemotactic response depended on the valency of the ligand; the response of the bacteria can be altered by varying chemoattractant ligand valency . Significantly, these differences in chemotactic responses were related to the ability of the multivalent ligands to cluster chemoreceptors at the plasma membrane . CONCLUSIONS: Our results demonstrate that ligand valency can be used to tune the chemotactic responses of bacteria . This mode of regulation may arise from changes in receptor occupation or changes in receptor clustering or both . Our data implicate changes in receptor clustering as one important mechanism for altering cellular responses . Given the diverse events modulated by changes in the spatial proximity of cell surface receptors, our results suggest a general strategy for tuning biological responses.

Int J Med Microbiol, 2000 May, 290(2), 123 - 34
Proteomics, DNA arrays and the analysis of still unknown regulons and unknown proteins of Bacillus subtilis and pathogenic gram-positive bacteria; Hecker M et al.; The complete sequence of the bacterial genomes provides new perspectives for the study of gene expression and gene function . By the combination of the highly sensitive 2-dimensional (2D) protein gel electrophoresis with the identification of the protein spots by microsequencing or mass spectrometry we established a 2D protein index of Bacillus subtilis that currently comprises almost 400 protein entries . A computer-aided evaluation of the 2D gels loaded with radioactively-labelled proteins from growing or stressed/starved cells proved to be a powerful tool in the analysis of global regulation of the expression of the entire genome . For the general stress regulon it is demonstrated how the proteomics approach can be used to analyse the regulation, structure and function of still unknown regulons . The application of this approach is illustrated for the sigmaB dependent general stress regulon . For the comprehensive description of proteins/genes belonging to stimulons or regulons it is generally recommended to complement the proteome approach with DNA array techniques in order to identify and allocate still undiscovered members of individual regulons . This approach is also very attractive to uncover the function of still unknown global regulators and regulons and to dissect the entire genome into its basic modules of global regulation . The same strategy can be used to analyse the regulation, structure and function of regulons encoding virulence factors of pathogenic bacteria for a comprehensive understanding of the pathogenicity and for the identification of new antibacterial targets.

J Biol Chem, 2001 Jan 19, 276(3), 2267 - 75 Epub 2000 Oct 23.
N-terminal amino acid residues mediate protein-protein interactions between DNA-bound alpha /beta -type small, acid-soluble spore proteins from Bacillus species; Hayes CS et al.; The binding of alpha/beta-type small, acid-soluble spore proteins (SASP) to DNA of spores of Bacillus species is the major determinant of DNA resistance to a variety of damaging treatments . The primary sequence of alpha/beta-type SASP is highly conserved; however, the N-terminal third of these proteins is less well conserved than the C-terminal two-thirds . To determine the functional importance of residues in the N-terminal region of alpha/beta-type SASP, variants of SspC (a minor alpha/beta-type SASP from Bacillus subtilis) with modified N termini were generated and their structural and DNA binding properties studied in vitro and in vivo . SspC variants with deletions of up to 14 residues ( approximately 20% of SspC residues) were able to bind DNA in vitro and adopted similar conformations when bound to DNA, as determined by circular dichroism spectroscopy and protein-protein cross-linking . Progressive deletion of up to 11 N-terminal residues resulted in proteins with progressively lower DNA binding affinity . However, SspC(Delta)(14) (in which 14 N-terminal residues have been deleted) showed significantly higher affinity for DNA than the larger proteins, SspC(Delta)(10) and SspC(Delta)(11) . The affinity of these proteins for DNA was shown to be largely dependent upon the charge of the first few N-terminal residues . These results are interpreted in the context of a model for DNA-dependent alpha/beta-type SASP protein-protein interaction involving the N-terminal regions of these proteins.

Prikl Biokhim Mikrobiol, 2000 Sep-Oct, 36(5), 549 - 54
{Effect of membrane-active microbial autoregulators on the growth of cultured ras-transformed fibroblasts}; Il'inskaia ON et al.; Differential effects on proliferation of individual vs . combined administration of high- and low-molecular-weight microbial autoregulators (extracellular RNase from Bacillus subtilis and anabiosis-inducing factor d1) are reported for the first time for cultured cells of higher eukaryotes . Proliferation of ras-transformed mouse fibroblasts was affected by both autoregulators dose-dependently . The cytotoxic activity of individual regulators was directly related to their concentration . Unlike RNase, factor d1 (which functions as a chemical chaperone) exerted reversible effects . Studies of the effects of combined administration of the autoregulators demonstrated that pretreatment of the cells with low-dose d1 decreased the toxicity of RNase . Higher doses of d1 were required to attenuate the effects of toxic agents with more pronounced membrane tropism . The results obtained suggest that a universal system regulating the physiological activity of cells is operative in taxonomically remote organisms . The operation of the system is based on sequential changes in the structural organization and function of subcellular structures, induced by low- and high-molecular-weight autoregulators.

Curr Opin Struct Biol, 2000 Oct, 10(5), 510 - 7
Glycosyltransferase structure and mechanism; Unligil UM et al.; The high-resolution X-ray crystal structures of a new form of bacteriophage T4 beta-glucosyltransferase, Escherichia coli MurG, Bacillus subtilis SpsA, bovine beta-1,4-galactosyltransferase 1 and rabbit N-acetylglucosaminyltransferase I have now been solved . These glycosyltransferase structures have provided the first detailed view of the structural basis of catalysis, as well as new insight into glycosyltransferase classification.

J Struct Biol, 2000 Aug, 131(2), 90 - 5
Structural characterization of penicillin-binding protein-related factor A (PrfA) from Bacillus species; Kelly SJ et al.; The prfA genes of Bacillus stearothermophilus and Bacillus subtilis are in an operon downstream of the ponA gene encoding penicillin-binding protein 1 (PBP1), a major enzyme involved in peptidoglycan synthesis . The specific function of the 23- to 24-kDa PrfA protein is unknown but this protein plays some role in nucleoid segregation and the functions of PrfA and PBP1 are interrelated . We overexpressed B . stearothermophilus and B . subtilis PrfA in Escherichia coli and purified the proteins to homogeneity by cation exchange and gel filtration chromatography . The protein is a monomer in solution, and circular dichroism spectroscopy revealed an abundance of beta-sheet secondary structure . Crystals of B . stearothermophilus PrfA were also obtained and diffracted X-rays to 1.8 A resolution .

Arch Microbiol, 2000 Sep, 174(3), 162 - 7
Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia; Laue H et al.; Alanine dehydrogenase {L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.} catalyses the reversible oxidative deamination of L-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate) . The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products . Alanine dehydrogenase seems to be induced during growth with taurine . The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification . SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa . The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure . The N-terminal amino acid sequence was determined . The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0-11.5 for oxidative deamination of alanine . The apparent Km values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively . The alanine dehydrogenase gene was sequenced . The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 33 - 8
The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins; Takamatsu H et al.; The synthesis and proteolysis of the spore coat proteins, SpoIVA and YrbA, of Bacillus subtilis were analyzed using antisera . Almost no intact full-length proteins of either type were extracted from wild-type spores, while yabG mutant spores contained intact SpoIVA and YrbA proteins . We purified recombinant YrbA and YabG proteins from Escherichia coli transformants and found that YrbA was cleaved to the smaller moiety in the presence of YabG in vitro . These observations indicate that YabG is a protease involved in the proteolysis and maturation of SpoIVA and YrbA proteins, conserved with the cortex and/or coat assembly by B . subtilis.

Res Microbiol, 2000 Sep, 151(7), 503 - 11
A tale of two genomes: resolution of dimeric chromosomes in Escherichia coli and Bacillus subtilis; Sciochetti SA et al.; Dimeric chromosomes can be formed during replication of circular bacterial chromosomes by an odd number of homologous recombination events between sister chromosomes . In the absence of a compensating recombination reaction such dimers cannot be segregated from each other as the cell divides . This review highlights the shared and divergent mechanisms employed by Escherichia coli and Bacillus subtilis in their effort to resolve and partition dimeric chromosomes safely . In particular, we discuss the Xer-type recombinases, RecA, FtsK/SpoIIIE, and dif.

Infect Immun, 2000 Nov, 68(11), 6281 - 8
Molecular cloning and analysis of a putative siderophore ABC transporter from Staphylococcus aureus; Morrissey JA et al.; From a mass-excised Staphylococcus aureus lambdaZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homology to bacterial siderophore transporter systems . The operon encodes four genes designated sstA, -B, -C, and -D encoding two putative cytoplasmic membrane proteins (sstA and sstB), an ATPase (sstC), and a membrane-bound 38-kDa lipoprotein (sstD) . The sst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dependent . SstD was overexpressed in Escherichia coli, purified by Triton X-114 phase partitioning, and used to generate monospecific antisera in rats . Immunoblotting studies located SstD in the membrane fraction of S . aureus and showed that expression of the lipoprotein was reduced under iron-rich growth conditions . Triton X-114 partitioning studies on isolated membranes provided additional biochemical evidence that SstD in S . aureus is a lipoprotein . Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detected in Bacillus subtilis . Expression of SstD in vivo was confirmed by immunoblotting studies with S . aureus recovered from a rat intraperitoneal chamber implant model . To further define the contribution of SstD in promoting growth of S . aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD . Expression of antisense sstD RNA in S . aureus resulted in a decrease in SstD expression under both iron-rich and iron-restricted growth conditions . However, this reduction in SstD levels did not affect the growth of S . aureus in vitro in an iron-limited growth medium or when grown in an intraperitoneal rat chamber implant model in vivo.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 195 - 201
Characterization of the relA/spoT gene from Bacillus stearothermophilus; Wendrich TM et al.; By use of degenerate primers, we amplified a fragment of a relA/spoT homologous gene from Bacillus stearothermophilus . Chromosomal walking enabled us to sequence the entire gene and its flanking regions . The primary sequence of the gene product is 78% identical to the RelA/SpoT homologue of Bacillus subtilis and both gene loci share a similar genetic organization . The B . stearothermophilus rel gene was analyzed in vivo by heterologous expression in the B . subtilis relA deletion strain TW30, and is shown to complement the growth defects of TW30 . The recombinant RelBst protein was detected by Western immunoanalysis, and synthesizes guanosine-3'-diphosphate-5'-(tri)diphosphate ((p)ppGpp) after amino acid stress and carbon starvation . These in vivo data, the genetic organization, and the primary structure compared to other RelA/SpoT homologues provide circumstantial evidence that the identified gene encodes the only (p)ppGpp synthetase in B . stearothermophilus presumed to serve also as (p)ppGpp hydrolase.

Genetika, 2000 Aug, 36(8), 1166 - 8
{Study of the phenotypic occurrence of ura gene inactivation in Bacillus subtilis}; Kreneva RA et al.; After inactivation of the ypaA gene in Bacillus subtilis, the phenotypic pattern obtained showed that this gene controls a system for active flavin transport and, possibly, riboflavin excretion under the conditions of constitutive synthesis.

EMBO J, 2000 Oct 16, 19(20), 5575 - 84
Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1; Bravo A et al.; The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells . The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures . Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro . Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner . We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer . This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase . We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.

EMBO J, 2000 Oct 16, 19(20), 5269 - 80
X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily; Unligil UM et al.; N:-acetylglucosaminyltransferase I (GnT I) serves as the gateway from oligomannose to hybrid and complex N:-glycans and plays a critical role in mammalian development and possibly all metazoans . We have determined the X-ray crystal structure of the catalytic fragment of GnT I in the absence and presence of bound UDP-GlcNAc/Mn(2+) at 1.5 and 1.8 A resolution, respectively . The structures identify residues critical for substrate binding and catalysis and provide evidence for similarity, at the mechanistic level, to the deglycosylation step of retaining beta-glycosidases . The structuring of a 13 residue loop, resulting from UDP-GlcNAc/Mn(2+) binding, provides an explanation for the ordered sequential 'Bi Bi' kinetics shown by GnT I . Analysis reveals a domain shared with Bacillus subtilis glycosyltransferase SpsA, bovine beta-1,4-galactosyl transferase 1 and Escherichia coli N:-acetylglucosamine-1-phosphate uridyltransferase . The low sequence identity, conserved fold and related functional features shown by this domain define a superfamily whose members probably share a common ancestor . Sequence analysis and protein threading show that the domain is represented in proteins from several glycosyltransferase families.

Eur J Biochem, 2000 Nov, 267(21), 6459 - 69
A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase; Eggert T et al.; A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence . We have cloned and overexpressed lipB in B . subtilis and Escherichia coli and have also purified the enzyme from a B . subtilis culture supernatant to electrophoretic homogeneity . Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique . LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of </= 10 carbon atoms . Triolein, which is a typical substrate for true lipases, was not hydrolysed at all . These results led us to classify LipB as an esterase rather than a lipase . The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis . The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases . All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine . We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif . When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7 . Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.

J Bacteriol, 2000 Nov, 182(21), 6250 - 3
Fate of the SpoIIAB*-ADP liberated after SpoIIAB phosphorylates SpoIIAA of Bacillus subtilis; Lee CS et al.; Phosphorylation of SpoIIAA catalyzed by SpoIIAB helps to regulate the first sporulation-specific sigma factor, sigma(F), of Bacillus subtilis . The steady-state rate of phosphorylation is known to be exceptionally slow and to be limited by the return of the protein kinase, SpoIIAB, to a catalytically active state . Previous work from this laboratory has suggested that, after catalyzing the phosphorylation, SpoIIAB is in a form (SpoIIAB*) that does not readily release ADP . We now show that the rate of release of ADP from the SpoIIAB*-ADP complex was much diminished by the presence of unreacted SpoIIAA, suggesting that SpoIIAA can form a long-lived ternary complex with SpoIIAB*-ADP in which the SpoIIAB* form is stabilized . In kinetic studies of the phosphorylation of SpoIIAA, the ternary complex SpoIIAA-SpoIIAB*-ADP could be distinguished from the short-lived complex SpoIIAA-SpoIIAB-ADP, which can be readily produced in the absence of an enzymatic reaction.

J Bacteriol, 2000 Nov, 182(21), 6099 - 105
Catabolite repression and induction of the Mg(2+)-citrate transporter CitM of Bacillus subtilis; Warner JB et al.; In Bacillus subtilis the citM gene encodes the Mg(2+)-citrate transporter . A target site for carbon catabolite repression (cre site) is located upstream of citM . Fusions of the citM promoter region, including the cre sequence, to the beta-galactosidase reporter gene were constructed and integrated into the amyE site of B . subtilis to study catabolic effects on citM expression . In parallel with beta-galactosidase activity, the uptake of Ni(2+)-citrate in whole cells was measured to correlate citM promoter activity with the enzymatic activity of the CitM protein . In minimal media, CitM was only expressed when citrate was present . The presence of glucose in the medium completely repressed citM expression; repression was also observed in media containing glycerol, inositol, or succinate-glutamate . Studies with B . subtilis mutants defective in the catabolite repression components HPr, Crh, and CcpA showed that the repression exerted by all these medium components was mediated via the carbon catabolite repression system . During growth on inositol and succinate, the presence of glutamate strongly potentiated the repression of citM expression by glucose . A reasonable correlation between citM promoter activity and CitM transport activity was observed in this study, indicating that the Mg(2+)-citrate uptake activity of B . subtilis is mainly regulated at the transcriptional level.

J Bacteriol, 2000 Nov, 182(21), 6027 - 35
RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro; Artsimovitch I et al.; Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals . Some of these signals are conserved throughout evolution, whereas others are species specific . In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays . We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals . Both enzymes responded similarly to other pause and termination signals and to the general E . coli elongation factors NusA and GreA . We also demonstrate that, although promoter recognition depends largely on the sigma subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme . We hypothesize that differences in signal recognition are due to the changes in contacts made between the beta and beta' subunits and the downstream DNA duplex.

J Bacteriol, 2000 Nov, 182(21), 5939 - 47
Role of TnrA in nitrogen source-dependent repression of Bacillus subtilis glutamate synthase gene expression; Belitsky BR et al.; Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism . In Bacillus subtilis, the gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression . In addition, the gltAB operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation . TnrA was found to bind directly to a site immediately downstream of the gltAB promoter . As is true for other genes, the activity of TnrA at the gltAB promoter was antagonized by glutamine synthetase under certain growth conditions.

Biochemistry, 2000 Oct 17, 39(41), 12717 - 22
Chain length determination of prenyltransferases: both heteromeric subunits of medium-chain (E)-prenyl diphosphate synthase are involved in the product chain length determination; Zhang YW et al.; Among prenyltransferases, medium-chain (E)-prenyl diphosphate synthases are unusual because of their heterodimeric structures . The larger subunit has highly conserved regions typical of (E)-prenyltransferases . The smaller one has recently been shown to be involved in the binding of allylic substrate as well as determining the chain length of the reaction product {Zhang, Y.-W., et al . (1999) Biochemistry 38, 14638-14643} . To better understand the product chain length determination mechanism of these enzymes, several amino acid residues in the larger subunits of Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase and Bacillus subtilis heptaprenyl diphosphate synthase were selected for substitutions by site-directed mutagenesis and examined by combination with the corresponding wild-type or mutated smaller subunits . Replacement of the Ala at the fifth position upstream to the first Asp-rich motif with bulky amino acids in both larger subunits resulted in shortening the chain lengths of the major products, and a double combination of mutant subunits of the heptaprenyl diphosphate synthase, I-D97A/II-A79F, yielded exclusively geranylgeranyl diphosphate . However, the combination of a mutant subunit and the wild-type, I-Y103S/II-WT or I-WT/II-I76G, produced a C(40) prenyl diphosphate, and the double combination of the mutants, I-Y103S/II-I76G, gave a reaction product with longer prenyl chain up to C(50) . These results suggest that medium-chain (E)-prenyl diphosphate synthases take a novel mode for the product chain length determination, in which both subunits cooperatively participate in maintaining and determining the product specificity of each enzyme.

Gene, 2000 Sep 19, 255(2), 297 - 305
Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action; Zhang L et al.; Selectively regulating gene expression in bacteria has provided an important tool for studying gene function . However, well-regulated gene control systems have been restricted primarily for use in laboratory non-pathogenic strains of bacteria (e.g . Escherichia coli, Bacillus subtilis) . The development of analogous systems for use in bacterial pathogens such as Staphylococcus aureus would significantly enhance our ability to examine the contribution of any given gene product to pathogen growth and viability . In this report, we adapt, examine and compare three regulated gene expression systems in S . aureus, which had previously been used in B . subtilis . We demonstrate that all three systems function and exhibit titratable induction, together covering a dynamic range of gene expression of approximately 3000-fold . This dynamic range correlates well with the physiological expression levels of cellular proteins . Importantly, we show that one of these systems, the Spac system, is particularly useful for examining gene essentiality and creating specific conditional lethal phenotypes . Moreover, we find that titration of selective target gene products using this system allows direct demonstration of antibiotic mode of action.

Nucleic Acids Res, 2000 Oct 15, 28(20), 4021 - 8
A heuristic graph comparison algorithm and its application to detect functionally related enzyme clusters; Ogata H et al.; The availability of computerized knowledge on biochemical pathways in the KEGG database opens new opportunities for developing computational methods to characterize and understand higher level functions of complete genomes . Our approach is based on the concept of graphs; for example, the genome is a graph with genes as nodes and the pathway is another graph with gene products as nodes . We have developed a simple method for graph comparison to identify local similarities, termed correlated clusters, between two graphs, which allows gaps and mismatches of nodes and edges and is especially suitable for detecting biological features . The method was applied to a comparison of the complete genomes of 10 microorganisms and the KEGG metabolic pathways, which revealed, not surprisingly, a tendency for formation of correlated clusters called FRECs (functionally related enzyme clusters) . However, this tendency varied considerably depending on the organism . The relative number of enzymes in FRECs was close to 50% for Bacillus subtilis and Escherichia coli, but was <10% for SYNECHOCYSTIS: and Saccharomyces cerevisiae . The FRECs collection is reorganized into a collection of ortholog group tables in KEGG, which represents conserved pathway motifs with the information about gene clusters in all the completely sequenced genomes.

J Exp Biol, 2000 Nov, 203 Pt 21, 3345 - 54
Bioconvective dynamics: dependence on organism behaviour; Czirok A et al.; Bioconvection occurs when a macroscopic nonuniformity of the concentration of microbial populations is generated and maintained by the directional swimming of the organisms . This study investigated the properties of the patterns near the onset of the instability and later during its evolution into a fully nonlinear convection regime . In suspensions of the bacteria Bacillus subtilis, which tend to swim upwards in a gradient of oxygen concentration that they create by consumption, we discovered that the dominant wavelength at the onset of the instability is determined primarily by the cell density and is influenced only weakly by the fluid depth . This observation contrasts strongly with previous observations on the gravitactic alga Chlamydomonas nivalis, in which the opposite dependence was found . Considerable differences were also found in the long-term evolution of the convection patterns . These results demonstrate the existence of readily distinguishable types of bioconvection systems, even at early stages of the instability . The observed differences are clearly and causally correlated with disparate reasons for upward swimming by these micro-organisms, leading to different geometric distributions of the density of the suspension.

Microbiology, 2000 Oct, 146 ( Pt 10), 2595 - 603
Expression of the ftsY gene, encoding a homologue of the alpha subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis; Kakeshita H et al.; Bacillus subtilis FtsY (Srb) is a homologue of the alpha subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth . The ftsY gene is expressed during both the exponential phase and sporulation . In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor sigma(A) . During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of sigma(K) and gerE mutants . Therefore, ftsY is solely expressed during sporulation from a sigma(K)- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene . To examine the role of FtsY during sporulation, the B . subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state . Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.

Microbiology, 2000 Oct, 146 ( Pt 10), 2583 - 94
Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system; Jensen CL et al.; A series of chimeric alpha-amylase genes derived from amyL, which encodes the liquefying alpha-amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques . The gene constructs were cloned in Bacillus subtilis, where their ability to direct the synthesis and secretion of active alpha-amylase was determined . Detectable alpha-amylase activity was observed for some, but not all, of the chimeric proteins . Studies on the secretion of wild-type AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion . The chimeric enzymes were degraded to a greater extent than the native enzyme . These findings suggest that cell-associated proteolysis is a significant problem affecting the use of B . subtilis as host bacterium for the production of heterologous proteins.

Int J Food Microbiol, 2000 Sep 10, 59(3), 235 - 9
Improvement of the traditional processing and fermentation of African oil bean (Pentaclethra macrophylla Bentham) into a food snack--'ugba'; Isu NR et al.; Inocula for the improvement of the traditional production of 'ugba' were developed by attaching pure cultures of Bacillus subtilis responsible for the natural fermentation of the African oil bean seeds on cowpea granules . Changes in pH, amino-nitrogen and protease activity were used as fermentation indicators . In comparison with the natural fermentation, changes in these process variables were more pronounced . Results also showed that the production time could be significantly reduced . In addition, the overall product quality of 'ugba' produced by the developed inocula was good and highly acceptable . The cultures were stable and viable for over 6 months on the granules of cowpea.

Int J Food Microbiol, 2000 Sep 25, 60(2-3), 163 - 9
In vitro digestibility of bacillus fermented soya bean; Kiers JL et al.; Bacillus fermented legume products include among others dawadawa and soumbala made from African locust bean, and natto and kinema made from soya bean . Bacillus subtilis is the dominant species involved in the fermentation . During Bacillus fermentation for 48 h of autoclaved soya bean the quantity of soluble and dialyzable matter increased from 22% and 6% up to 65% and 40%, respectively . Protein and carbohydrate degradation during fermentation of soya bean with several Bacillus spp . was investigated and appeared to be substantial during the first 18 h of fermentation resulting in the release of high levels of peptides and oligosaccharides . In vitro digestibility was increased from 29% up to 33-43% after Bacillus fermentation for 48 h . It was shown that Bacillus spp . were able to degrade soya bean macromolecules to a large extent resulting in water-soluble low molecular weight compounds . In vitro digestion of Bacillus fermented soya bean using gastrointestinal enzymes only slightly increased the amount of dialyzable matter, which clearly demonstrated the beneficial effect of Bacillus fermentation on food nutrient availability.

Phytochemistry, 2000 Aug, 54(8), 777 - 82
Highly oxidized cuparene-type sesquiterpenes from a mycelial culture of Flammulina velutipes; Ishikawa NK et al.; Cuparene-type sesquiterpenes were isolated from a culture broth of Flammulina velutipes (Curt.:Fr.) Sing . Using spectroscopic methods (HR-MS, 1H and 13C NMR, and 2D NMR, spectroscopy), their structures were determined to be 2,3,4,5-tetrahydro-2,7-dihydroxy-5,8,10,10-tetramethyl-2,5-methano-1- benzoxepin and 5-methyl-2-(3-oxo-1,2,2-trimethylcyclopentyl)benzoquinone . Both showed antimicrobial activity against Cladosporium herharum and Bacillus subtilis.

Int J Pharm, 2000 Aug 25, 204(1-2), 43 - 6
Fabrication of oral acid resistant Fermosorb (Ferment + sorbent)-type preparations can be simplified using industrial Bacillus subtilis culture filtrates as an enzyme source; Biziulevicius GA et al.; In our recent article {Biziulevicius, G.A . and Zukaite, V., 1999 . Int . J . Pharm . 189, 43-55} we described a novel approach in design of oral preparations intented for intestinal delivery of enzymes, based on reversible immobilization of the latter onto the polymer matrix . Fermosorb (ferment + sorbent)-type preparations, produced in such a way, are characterized as two-component delayed-release enzyme formulations being stable at acidic pH and thus ensuring the protection of an active substance in the environment of the gastric region and liberating the active substance through dissociation of the enzyme-polymer complex at neutral pH values characteristic for the intestines . In the present paper we report our updated findings showing that the technology of fabrication of two Fermosorb-type preparations manufactured (namely Fermosorb and Polyferm) can be simplified (as well as their production cost reduced) substituting the acetone precipitated enzyme preparation solutions, currently used as an enzyme source, by their precursors--industrial Bacillus subtilis culture filtrates . Moreover, we give a description of how one more Fermosorb-type preparation aimed at intestinal delivery of amylolytic enzymes can be produced in a similar manner . Determination of immobilization conditions has revealed, that irrespectively of the enzyme origin (lytic, proteolytic or amylolytic) and its source (1%, acetone precipitated preparation solution or culture filtrate), optimal for immobilization v/w ratio of the liquid phase and the polymer matrix (Biocarb L) remains the same and is equal to 10:1 (approximate) . The main differences have been found to be in optimal for immobilization pH values as well as process duration in regard to the two enzyme sources applied . In case of proteolytic and amylolytic enzymes only one of the variables was different (process duration for the first ones, optimal pH for the second ones), while in case of lytic enzymes both variables were different . The percentage of the enzymes activity uptaken from the reaction mixture formed by either of the enzyme sources and the polymer matrix (approximately 60%) as well as activity losses at drying the enzyme-polymer complexes ( approximately 20%), tolling in the final activity yield of near 40%, were found to be very similar.

J Biochem (Tokyo), 2000 Oct, 128(4), 655 - 63
Synthesis and characterization of the spore proteins of Bacillus subtilis YdhD, YkuD, and YkvP, which carry a motif conserved among cell wall binding proteins; Kodama T et al.; We have previously reported that YaaH and YrbA are spore proteins of Bacillus subtilis that are required for spore resistance and/or germination and that they have a motif conserved among so-called cell wall binding proteins {Kodama et al . (1999) J . Bacteriol . 181, 4584-4591, Takamatsu et al . (1999) J . Bacteriol . 181, 4986-4994} . In this study, we analyzed the expression of ydhD, ykuD, and ykvP genes, which encode putative proteins containing the same motif . Transcription of ydhD was dependent on SigE, and the mRNA was detectable from 2 h after the cessation of logarithmic growth (T(2) of sporulation) . ykuD was transcribed by SigK RNA polymerase from T(4) of sporulation . Both SigK and GerE were essential for ykvP expression, and this gene was transcribed from T(5) of sporulation . Inactivation of these genes by insertion of an erythromycin resistance gene did not affect vegetative growth, spore resistance to heat, chloroform, and lysozyme, or spore germination in the presence of L-alanine or in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride . The His tag fusions of YdhD, YkuD, and YkvP downstream of their natural promoter regions were introduced into a multicopy plasmid . These fusion proteins were produced during sporulation in B . subtilis transformants and were detected in mature spores, indicating that YdhD, YkuD, and YkvP are all proteins intrinsic to spores . Excessive YkuD and YkvP in the sporulating cells did not affect spore resistance or germination . The cells producing excessive YdhD also did not show impaired spore resistance, but their germination properties were changed: the spores revealed reduced response to L-alanine and some of them germinated even without germinants . Escherichia coli b-lactamase, whose signal sequence had been genetically replaced by the cell wall binding motif of YaaH, was produced in sporulating cells, and Western blot analysis indicated that the fused protein was assembled into spores . We speculate that the conserved motif functions as a kind of signal sequence involved in assembly of these proteins on forespores.

J Biochem (Tokyo), 2000 Oct, 128(4), 585 - 9
Enhanced thermostability of the single-Cys mutant subtilisin E under oxidizing conditions; Takagi H et al.; We obtained enhanced thermostability by replacing Ser161 with Cys in subtilisin E from Bacillus subtilis, a cysteine-free alkaline serine protease . The Ser161Cys mutant subtilisin E was purified from the culture supernatant of the recombinant B . subtilis in an oxidizing environment . SDS-polyacrylamide gel electrophoresis and mass spectrometry under oxidizing conditions indicated that the mutant enzyme in part formed an oligomeric protein, which may contain an intermolecular disulfide bond between two surface Cys residues at position 161 . Further, no free sulfhydryl groups were detected in the mutant enzyme, suggesting the sulfhydryl modification in a monomeric form under oxidizing conditions . The Ser161Cys mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme . The half-life of thermal inactivation of the mutant was found to be 2-4 times longer than that of the wild-type enzyme . The optimum temperature of the mutant was 55 degrees C, which was 5 degrees C higher than that of the wild-type enzyme . Under reducing conditions, however, the characteristics of the mutant enzyme reverted to those of the wild-type enzyme . Similar results were obtained for another Cys mutant as to position 194 (wild-type, Ser), which is the same surface residue as Ser161 . Possible reasons for the enhanced thermostability of the single-Cys mutant subtilisins E under oxidizing conditions are discussed in terms of two different mechanisms.

J Biol Chem, 2000 Dec 22, 275(51), 40128 - 33
The enoyl-{acyl-carrier-protein} reductases FabI and FabL from Bacillus subtilis; Heath RJ et al.; Enoyl-{acyl-carrier-protein} (ACP) reductase is a key enzyme in type II fatty-acid synthases that catalyzes the last step in each elongation cycle . The FabI component of Bacillus subtilis (bsFabI) was identified in the genomic data base by homology to the Escherichia coli protein . bsFabI was cloned and purified and exhibited properties similar to those of E . coli FabI, including a marked preference for NADH over NADPH as a cofactor . Overexpression of the B . subtilis fabI gene complemented the temperature-sensitive growth phenotype of an E . coli fabI mutant . Triclosan was a slow-binding inhibitor of bsFabI and formed a stable bsFabI.NAD(+) . triclosan ternary complex . Analysis of the B . subtilis genomic data base revealed a second open reading frame (ygaA) that was predicted to encode a protein with a relatively low overall similarity to FabI, but contained the Tyr-Xaa(6)-Lys enoyl-ACP reductase catalytic architecture . The purified YgaA protein catalyzed the NADPH-dependent reduction of trans-2-enoyl thioesters of both N-acetylcysteamine and ACP . YgaA was reversibly inhibited by triclosan, but did not form the stable ternary complex characteristic of the FabI proteins . Expression of YgaA complemented the fabI(ts) defect in E . coli and conferred complete triclosan resistance . Single knockouts of the ygaA or fabI gene in B . subtilis were viable, but double knockouts were not obtained . The fabI knockout was as sensitive as the wild-type strain to triclosan, whereas the ygaA knockout was 250-fold more sensitive to the drug . YgaA was renamed FabL to denote the discovery of a new family of proteins that carry out the enoyl-ACP reductase step in type II fatty-acid synthases.

J Biol Chem, 2000 Dec 22, 275(51), 40547 - 53
Sequence requirements for protein-primed initiation and elongation of phage O29 DNA replication; Gonzalez-Huici V et al.; The double-stranded linear DNA of Bacillus subtilis phage O29 is replicated by a mechanism in which a terminal protein (TP) acts as a primer . The second 3'-terminal nucleotide of the template directs the incorporation of the 5'-terminal nucleotide into the TP, giving rise to the initiation complex TP-dAMP . Elongation then proceeds by a sliding-back mechanism in which the dAMP covalently linked to the TP pairs to the 3'-terminal nucleotide of the template strand to recover full-length DNA . We have studied the sequence requirements for efficient initiation of replication using mutated TP-free double-stranded DNA fragments . Efficient initiation only requires the terminal repetition 5'-AA . The 3'-terminal T, although not used as template, increases the affinity of DNA polymerase for the initiator nucleotide; in addition, although to a minor extent, the third 3'-terminal position also directs the formation of the initiation complex and modulates the initiation rate at the second position . Efficient elongation requires a previous sliding-back, demanding again a repetition of two nucleotides at the 3' end; if the sliding-back is prevented, a residual elongation can proceed directly from the second position or after jumping back from the third to the first position.

J Bacteriol, 2000 Oct, 182(20), 5931 - 4
Identification in Listeria monocytogenes of MecA, a homologue of the Bacillus subtilis competence regulatory protein; Borezee E et al.; We identified in Listeria monocytogenes a gene encoding a protein homologous to MecA, a regulatory protein acting with ClpC and ComK in the competence pathway of Bacillus subtilis . In L . monocytogenes, MecA is involved, along with ClpC and ClpP, in the downregulation of a 64-kDa secreted protein . In B . subtilis, the MecA protein of L . monocytogenes behaves as a regulatory protein, controlling the transcription of comK and comG . Complete or disrupted ComK homologues were also found in L . monocytogenes . However, we failed to detect competence in various strains of L . monocytogenes, including those with intact ComK . Our results suggest that the functions of MecA in the saprophytes L . monocytogenes and B . subtilis have presumably diverged in response to their respective ecological niches.

J Bacteriol, 2000 Oct, 182(20), 5885 - 92
Transcriptional control of the sulfur-regulated cysH operon, containing genes involved in L-cysteine biosynthesis in Bacillus subtilis; Mansilla MC et al.; The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in Bacillus subtilis and other gram-positive bacteria . In this study we describe the expression pattern of the B . subtilis cysH operon in response to sulfur starvation . A 6.1-kb polycistronic transcript which includes the cysH, cysP, ylnB, ylnC, ylnD, ylnE, and ylnF genes was identified . Its synthesis was induced by sulfur limitation and strongly repressed by cysteine . The cysH operon contains a 5' leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system . Here we show that induction of B . subtilis cysH operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription . Deletion of a 46-bp region adjacent to the -35 region of the cysH promoter led to high-level expression of the operon, even in the presence of cysteine . We also found that O-acetyl-L-serine (OAS), a direct precursor of cysteine, renders cysH transcription independent of sulfur starvation and insensitive to cysteine repression . We propose that transcription of the cysH operon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS . Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of cysH expression . These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.

J Bacteriol, 2000 Oct, 182(20), 5663 - 70
Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na(+) and alkali but not cholate resistance; Ito M et al.; The Bacillus subtilis mrp (multiple resistance and pH) operon supports Na(+) and alkali resistance via an Na(+)/H(+) antiport, as well as cholate efflux and resistance . Among the individual mutants with nonpolar mutations in each of the seven mrp genes, only the mrpF mutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted gene in trans . Expression of mrpF in the mrp null (VKN1) strain also restored cholate transport and increased Na(+) efflux, indicating that MrpF does not require even low levels of other mrp gene expression for its own function . In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of other mrp genes, i.e., mrpA restored Na(+) resistance and efflux to strain VK6 (a polar mrpA mutant which expresses low levels of mrpB to -G) but not to the null strain VKN1 . In a wild-type background, each nonpolar mutation in individual mrp genes caused profound Na(+) sensitivity at both pH 7.0 and 8.3 . The mrpA and mrpD mutants were particularly sensitive to alkaline pH even without added Na(+) . While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiple mrp gene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters . Northern analyses indicated regulatory relationships among mrp genes as well . All the mrp mutants, especially the mrpA, -B, -D, -E, and -G mutants, had elevated levels of mrp RNA relative to the wild type . Expression of an upstream gene, maeN, that encodes an Na(+)/malate symporter, was coordinately regulated with mrp, although it is not part of the operon.

Biochim Biophys Acta, 2000 Jul 26, 1475(3), 353 - 9
Structural and functional studies on an FtsH inhibitor from Bacillus subtilis; Prajapati RS et al.; The small 3 kDa SpoVM protein is essential for development of the spore in Bacillus subtilis . Genetic and biochemical experiments have shown that the function of SpoVM is to inhibit the proteolytic activity of FtsH during sporulation . We have used a combination of genetic and biophysical techniques to characterise the role of this small polypeptide . SpoVM was found to be widespread in Bacillus as well as in two Clostridia species, suggesting that SpoVM provides a common mechanism for inactivating the FtsH protease during spore differentiation . Using site-specific mutagenesis, we have identified C-terminal residues of SpoVM essential for biological activity . Analysis of SpoVM's structure showed that it is able to assume an alpha-helical conformation in the presence of a lipid interface which may be important in interacting with FtsH.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 422 - 31
Succinate: quinone oxidoreductases: new insights from X-ray crystal structures; Lancaster CR et al.; Membrane-bound succinate dehydrogenases (succinate:quinone reductases, SQR) and fumarate reductases (quinol:fumarate reductases, QFR) couple the oxidation of succinate to fumarate to the reduction of quinone to quinol and also catalyse the reverse reaction . SQR (respiratory complex II) is involved in aerobic metabolism as part of the citric acid cycle and of the aerobic respiratory chain . QFR is involved in anaerobic respiration with fumarate as the terminal electron acceptor, and is part of an electron transport chain catalysing the oxidation of various donor substrates by fumarate . QFR and SQR complexes are collectively referred to as succinate:quinone oxidoreductases (EC 1.3.5.1), have very similar compositions and are predicted to share similar structures . The complexes consist of two hydrophilic and one or two hydrophobic, membrane-integrated subunits . The larger hydrophilic subunit A carries covalently bound flavin adenine dinucleotide and subunit B contains three iron-sulphur centres . QFR of Wolinella succinogenes and SQR of Bacillus subtilis contain only one hydrophobic subunit (C) with two haem b groups . In contrast, SQR and QFR of Escherichia coli contain two hydrophobic subunits (C and D) which bind either one (SQR) or no haem b group (QFR) . The structure of W . succinogenes QFR has been determined at 2.2 A resolution by X-ray crystallography (C.R.D . Lancaster, A . Kroger, M . Auer, H . Michel, Nature 402 (1999) 377-385) . Based on this structure of the three protein subunits and the arrangement of the six prosthetic groups, a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b to the site of fumarate reduction and a mechanism of fumarate reduction was proposed . The W . succinogenes QFR structure is different from that of the haem-less QFR of E . coli, described at 3.3 A resolution (T.M . Iverson, C . Luna-Chavez, G . Cecchini, D.C . Rees, Science 284 (1999) 1961-1966), mainly with respect to the structure of the membrane-embedded subunits and the relative orientations of soluble and membrane-embedded subunits . Also, similarities and differences between QFR transmembrane helix IV and transmembrane helix F of bacteriorhodopsin and their implications are discussed.

Nucleic Acids Res, 2000 Oct 1, 28(19), 3785 - 92
UV-induced crosslinks in the 16S rRNAs of Escherichia coli, Bacillus subtilis and Thermus aquaticus and their implications for ribosome structure and photochemistry; Noah JW et al.; Sixteen long-range crosslinks are induced in Escherichia coli 16S rRNA by far-UV irradiation . Crosslinking patterns in two other organisms, Bacillus subtilis and Thermus aquaticus, were investigated to determine if the number and location of crosslinks in E.coli occur because of unusually photoreactive nucleotides at particular locations in the rRNA sequence . Thirteen long-range crosslinks in B.subtilis and 15 long-range crosslinks in T.aquaticus were detected by gel electrophoresis and 10 crosslinks in each organism were identified completely by reverse transcription analysis . Of the 10 identified crosslinks in B.subtilis, eight correspond exactly to E.coli crosslinks and two crosslinks are formed close to sites of crosslinks in E.coli . Of the 10 identified crosslinks in T.aquaticus, five correspond exactly to E.coli crosslinks, three are formed close to E.coli crosslinking sites, one crosslink corresponds to a UV laser irradiation-induced crosslink in E.coli and the last is not seen in E.coli . The overall similarity of crosslink positions in the three organisms suggests that the crosslinks arise from tertiary interactions that are highly conserved but with differences in detail in some regions.

Anal Biochem, 2000 Oct 1, 285(1), 50 - 7
Detection and analysis of Bacillus subtilis growth with piezoelectric quartz crystal impedance based on starch hydrolysis; Wu Y et al.; A piezoelectric quartz crystal (PQC) impedance method based on the alpha-amylase-catalyzed hydrolysis of starch present in a culture medium has been developed for in situ monitoring of the whole growth process of Bacillus subtilis and the variation in the activity of alpha-amylase during bacterial growth . An S-shaped response behavior was observed for Deltaf(0), and simultaneously inverse S-shaped responses were found for DeltaR(1) and DeltaL(1) . The ratio of DeltaR(1) to Deltaf(0) or DeltaL(1) coincided well with that calculated from Martin's equations reflecting the solution density-viscosity effect, suggesting that the continuing change in liquid loading onto the PQC surface causes significant variation in Deltaf(0), DeltaR(1), and DeltaL(1) . Bacterial growth equations were derived from the kinetics of the enzyme-catalyzed hydrolysis of starch, which fit well with the experimental responses of Deltaf(0), DeltaR(1), and DeltaL(1) . Kinetic parameters of bacterial growth, including the asymptote (A), the maximum specific growth rate (microm), and the lag time (lambda), were obtained and were in good agreement with those obtained from the pour plate count method . The variation in the activity of alpha-amylase exhibited peak-type behavior with its maximum value at the later stage of the log phase . In addition, the influence of initial bacterial concentration was also investigated .

Biochemistry, 2000 Sep 12, 39(36), 11107 - 13
Mg2+-dependent compaction and folding of yeast tRNAPhe and the catalytic domain of the B . subtilis RNase P RNA determined by small-angle X-ray scattering; Fang X et al.; We apply synchrotron-based small-angle X-ray scattering to investigate the relationship between compaction, metal binding, and structure formation of two RNAs at 37 degrees C: the 76 nucleotide yeast tRNA(Phe) and the 255 nucleotide catalytic domain of the Bacillus subtilis RNase P RNA . For both RNAs, this method provides direct evidence for the population of a distinct folding intermediate . The relative compaction between the intermediate and the native state does not correlate with the size of the RNA but does correlate well with the amount of surface burial as quantified previously by the urea-dependent m-value . The total compaction process can be described in two major stages . Starting from a completely unfolded state (4-8 M urea, no Mg(2+)), the major amount of compaction occurs upon the dilution of the denaturant and the addition of micromolar amounts of Mg(2+) to form the intermediate . The native state forms in a single transition from the intermediate state upon cooperative binding of three to four Mg(2+) ions . The characterization of this intermediate by small-angle X-ray scattering lends strong support for the cooperative Mg(2+)-binding model to describe the stability of a tertiary RNA.

Structure Fold Des, 2000 Aug 15, 8(8), 883 - 95
Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites; Parris KD et al.; BACKGROUND: Holo-(acyl carrier protein) synthase (AcpS), a member of the phosphopantetheinyl transferase superfamily, plays a crucial role in the functional activation of acyl carrier protein (ACP) in the fatty acid biosynthesis pathway . AcpS catalyzes the attachment of the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to the sidechain of a conserved serine residue on apo-ACP . RESULTS: We describe here the first crystal structure of a type II ACP from Bacillus subtilis in complex with its activator AcpS at 2.3 A . We also have determined the structures of AcpS alone (at 1.8 A) and AcpS in complex with CoA (at 1.5 A) . These structures reveal that AcpS exists as a trimer . A catalytic center is located at each of the solvent-exposed interfaces between AcpS molecules . Site-directed mutagenesis studies confirm the importance of trimer formation in AcpS activity . CONCLUSIONS: The active site in AcpS is only formed when two AcpS molecules dimerize . The addition of a third molecule allows for the formation of two additional active sites and also permits a large hydrophobic surface from each molecule of AcpS to be buried in the trimer . The mutations Ile5-->Arg, Gln113-->Glu and Gln113-->Arg show that AcpS is inactive when unable to form a trimer . The co-crystal structures of AcpS-CoA and AcpS-ACP allow us to propose a catalytic mechanism for this class of 4'-phosphopantetheinyl transferases.

Structure Fold Des, 2000 Aug 15, 8(8), 851 - 62
A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction; Zapf J et al.; BACKGROUND: Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli . Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis . Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F . Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems . RESULTS: Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B . subtilis was carried out using X-ray crystallographic techniques . The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer . Upon complex formation, the Spo0B conformation remains unchanged . Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations . CONCLUSIONS: The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions . The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins . The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs . The bulk of the interactions outside this patch are through nonconserved residues . Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.

Genetika, 2000 Jul, 36(7), 1000 - 2
{A large conjugative plasmid from a soil strain of Bacillus subtilis}; Poluektova EU et al.; A large plasmid 35.5 kb in size was found in the soil Bacillus subtilis strain . This plasmids was shown to be capable of conjugal mobilization of small plasmids.

Mol Cells, 2000 Aug 31, 10(4), 423 - 31
The biochemical and molecular characterization of recombinant Bacillus subtilis tripeptidase (PepT) as a zinc-dependent metalloenzyme; Cha MH et al.; Aminopeptidases catalyze the release of N-terminal amino acid residue from polypeptides and peptides, and most of them are known to be metalloenzymes . A tripeptidase gene (pepT) of Bacillus subtilis was expressed in Escherichia coli, and the resulting recombinant PepT was purified in an active form through sequential chromatographies . The addition of Zn2+ or Co2+ increased the enzymatic activity by approximately two fold . The points at which Zn2+ and Co2+ stimulated a half-maximum activity of the PepT were 650 nM and 1,700 nM, respectively . The measurement of the metal content showed that this enzyme contained 0.26 atom of Zn2+ per molecule with essentially the absence of Co2+ and others, and 0.53 atom of Zn2+ with 1.5-fold increase of activity when reconstituted with Zn2+ . Consistent with this result, this enzyme is much readily refolded in the presence of Zn2+ than Co2+ . To further delineate the structure and function relations, we made serial deletion mutants and analyzed their enzymatic activities . Of eight deletion mutants, only a mutant lacking the N-terminal 66 amino acid residues retained enzymatic activity . The mutant enzyme, however, required a concentration of Zn2+ ion at least ten-fold higher to reach maximum activity without significantly affecting kinetic parameters such as Km and Vmax compared to the full length PepT . Taken together, these data suggest that the B . subtilis PepT is likely to be a Zn2+-dependent metalloenzyme and that the N-terminal region of the PepT stabilizes Zn2+-binding.

J Bacteriol, 2000 Oct, 182(19), 5634 - 8
Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family; Obuchowski M et al.; We cloned the yloO gene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC . This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure . PrpC has phosphatase activity in vitro against a synthetic substrate, p-nitrophenol phosphate, and endogenous Bacillus subtilis proteins . The prkC and prpC genes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC . These findings suggest that PrkC and PrpC may function as a couple in vivo.

J Bacteriol, 2000 Oct, 182(19), 5611 - 4
Bacillus subtilis ccpA gene mutants specifically defective in activation of acetoin biosynthesis; Turinsky AJ et al.; A large number of carbon source utilization pathways are repressed in Bacillus subtilis by the global regulator CcpA, which also acts as an activator of carbon excretion pathways during growth in media containing glucose . In this study, CcpA mutants defective in transcriptional activation of the alsSD operon, which is involved in acetoin biosynthesis, were identified . These mutants retained normal glucose repression of amyE, encoding alpha-amylase, and acsA, encoding acetyl-coenzyme A synthetase, and normal activation of ackA, which is involved in acetate excretion; in these ccpA mutants the CcpA functions of activation of the acetate and acetoin excretion pathways appear to be separated.

J Bacteriol, 2000 Oct, 182(19), 5572 - 9
Analysis of the essential cell division gene ftsL of Bacillus subtilis by mutagenesis and heterologous complementation; Sievers J et al.; The ftsL gene is required for the initiation of cell division in a broad range of bacteria . Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus . The external C-terminal domain has features of an alpha-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC . To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis . Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon . It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect . Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function . ftsL homologues cloned from three diverse Bacillus species, Bacillus licheniformis, Bacillus badius, and Bacillus circulans, could complement an ftsL null mutation in B . subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different from B . subtilis FtsL . However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B . subtilis ftsL product) was not functional . We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.

J Bacteriol, 2000 Oct, 182(19), 5556 - 62
Analysis of nucleoid morphology during germination and outgrowth of spores of Bacillus species; Ragkousi K et al.; After a few minutes of germination, nucleoids in the great majority of spores of Bacillus subtilis and Bacillus megaterium were ring shaped . The major spore DNA binding proteins, the alpha/beta-type small, acid-soluble proteins (SASP), colocalized to these nucleoid rings early in spore germination, as did the B . megaterium homolog of the major B . subtilis chromosomal protein HBsu . The percentage of ring-shaped nucleoids was decreased in germinated spores with lower levels of alpha/beta-type SASP . As spore outgrowth proceeded, the ring-shaped nucleoids disappeared and the nucleoid became more compact . This change took place after degradation of most of the spores' pool of major alpha/beta-type SASP and was delayed when alpha/beta-type SASP degradation was delayed . Later in spore outgrowth, the shape of the nucleoid reverted to the diffuse lobular shape seen in growing cells.

J Bacteriol, 2000 Oct, 182(19), 5505 - 12
Characterization of spores of Bacillus subtilis which lack dipicolinic acid; Paidhungat M et al.; Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail . However, the spores of a strain lacking the three major germinant receptors (termed Deltager3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Deltager3 spores . The Deltager3 spoVF spores lack DPA and have higher levels of core water than Deltager3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to Deltager3 spoVF spores . The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA . The Deltager3 spoVF spores also contain minimal levels of the processed active form (termed P(41)) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing . However, any P(41) formed in Deltager3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-gamma . Analysis of the resistance of wild-type, Deltager3, and Deltager3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Deltager3 spores to Betadine and glutaraldehyde . These results are discussed in view of current models of spore resistance and spore germination.

J Bacteriol, 2000 Oct, 182(19), 5454 - 61
An operon for a putative ATP-binding cassette transport system involved in acetoin utilization of Bacillus subtilis; Yoshida KI et al.; The ytrABCDEF operon of Bacillus subtilis was deduced to encode a putative ATP-binding cassette (ABC) transport system . YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding . Expression of the operon was examined in cells grown in a minimal medium . The results indicate that the expression was induced only early in the stationary phase . The six ytr genes form a single operon, transcribed from a putative sigma(A)-dependent promoter present upstream of ytrA . YtrA, which possesses a helix-turn-helix motif of the GntR family, acts probably as a repressor and regulates its own transcription . Inactivation of the operon led to a decrease in maximum cell yield and less-efficient sporulation, suggesting its involvement in the growth in stationary phase and sporulation . It is known that B . subtilis produces acetoin as an external carbon storage compound and then reuses it later during stationary phase and sporulation . When either the entire ytr operon or its last gene, ytrF, was inactivated, the production of acetoin was not affected, but the reuse of acetoin became less efficient . We suggest that the Ytr transport system plays a role in acetoin utilization during stationary phase and sporulation.

J Biol Chem, 2000 Dec 8, 275(49), 38813 - 22
The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection; Carlos JL et al.; Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export . Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region . Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity . Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein . One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane . This limits the enzyme-substrate interactions such that cleavage occurs at only one site . In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2) . With purified full-length as well as truncated constructs of both B . subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate . In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity . In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 133 - 9
A chromosomal locus encoding a phosphoserine phosphatase- and a truncated MinD-like protein affects differentiation in Streptomyces azureus ATCC14921; Nishiyama T et al.; We isolated BalA1, a representative transformant of thiostrepton-producing strain Streptomyces azureus ATCC14921, which carries an approximately 2.5-kb chromosomal DNA fragment on a high-copy-number plasmid . While strain BalA1 formed little aerial hyphae, its morphological defect was restored by cultivation with S . azureus, S . laurentii, etc . Strain BalA1 strongly inhibited the growth of Bacillus subtilis more than its parent strain, and also inhibited the development of its parent and some Streptomyces strains with thiostrepton resistance . Furthermore, it induced Streptomyces coelicolor A3(2) to produce undecylprodigiosin, at an early stage of growth . The 2.5-kb fragment contained two orfs, orf1 and truncated orf2 . The deduced products were somewhat similar to phosphoserine phosphatase-like protein and the N-terminal region of MinD-like protein, respectively . The individual function of orf1 or the function of both orf1 and truncated orf2 seems to induce particular phenotypes or properties in strain BalA1.

FEMS Microbiol Rev, 2000 Oct, 24(4), 449 - 67
The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis; Braibant M et al.; We have undertaken the inventory and assembly of the typical subunits of the ABC transporters encoded by the complete genome of Mycobacterium tuberculosis . These subunits, i.e . the nucleotide binding domains (NBDs), the membrane-spanning domains (MSDs) and the substrate binding proteins (SBPs), were identified on the basis of their characteristic stretches of amino acids and/or conserved structure . A total of 45 NBDs present in 38 proteins, of 47 MSDs present in 44 proteins and of 15 SBPs were found to be encoded by M . tuberculosis . Analysis of transcriptional clusters and searches of homology between the identified subunits of the transporters and proteins characterized in other organisms allowed the reconstitution of at least 26 complete (including at least one NBD and one MSD) and 11 incomplete ABC transporters . Sixteen of them were unambiguously classified as importers whereas 21 were presumed to be exporters . By searches of homology with already known transporters from other organisms, potential substrates (peptides, macrolides, carbohydrates, multidrugs, antibiotics, iron, anions) could be attributed to 30 of the ABC transporters identified in M . tuberculosis . The ABC transporters have been further classified in nine different sub-families according to a tree obtained from the clustering of their NBDs . Contrary to Escherichia coli and similarly to Bacillus subtilis, there is an equal representation of extruders and importers . Many exporters were found to be potentially implicated in the transport of drugs, probably contributing to the resistance of M . tuberculosis to many antibiotics . Interestingly, a transporter (absent in E . coli and in B . subtilis) potentially implicated in the export of a factor required for the bacterial attachment to the eukaryotic host cells was also identified . In comparison to E . coli and B . subtilis, there is an under-representation of the importers (with the exception of the phosphate importers) in M . tuberculosis . This may reflect the capacity of this bacterium to synthesize many essential compounds and to grow in the presence of few external nutrients . The genes encoding the ABC transporters occupy about 2.5% of the genome of M . tuberculosis.

Microbiol Mol Biol Rev, 2000 Sep, 64(3), 548 - 72
Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments; Nicholson WL et al.; Endospores of Bacillus spp., especially Bacillus subtilis, have served as experimental models for exploring the molecular mechanisms underlying the incredible longevity of spores and their resistance to environmental insults . In this review we summarize the molecular laboratory model of spore resistance mechanisms and attempt to use the model as a basis for exploration of the resistance of spores to environmental extremes both on Earth and during postulated interplanetary transfer through space as a result of natural impact processes.

Microbiol Mol Biol Rev, 2000 Sep, 64(3), 515 - 47
Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome; Tjalsma H et al.; One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations . This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes . The recent sequencing of the genome of B . subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism . Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins . The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported . By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion . In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported . The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed . The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B . subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae . Thus, they may serve as a lead for future research and applications.

Microbiology, 2000 Sep, 146 ( Pt 9), 2333 - 42
Regulation of the glucose-specific phosphotransferase system (PTS) of Staphylococcus carnosus by the antiterminator protein GlcT; Knezevic I et al.; The ptsG operon of Staphylococcus carnosus consists of two adjacent genes, glcA and glcB, encoding glucose- and glucoside-specific enzymes II, respectively, the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) . The expression of the ptsG operon is glucose-inducible . Putative RAT (ribonucleic antiterminator) and terminator sequences localized in the promoter region of glcA suggest regulation via antitermination . The glcT gene was cloned and the putative antiterminator protein GlcT was purified . Activity of this protein was demonstrated in vivo in Escherichia coli and Bacillus subtilis . In vitro studies led to the assumption that phosphoenolpyruvate-dependent phosphorylation of residue His105 via the general PTS components enzyme I and HPr facilitates dimerization of GlcT and consequently activation . Because of the high similarity of the two ptsG-RAT sequences of B . subtilis and S . carnosus, in vivo studies were performed in B . subtilis . These indicated that GlcT of S . carnosus is able to recognize ptsG-RAT sequences of B . subtilis and to cause antitermination . The specific interaction between B . subtilis ptsG-RAT and S . carnosus GlcT demonstrated by surface plasmon resonance suggests that only the dimer of GlcT binds to the RAT sequence . HPr-dependent phosphorylation of GlcT facilitates dimer formation and may be a control device for the proper function of the general PTS components enzyme I and HPr necessary for glucose uptake and phosphorylation by the corresponding enzyme II.

Mol Microbiol, 2000 Sep, 37(5), 1208 - 19
ResD signal transduction regulator of aerobic respiration in Bacillus subtilis: ctaA promoter regulation; Zhang X et al.; A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, encoded by resD and resE genes of the res operon (resABCDE), has a regulatory role in both aerobic and anaerobic respiration . In terms of aerobic respiration, resD functions upstream of ctaA, a gene required for haem A biogenesis and hence for the synthesis of haem A-containing cytochrome terminal oxidases . Although ResD is probably a transcription factor, there was no direct evidence that ResD protein, either phosphorylated or unphosphorylated, interacts directly with regulatory regions of ResD-controlled genes . Here, we report the overexpression and purification of ResD and ResE and their role in gene activation . ResD can be phosphorylated by ResE in vitro and is a monomer in solution in either the phosphorylated or unphosphorylated state . The binding activity of ResD to the ctaA promoter was examined by gel shift assays and DNase I footprinting assays . DNase I footprinting showed both unphosphorylated and phosphorylated ResD binding to the ctaA promoter and showed that there are three binding sites (A1, A2 and A3), two (A1 and A2) upstream of the -35 promoter region and one (A3) downstream of the -10 of the promoter . The role of each site in ctaA promoter activity and ResD binding was characterized using deletion analysis, followed by the DNase I footprinting and in vivo transcription assays of promoter-lacZ fusions . Our results showed that the concentration of ResD required to bind at each site is different and that ResD binding at the A1 site is independent of the other two ResD binding sites, but that the concentration of ResD approximately P required to protect site A2 is reduced when site A3 is present . In vivo transcription assays from promoter-lacZ fusion constructs showed that DNA containing ResD-binding site A2 was essential for promoter activity and that promoter constructs containing both binding sites A2 and A3 were sufficient for full promoter activity.

Mol Microbiol, 2000 Sep, 37(5), 1198 - 207
Interaction of ResD with regulatory regions of anaerobically induced genes in Bacillus subtilis; Nakano MM et al.; The two-component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis . ResD, when it undergoes ResE-dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD . In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis-acting sequences required for ResDE-dependent transcription . The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE-dependent regulation and that fnr has a single target site . Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes . Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding . In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed . Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters . Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.

Mol Microbiol, 2000 Aug, 37(4), 898 - 912
The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilis; Yamamoto H et al.; citS and citT genes encoding a new two-component system were identified in the 71 degrees region between the pel and citM loci on the Bacillus subtilis chromosome . citS- and citT-deficient strains were unable to grow on minimal plates including citrate as a sole carbon source . In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg-citrate complex, exhibited the same phenotype on this medium . Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM-yflN operon . Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant . Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose . We found a sequence exhibiting homology to a catabolite-responsive element (cre) in the citM promoter region . Glucose repression was lost in ccpA and citM-cre mutants . From the result of a citM-promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from -62 to -74 and from -149 to -189, relative to the citM start point . Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from -36 to -84 and from -168 to -194 . From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA.

Mol Microbiol, 2000 Aug, 37(4), 885 - 97
The ClpX protein of Bacillus subtilis indirectly influences RNA polymerase holoenzyme composition and directly stimulates sigma-dependent transcription; Liu J et al.; In Bacillus subtilis, several processes associated with the onset of stationary phase, including the initiation of sporulation, require the activity of the minor sigmaH form of RNA polymerase (RNAP) . The induction of sigmaH-dependent gene transcription requires the regulatory ATPase, ClpX . The ClpX-dependent post-exponential increase in sigmaH activity is not dependent on the activator of sporulation gene expression, Spo0A . By determining the level of sigmaH and sigmaA in whole-cell extracts and RNAP preparations, evidence is presented that clpX does not influence the concentration of sigma subunits, but is required for the stationary phase reduction in sigmaA-RNAP holoenzyme . This is probably an indirect consequence of ClpX activity, because the ClpX-dependent decrease in sigmaA-RNAP concentration does not occur in a spo0A abrB mutant . The addition of ClpX to in vitro transcription reactions resulted in the stimulation of RNAP holoenzyme activity, but sigmaH-RNAP was observed to be more sensitive to ClpX-dependent stimulation than sigmaA-RNAP . No difference in transcriptional activity was observed in single-cycle in vitro transcription reactions, suggesting that ClpX acted at a step in transcription initiation after closed- and open-promoter complex formation . ClpX is proposed to function indirectly in the displacement of sigmaA from core RNAP and to act directly in the stimulation of sigmaH-dependent transcription in sporulating B . subtilis cells.

Mol Microbiol, 2000 Aug, 37(4), 869 - 84
Mutations conferring amino acid residue substitutions in the carboxy-terminal domain of RNA polymerase alpha can suppress clpX and clpP with respect to developmentally regulated transcription in Bacillus subtilis; Nakano MM et al.; The Bacillus subtilis clpX and clpP genes are the sites of pleiotropic mutations that adversely affect growth on a variety of media and impair developmental processes such as sporulation and competence development . ClpX is necessary for the post-exponential induction of genes that require the sigmaH form of RNA polymerase for transcription . Both ClpX and ClpP are required for the activation of sigmaA-dependent transcription of the srf operon that encodes surfactin synthetase and the regulatory peptide ComS, required for the development of genetic competence . Transcription of srf is activated by the two-component regulatory system ComPA in response to the peptide pheromone, ComX, which mediates cell density-dependent control . A clpX mutant, although able to produce ComX, is unable to respond to the pheromone . A mutant allele of comP, encoding a product whose activity is independent of ComX, is not able to suppress clpX with respect to srf expression, suggesting that ClpXP acts at the level of ComA-dependent activation of srf transcription initiation . Suppressor mutations of clpX (cxs-1 and cxs-2) were isolated in screens for pseudorevertants exhibiting high levels of srf expression and sigmaH-dependent transcription respectively . One mutation, cxs-1, suppressed a clpP null mutation with respect to srf transcription, but did not overcome the block conferred by clpP on competence development and sporulation . Both cxs-1 and cxs-2 mutations map to the region of the rpoA gene encoding the RNA polymerase alpha C-terminal domain (alphaCTD) . The reconstruction of the cxs-1 and cxs-2 alleles of rpoA confirmed that these mutations confer the suppressor phenotype . These findings provide further support for the hypothesis that ClpX and ClpP might be intimately associated with transcription initiation in B . subtilis.

Mol Microbiol, 2000 Aug, 37(4), 828 - 38
Regulation by external pH and stationary growth phase of the acetolactate synthase from Synechocystis PCC6803; Maestri O et al.; Several characteristics identify the protein encoded by the alsS gene {sll1981 in Cyanobase } of Synechocystis PCC6803 as an acetolactate synthase . The AlsS protein is about 60% homologous to the AlsS from Bacillus subtilis or other bacteria . These enzymes condense two pyruvates to form acetolactate, implicated in pH homeostasis via the acetoin-2, 3-butanediol pathway or in valine biosynthesis . Transcriptional fusions revealed that alsS was induced at the onset of stationary phase, as in B . subtilis, a situation leading to an increase in the pHout to above 11 in Synechocystis . This is the first cyanobacterial gene showing a dependence on pH for its expression . Induction was also obtained by the presence of > 100 mM Na+, the effect being prevented by amiloride, in agreement with Na+/H+ exchange in the pH homeostasis process . Homology of the Synechocystis AlsS protein to the close family of acetohydroxy acid synthases (including one in Synechocystis) is around 30% . These enzymes are involved in the parallel routes for valine/leucine and isoleucine biosynthesis . No phenotype of auxotrophy for any of these amino acids was associated with a null mutation in the Synechocystis alsS gene . The AlsS enzyme did not complement the isoleucine deficiency of an acetohydroxy acid synthase-deficient Escherichia coli mutant.

J Appl Microbiol, 2000 Aug, 89(2), 330 - 8
Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid; Tennen R et al.; Treatment of wild-type spores of Bacillus subtilis with glutaraldehyde or an iodine-based disinfectant (Betadine) did not cause detectable mutagenesis, and spores (termed alpha-beta-) lacking the major DNA-protective alpha/beta-type, small, acid-soluble proteins (SASP) exhibited similar sensitivity to these agents . A recA mutation did not sensitize wild-type or alpha-beta- spores to Betadine or glutaraldehyde, nor did spore treatment with these agents result in significant expression of a recA-lacZ fusion when the treated spores germinated . Spore glutaraldehyde sensitivity was increased dramatically by removal of much spore coat protein, but this treatment had no effect on Betadine sensitivity . In contrast, nitrous acid treatment of wild-type and alpha-beta- spores caused significant mutagenesis, with alpha-beta- spores being much more sensitive to this agent . A recA mutation further sensitized both wild-type and alpha-beta- spores to nitrous acid, and there was significant expression of a recA-lacZ fusion when nitrous acid-treated spores germinated . These results indicate that: (a) nitrous acid kills B . subtilis spores at least in part by DNA damage, and alpha/beta-type SASP protect against this DNA damage; (b) killing of spores by glutaraldehyde or Betadine is not due to DNA damage; and (c) the spore coat protects spores against killing by glutaraldehyde but not Betadine . Further analysis also demonstrated that spores treated with nitrous acid still germinated normally, while those treated with glutaraldehyde or Betadine did not.

Dermatology, 2000, 201(1), 44 - 5
Extreme UV exposure of professional cyclists; Moehrle M et al.; BACKGROUND: The most important risk factor for the development of melanoma and non-melanoma skin cancer is thought to be ultraviolet (UV) radiation . To date there is no quantification of the UV exposure of outdoor sports professionals training and competing at high solar UV levels . METHODS: During eight stages of the 'Tour de Suisse' cycling race, the UV exposure of 6 professional cyclists was monitored with Bacillus subtilis spore film dosimeters . RESULTS: The measurements showed a personal UV exposure between 0.2 minimal erythema dose (MED) during the prologue and 17.2 MED during a mountain stage . The mean daily personal exposure of all full stages (prologue excluded) was 8.1 MED . The personal exposure level determined during these races exceeded international exposure limits by more than 30 times . CONCLUSION: Therefore UV exposure of sports professionals should be limited by application of sun screens, protective clothing and training/competition at low insolation .

Infect Control Hosp Epidemiol, 2000 Aug, 21(8), 499 - 504
Cleaning of blood-contaminated reprocessed angiographic catheters and spinal needles; Penna TC et al.; OBJECTIVES: To evaluate the efficacy of a multistep cleaning method using a cleaner and a chemical disinfectant on blood-contaminated angiographic catheters and spinal needles intended to be sterilized by hydrogen peroxide gas plasma . METHOD: A mixture of radiopaque iodine contrast, bovine blood (plus ethylenediaminetetraacetic acid), and a suspension of Bacillus subtilis spores was used to simulate catheterization and needle use . The mixture was a 1:1 proportion of contrast and blood, inoculated so that there was a final concentration of B subtilis spores of 1.0x10(6) colony-forming units (CFU)/mL . The inoculated devices were cleaned using a hydrogen peroxide solution at a concentration of 1.5+/-0.5 percent by weight, followed by distilled water with enzymatic detergent . After drying, the devices were sterilized with hydrogen peroxide gas plasma . RESULTS: The initial B subtilis spore concentration inoculated into catheters and needles varied from 2.12x10(4) to 2.74x10(7) CFU/mL . The residual load of B . subtilis spores after cleaning varied from zero (no count) to a maximum of 200 CFU/device . The multistep cleaning procedure was responsible for an average 5log10 reduction of B . subtilis spores in the catheter and needle lumens . CONCLUSIONS: The hydrogen peroxide and enzymatic detergent aqueous solutions were shown to be efficacious when used as part of a multistep cleaning method . The low level of microbial contamination prior to sterilization with hydrogen peroxide gas plasma assured that the intended sterility assurance level was reached.

J Ethnopharmacol, 2000 Sep, 72(1-2), 313 - 6
Antibacterial activity of water and acetone extracts of the roots of Euclea natalensis; Lall N et al.; Water and acetone extracts of the roots of Euclea natalensis A.DC . were investigated for their in vitro antibacterial properties . The Gram-positive bacteria tested appeared to be more susceptible to the extracts than the Gram-negative bacteria . The water and acetone extracts inhibited the growth of Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Micrococcus kristinae and Staphylococcus aureus at concentrations ranging between 0.1 and 6.0 mg/ml . The water extract did not exert any inhibitory action on Gram-negative bacteria while the acetone extract showed inhibitory activity at a concentration of 5.0 mg/ml against all the Gram-negative bacteria investigated . The antibacterial activity of acetone extract was also investigated by a direct bioassay on TLC plates against S . aureus.

Appl Environ Microbiol, 2000 Sep, 66(9), 4045 - 9
Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant; Fry B et al.; Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media . Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero . In this paper, we report the construction and characterization of a pyk mutant of B . subtilis . Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium . The substantial reduction in acid production is accompanied by increased CO(2) production and a reduced rate of growth . Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant . The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant . The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants . The very high concentration of PEP which accumulates in the B . subtilis pyk mutant could be exploited for production of various aromatics.

Appl Environ Microbiol, 2000 Sep, 66(9), 3735 - 42
Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores; Ciarciaglini G et al.; In this work, we have used spores of Bacillus subtilis that specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination . Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0 . 5%), and a pasteurization step (90 degrees C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone . Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the L-alanine germination apparatus of the spore . However, we were able to restore germination of the preservative-damaged spores unable to germinate on L-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid . The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.

Appl Environ Microbiol, 2000 Sep, 66(9), 3727 - 34
Thermostable chitosanase from Bacillus sp . Strain CK4: cloning and expression of the gene and characterization of the enzyme; Yoon HG et al.; A thermostable chitosanase gene from the environmental isolate Bacillus sp . strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined . The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme . The deduced amino acid sequence of the chitosanase from Bacillus sp . strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively . C-terminal homology analysis shows that Bacillus sp . strain CK4 belongs to cluster III with B . subtilis . The gene was similar in size to that of the mesophile B . subtilis but showed a higher preference for codons ending in G or C . The enzyme contains 2 additional cysteine residues at positions 49 and 211 . The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography . The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications . The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.

Arzneimittelforschung, 2000 Jul, 50(7), 652 - 5
Synthesis and antibacterial activity of new arylamido derivatives of 6 beta-aminopenicillanic, 7 beta-aminocephalosporanic and 7 beta-aminodesacetoxycephalosporanic acids; Kaloyanov N et al.; New semisynthetic penicillins and cephalosporins have been synthesized by acylation of 6 beta-aminopenicillanic, 7 beta-aminocephalosporanic and 7 beta-aminodesacetoxycephalosporanic acids with ortho-substituted aromatic acids, using the method of mixed anhydrides . The chemical structures of the compounds obtained were confirmed by elemental analysis and by IR- and 1H-NMR spectra . Antibacterial activities of the compounds were determined by the macrodilution susceptibility test in brain-heart infusion broth . Test organisms producing beta-lactamases: Bacillus subtilis L2, Bacillus subtilis HB2, Bacillus cereus 30, Bacillus subtilis 6633 ATCC, Bacillus mycoides 924; Staphylococcus aureus 1/45 "Oxford" as Gram-positive bacteria, and Escherichia coli 111, Escherichia coli K12/F- inverted question marklac-/, Escherichia coli K12/F- inverted question marklambda-/, Escherichia coli K12/F- inverted question marklambda- inverted question marklac+/ as Gram-negative bacteria . In general, the derivatives of 6 beta-aminopenicillanic acid were more active than 7 beta-aminocephalosporanic and 7 beta-aminodesacetoxycephalosporanic acid derivatives . Among all the compounds synthesized 6 beta-{4'-(dimethylamino)-azobenzene-2-amido}penicillanic acid and 6 beta-(N-phenylanthranilamido)penicillanic acid showed the best activity and were with the broadest spectrum of action.

Biochem Soc Trans, 2000, 28(4), 517 - 20
Recognition of multiple drugs by a single protein: a trivial solution of an old paradox; Vazquez-Laslop N et al.; Multidrug-efflux transporters recognize scores of structurally dissimilar toxic compounds and expel them from cells . The broad chemical specificity of these transporters challenges some of the basic dogmas of biochemistry and remains unexplained . To understand, at least in principle, how a protein can recognize multiple compounds, we analysed the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr . This regulator, BmrR, binds multiple dissimilar hydrophobic cations and, by activating the expression of the Bmr transporter, causes their expulsion from the cell . Crystallographic analysis of the complexes of the inducer-binding domain of BmrR with some of its inducers revealed that ligands cause disordering of the surface alpha-helix and penetrate the hydrophobic core of the protein, where they form multiple van der Waals and stacking interactions with hydrophobic amino acids and an electrostatic bond with the buried glutamic residue . Mutational analysis of the binding site suggests that each ligand forms a unique set of atomic contacts with the protein: each tested mutation exerted disparate effects on the binding of different ligands . The example of BmrR demonstrates that a protein can bind multiple compounds with micromolar affinities by using only electrostatic and hydrophobic interactions . Its ligand specificity can be broadened by the flexibility of the binding site . It therefore seems that the commonly expressed fascination with the broad specificity of multidrug transporters is misdirected and originates from an almost exclusive familiarity with the more sophisticated processes of specific molecular recognition that predominate among existing proteins.

Biochem Soc Trans, 2000, 28(4), 513 - 7
Expression, purification and properties of multidrug efflux proteins; Henderson PJ et al.; A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E . coli . They all catalyse drug/H(+) antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins . The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification . Growth conditions were optimized in 1-25-litre cultures of E . coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates . Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni(2+)-nitrilotriacetate-affinity column chromography, yielding 5-25 mg per 25 litres of original culture . All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag . Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of alpha-helix (more than 75%) . Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass . The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays . The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution . Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses . In addition, ligands for efflux proteins labelled with (13)C or (15)N have been synthesized to implement solid-state NMR studies of the ligand-binding site.

Res Microbiol, 2000 Jul-Aug, 151(6), 481 - 6
Bacillus subtilis homologous recombination: genes and products; Fernandez S et al.; Homologous recombination plays a critical role in maintaining gene diversification and genome stability . Fourteen Bacillus subtilis recombination gene products have been genetically characterised and classified into five different epistatic groups . At least seven other recombination genes could be predicted . Recombination gene products which define activities that help RecA to process DNA repair and recombination have been studied, but those that processed recombination intermediates into products (post-synaptic stage) await elucidation.

Res Microbiol, 2000 Jul-Aug, 151(6), 475 - 80
Internalizing DNA; Dubnau D et al.; The steps involved in the transformation of Bacillus subtilis are reviewed . These include the initial binding, processing and passage of DNA across the cell wall and transport across the plasma membrane . Our understanding of the roles of the proteins known to be required for these steps is reviewed.

J Biol Chem, 2000 Nov 24, 275(47), 36832 - 8
A strategically positioned cation is crucial for efficient catalysis by chorismate mutase; Kast P et al.; Combinatorial mutagenesis and in vivo selection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg(90) . Here, we present a detailed kinetic and crystallographic study of several such variants . Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude . Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat) . Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site . Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its epsilon-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state . These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation . The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed.

J Bacteriol, 2000 Sep, 182(18), 5202 - 10
Mutations in multidrug efflux homologs, sugar isomerases, and antimicrobial biosynthesis genes differentially elevate activity of the sigma(X) and sigma(W) factors in Bacillus subtilis; Turner MS et al.; The sigma(X) and sigma(W) extracytoplasmic function sigma factors regulate more than 40 genes in Bacillus subtilis . sigma(W) activates genes which function in detoxification and the production of antimicrobial compounds, while sigma(X) activates functions that modify the cell envelope . Transposon mutagenesis was used to identify loci which negatively regulate sigma(W) or sigma(X) as judged by up-regulation from the autoregulatory promoter site P(W) or P(X) . Fourteen insertions that activate P(W) were identified . The largest class of insertions are likely to affect transport . These include insertions in genes encoding two multidrug efflux protein homologs (yqgE and yulE), a component of the oligopeptide uptake system (oppA), and two transmembrane proteins with weak similarity to transporters (yhdP and yueF) . Expression from P(W) is also elevated as a result of inactivation of at least one member of the sigma(W) regulon (ysdB), an ArsR homolog (yvbA), a predicted rhamnose isomerase (yulE), and a gene (pksR) implicated in synthesis of difficidin, a polyketide antibiotic . In a parallel screen, we identified seven insertions that up-regulate P(X) . Remarkably, these insertions were in functionally similar genes, including a multidrug efflux homolog (yitG), a mannose-6-phosphate isomerase gene (yjdE), and loci involved in antibiotic synthesis (srfAB and possibly yogA and yngK) . Significantly, most insertions that activate P(W) have little or no effect on P(X), and conversely, insertions that activate P(X) have no effect on P(W) . This suggests that these two regulons respond to distinct sets of molecular signals which may include toxic molecules which are exported, cell density signals, and antimicrobial compounds.

Anal Chem, 2000 Aug 15, 72(16), 3739 - 44
Testing the significance of microorganism identification by mass spectrometry and proteome database search; Pineda FJ et al.; We derive and validate a simple statistical model that predicts the distribution of false matches between peaks in matrix-assisted laser desorption/ionization mass spectrometry data and proteins in proteome databases . The model allows us to calculate the significance of previously reported microorganism identification results . In particular, for deltam = +/-1.5 Da, we find that the computed significance levels are sufficient to demonstrate the ability to identify microorganisms, provided the number of candidate microorganisms is limited to roughly three Escherichia coli-like or roughly 10 Bacillus subtilis-like microorganisms (in the sense of having roughly the same number of proteins per unit-mass interval) . We conclude that, given the cluttered and incomplete nature of the data, it is likely that neither simple ranking nor simple hypothesis testing will be sufficient for truly robust microorganism identification over a large number of candidate microorganisms.

Biochem, Eng . J. . 2000 Oct 1, 6(2), 81 - 86
Efficient production of protopectinases by Bacillus subtilis using medium based on soybean flour; Matsumoto T et al.; We have developed a culture system for efficient production of protopectinases (PPases) by Bacillus subtilis . PPase shows the pectin-releasing activity and is expected to be utilized in the enzymatic cotton scouring . B . subtilis IFO3134 was cultivated using defatted soybean flour as a main component of culture media . This strain produced three different types of PPases, namely, PPase-C, -N and -R performing endo-arabinase activity, pectate-lyase activity and pectin-lyase activity, respectively . The effects of alkaline solubilization and autoclave treatments to extract nutrients from soybean flour and initial soybean flour concentration (20-80g/l) on production of PPases in batch fermentation were investigated . Alkaline solubilization of soybean flour with NaOH remarkably reduced enzyme productivity . In addition, a higher initial concentration of soybean flour reduced the enzyme productivity of cells . The pectin-releasing activity was the largest and reached up to 2200-2400U/ml, when the culture medium containing an initial soybean flour concentration of 40g/l was autoclaved for 45-60min without alkaline solubilization treatment.

J Agric Food Chem, 2000 Aug, 48(8), 3210 - 6
Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1; Chang CT et al.; A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium . In addition, a strong fibrinolytic enzyme was purified from the cultivation media . The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis . The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis . The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3 . The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates . Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA . PMSF and NBS almost completely inhibited the activity of the enzyme . These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.

Biochemistry, 2000 Aug 29, 39(34), 10514 - 20
Substrate specificity of the loading didomain of the erythromycin polyketide synthase; Lau J et al.; The priming of many modular polyketide synthases is catalyzed by a loading acyltransferase-acyl carrier protein (AT(L)-ACP(L)) didomain which initiates polyketide biosynthesis by transferring a primer unit to the ketosynthase domain of the first module . Because the AT(L) domain influences the choice of the starter unit incorporated into the polyketide backbone, its specificity is of considerable interest . The AT(L)-ACP(L) didomain of the 6-deoxyerythronolide B synthase (DEBS) was functionally expressed in Escherichia coli . Coexpression of the Sfp phosphopantetheinyl transferase from Bacillus subtilis in E . coli leads to efficient posttranslational modification of the ACP(L) domain with a phosphopantetheine moiety . Competition experiments were performed with the holo-protein to determine the relative rates of incorporation of a variety of unnatural substrates in the presence of comparable concentrations of labeled acetyl-CoA . Our results showed that the loading didomain of DEBS can accept a surprisingly broad range of substrates, although it exhibits a preference for unbranched alkyl chain substrates over branched alkyl chain, polar, aromatic, and charged substrates . In particular, its tolerance toward acetyl- and butyryl-CoA is unexpectedly strong . The studies described here present an attractive prototype for the expression, analysis, and engineering of acyltransferase domains in modular polyketide synthases.

Mol Gen Genet, 2000 Jul, 263(6), 1053 - 60
Identification and characterization of the mre gene region of Streptomyces coelicolor A3(2); Burger A et al.; During a search for new differentiation factors in Streptomyces coelicolor A3(2), a locus at 11 o'clock on the S . coelicolor map was identified which harbours several genes that show extensive similarity to cell division and differentiation genes from Escherichia coli and Bacillus subtilis . From the sequence data it was concluded that the region contains the genes mireB, mreC, mreD (murein formation gene cluster E), pbp83 (high-molecular-weight penicillin-binding protein) and sfr (member of the spoVE/ftsW/rodA family) . Mre gene products are reported to be responsible for determining cell shape in E . coli and Bacillus . The S . coelicolor mreC gene was inactivated by gene disruption, resulting in mutants which showed significant growth retardation in comparison to the wild type . Inactivation of the mreB gene was incompatible with viability, and thus mreB represents a Streptomyces cell division gene that is essential for survival . Promoter-probe experiments led to the identification of an operon structure, with promoters located upstream of mreB, pbp83 and sfr . Detailed studies of mreB transcription revealed the existence of three promoters; two of them are constitutively transcribed, whereas the third is developmentally regulated.

Amino Acids, 2000, 18(4), 375 - 87
Isozymic nature of spore coat-associated alanine racemase of Bacillus subtilis; Kanda-Nambu K et al.; Spore coat-associated alanine racemase of Bacillus subtilis, which converts L-alanine to D-alanine, that is, the germinant to the competitive inhibitor, to regulate spore germination for survival of the organism under unfavorable growth conditions, was examined . The dormant spores, L-alanine-initiated germination of which is inhibited by diphenylamine, were used to characterize the enzyme in the native form because of its unextractablility from dormant spores . The presence of isozymes, Enz-I and Enz-II with Km for L-alanine of about 20mM and 50mM and optimum activity at around 40 degrees C and 65 degrees C, respectively, was proposed . The enzymes were selectively used depending on the L-alanine concentration and the temperature . The pH profiles of the activity (optimun at pH 9.0) and the stability (stable between pH 6-11 at 60 degrees C) were similar, but Enz-II was more heat-stable than Enz-I and the denaturation curve demonstrated a two-domain structure for Enz-II . Sensitivity to D-penicillamine, hydroxylamine and HgCl2 was similar between Enz-I and Enz-II, while that to D-cycloserine, L- and D-aminoethylphosphonic acid, monoiodoacetate and N-ethylmaleimide was different; HgCl2 was the most effective inhibitor among these compounds.

Genes Cells, 2000 Aug, 5(8), 627 - 35
Requirement of transfer-messenger RNA for the growth of Bacillus subtilis under stresses; Muto A et al.; BACKGROUND: Bacterial transfer-messenger RNA (tmRNA, 10Sa RNA) is involved in a trans-translation reaction which contributes to the degradation of incompletely synthesized peptides and to the recycling of stalled ribosomes . However, its physiological role in the cell remains elusive . In this study, an efficient system for controlling the expression of the gene for tmRNA (ssrA), as well as a tmRNA gene-defective strain (ssrA:cat), were constructed in Bacillus subtilis . The effects of tmRNA on the growth of the cells were investigated under various physiological culture conditions using these strains . RESULTS: The cells were viable in the absence of ssrA expression under the usual culture conditions . However, the growth rate of cells without tmRNA expression, relative to that of the expressed cells, decreased with elevating temperature (> 45 degrees C), and at 52 degrees C, the highest temperature for growth of the wild-type, cells grew depending on the expression level of tmRNA . Furthermore, the transcription level of the ssrA from the authentic promoter at a high temperature (51 degrees C) was about 10-fold higher than that at a lower temperature (37 degrees C) . tmRNA-dependent growth and an increase in tmRNA amount were also observed in cells under other stresses, such as high concentrations of ethanol or cadmium chloride . It is also shown that alanylated tmRNA rather than tmRNA-mediated proteolysis is required for growth at high temperature . CONCLUSION: The expression of tmRNA gene (ssrA) is required for the efficient growth of B . subtilis under several strong stresses . The transcription of ssrA increases under several stressful conditions, suggesting that it is a stress-response gene . Alanyl-tmRNA, probably via its ability of recycling stalled ribosomes via trans-translation, is involved in the stress tolerance of bacteria.

J Appl Microbiol, 2000 Jul, 89(1), 152 - 7
High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant; Panbangred W et al.; By marker exchange mutagenesis, Bacillus megaterium strain UN-1 (Bm-UN1) was used to prepare a mutant strain B . megaterium UN-cat (Bm-UNcat) lacking the penicillin G acylase gene (pac) . The pac gene from Bm-UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm-UNcat and Bacillus subtilis . Bm-UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13.7, 19.5 and 20.4 U ml(-1) at 24, 36 and 48 h of culture, respectively . This was two- to fivefold higher than PAC produced by B . subtilis harbouring pBA402 and about 20-fold higher than PAC produced by the parent strain, Bm-UN1.

Biosens Bioelectron, 2000 Jan, 14(10-11), 761 - 70
Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system; Uithoven KA et al.; An instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system has been developed for the rapid and specific identification of biological warfare (BW) agents . The system has been designed to assay for up to eight agents simultaneously and provides an indication of the absence or presence of a threat within 15 min . Parameters affecting the mixing of the reagents within the instrument's fluidic lines were investigated and optimized . Measurements of blank samples and samples containing Bacillus subtilis spores in the concentration range of 10(4) to 10(6) cfu/ml indicate the limit of detection (LOD) is 3 x 10(3) cfu/ml for B . subtilus . Although the LOD is higher than that of several technologies currently under development, this instrument offers an immediate interim approach for addressing the need to rapidly detect biological warfare agents in the field.

Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1522 - 5
Overexpression, purification, and characterization of Bacillus subtilis N-acetylmuramoyl-L-alanine amidase CwlC; Shida T et al.; N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold . The amidase hydrolyzed type A cell walls such as B . subtilis . The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it . The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.

RNA, 2000 Aug, 6(8), 1131 - 41
tRNA determinants for transcription antitermination of the Bacillus subtilis tyrS gene; Grundy FJ et al.; Transcriptional regulation of the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria is mediated by a conserved transcription antitermination system, in which readthrough of a termination site in the leader region of the mRNA is directed by a specific interaction with the cognate uncharged tRNA . The specificity of this interaction is determined in part by pairing of the anticodon of the tRNA with a "specifier sequence" in the leader, a codon representing the appropriate amino acid, as well as by pairing of the acceptor end of the tRNA with an unpaired region of the antiterminator . Previous studies have indicated that although these interactions are necessary for antitermination, they are unlikely to be sufficient . In the current study, the effect of multiple mutations in tRNA(Tyr) on readthrough of the tyrS leader region terminator, independent of other tRNA functions, was assessed using a system for in vivo expression of pools of tRNA variants; this system may be generally useful for in vivo expression of RNAs with defined end points . Although alterations in helical regions of tRNA(Tyr) that did not perturb base pairing were generally permitted, substitutions affecting conserved features of tRNAs were not . The long variable arm of tRNA(Tyr) could be replaced by either a short variable arm or a long insertion of a stable stem-loop structure . These results indicate that the tRNA-leader RNA interaction is highly constrained, and is likely to involve recognition of the overall tertiary structure of the tRNA.

J Mol Microbiol Biotechnol, 1999 Nov, 1(2), 203 - 9
Cold shock response in Bacillus subtilis; Graumann PL et al.; Following a rapid decrease in temperature, the physiology of Bacillus subtilis cells changes profoundly . Cold shock adaptation has been monitored at the level of membrane composition, adjustment in DNA topology, and change in cytosolic protein synthesis/composition . Some major players in these processes (cold-stress induced proteins and cold acclimatization proteins, CIPs and CAPs) have been identified and mechanisms in cold shock acclimatization begin to emerge; however, important questions regarding their cellular function still need to be answered.

J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 141 - 8
The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis; Faires N et al.; Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA . An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired . We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport . We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants) . Catabolite repression of beta-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant . A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen . An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase . This enzyme is necessary for the assimilation of ammonium . In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.

Can J Microbiol, 2000 Aug, 46(8), 770 - 3
Clay minerals protect bacteriophage PBS1 of Bacillus subtilis against inactivation and loss of transducing ability by UV radiation; Vettori C et al.; The effect of UV radiation on the survival of and transduction by phage PBS1 of Bacillus subtilis, free or adsorbed on the clay minerals montmorillonite (M) and kaolinite (K), was studied . After free or clay-associated phage (approximately 10(7) PFU.mL-1) was irradiated with UV light (254 nm) for 0, 1, 2, 5, 10, and 30 min and then allowed to infect B . subtilis FB300 (thiB4 metA29 argF4 Rfmr), the phage was titered, and Met+ transductants were enumerated on selective media . After 1 min of irradiation, the titer of free and clay-associated phage decreased significantly (approximately 1.6 times for free phage, and approximately 4.9 and 6.8 times for M and K, respectively), whereas the transduction frequency increased significantly (approximately 3 times for free phage and approximately 1.4 and 2.2 times for M and K, respectively) . The titer and transduction frequency of clay-associated phage remain essentially constant between 1 and 10 min of irradiation, whereas the titer of free phage decreased by approximately 1 order of magnitude after 5 min of irradiation . When free phage was irradiated for 10 min, the titer and transduction frequency decreased by approximately 2 and 0.5 orders of magnitude, respectively, whereas 30 min of irradiation was necessary to obtain comparable decreases with clay-associated phage . These results indicated that phages are protected to some extent from UV radiation when adsorbed on clay minerals.

J Bacteriol, 2000 Sep, 182(17), 5009 - 12
Analysis of tnrA alleles which result in a glucose-resistant sporulation phenotype in Bacillus subtilis; Shin BS et al.; Bacillus subtilis cells cannot sporulate in the presence of catabolites such as glucose . During the analysis of Tn10-generated mutants, we found that deletion of the C-terminal region of the tnrA gene, which encodes a global regulator that positively regulates a number of genes in response to nitrogen limitation, results in a catabolite-resistant sporulation phenotype . Analyses of nrg-lacZ and nasB-lacZ, which are activated by TnrA under nitrogen limitation, showed that C-terminally truncated TnrA activates nitrogen-regulated genes constitutively . The relief of catabolite repression of sporulation may result from the uncontrolled expression of the TnrA-regulated genes.

J Bacteriol, 2000 Sep, 182(17), 4992 - 4
Bacillus subtilis comZ (yjzA) negatively affects expression of comG but not comK; Ogura M et al.; The yjzA open reading frame, along with med, constitutes an operon . Disruption of yjzA caused a five-fold enhancement of comG expression, thereby leading to a three-fold-higher transformation efficiency . The expression of comK and the other three late competence operons was not affected significantly in the yjzA-deficient mutant.

J Bacteriol, 2000 Sep, 182(17), 4841 - 8
Relative roles of the fla/che P(A), P(D-3), and P(sigD) promoters in regulating motility and sigD expression in Bacillus subtilis; West JT et al.; Three promoters have been identified as having potentially important regulatory roles in governing expression of the fla/che operon and of sigD, a gene that lies near the 3' end of the operon . Two of these promoters, fla/che P(A) and P(D-3), lie upstream of the >26-kb fla/che operon . The third promoter, P(sigD), lies within the operon, immediately upstream of sigD . fla/che P(A), transcribed by E sigma(A), lies >/=24 kb upstream of sigD and appears to be largely responsible for sigD expression . P(D-3), transcribed by E sigma(D), has been proposed to participate in an autoregulatory positive feedback loop . P(sigD), a minor sigma(A)-dependent promoter, has been implicated as essential for normal expression of the fla/che operon . We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of fla/che P(A), P(D-3), or P(sigD) . Our analysis of these strains indicates that fla/che P(A) is absolutely essential for motility, that P(D-3) does not function in positive feedback regulation of sigD expression, and that P(sigD) is not essential for normal fla/che expression . Further, our results suggest that an additional promoter(s) contributes to sigD expression.

J Bacteriol, 2000 Sep, 182(17), 4758 - 63
A two-dimensional protein gel electrophoresis study of the heat stress response of Bacillus subtilis cells during sporulation; Movahedi S et al.; The heat resistance of spores of Bacillus subtilis formed at 30 degrees C was enhanced by pretreatment at 48 degrees C for 30 min, 60 min into sporulation, for all four strains examined . High-resolution two-dimensional gel electrophoresis showed the generation and/or overexpression of 60 proteins, 11 of which were specific to heat shock, concurrent to this acquired thermotolerance . The greatest number of new proteins was observed between 30 and 60 min after heat shock, and the longer the time between exponential growth and heat treatment, the fewer differences were observed on corresponding protein profiles . The time at which heating produced the maximum increase in spore resistance and the most new proteins on two-dimensional gels occurred before alkaline phosphatase and dipicolinic acid production and corresponded to stage I or II of sporulation . The stress proteins formed disappeared later in sporulation, suggesting that heat shock proteins increase spore heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth.

J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 217 - 24
Recombinant acylheptapeptide lichenysin: high level of production by Bacillus subtilis cells; Yakimov MM et al.; Peptide synthetases are multi-domain proteins that catalyze the assembly, from amino acids and amino acid derivatives, of peptides and lipopeptides, some of which exhibit activities (pharmaceutical, surfactant, etc.) of considerable biotechnological importance . Although there is substantial interest in the generation of greater peptide diversity, in order to create new biotechnologically interesting products, attempts reported so far to exchange amino acid-activating minimal modules between enzymes have only yielded hybrid catalysts with poor activities . We report here the replacement of an entire first, L-Glu-, and fifth, L-Asp-incorporating modules of surfactin synthetase, to create a fully active hybrid enzyme that forms a novel peptide in high yields . Whole encoding regions of lichenysin A synthetase modules were introduced into surfactin biosynthesis operon between His140/His1185 of SrfAA and His1183/His2226 of SrfAB, the amino acid residues of a proposed active-site motif (HHXXXDG) of the condensation domains which is involved in the catalysis of nonribosomal peptide bond formation (Stachelhaus et al., 1998) . When the lipopeptides produced by the recombinant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical by their fatty acid profiles . We thereby demonstrate the utility of whole module swapping for designing novel peptides, for creating peptide diversity, and for redesigning existing peptides produced in performant production strains in high yields to correspond to desired peptides produced in low yields, or from strains unsuitable for production purposes.

Metab Eng, 1999 Jan, 1(1), 26 - 34
Metabolic flux analysis of Escherichia coli deficient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase; Yang YT et al.; Several approaches to reduce acetate accumulation in Escherichia coli cultures have recently been reported . This reduction subsequently led to a significant enhancement in recombinant protein production . In those studies, metabolically engineered E . coli strains with reduced acetate synthesis rates were constructed through the modification of glucose uptake rate, the elimination of critical enzymes that are involved in the acetate formation pathways, and the redirection of carbon flux toward less inhibitory byproducts . In particular, it has been shown that strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene not only produce less acetate but also have a higher ATP yield . Metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point revealed that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin . The main focus of this study is the systematic analysis of the effects of small perturbations in the host's existing pathways on the redistribution of carbon fluxes . Specifically, a mutant with deleted acetate kinase (ACK) and acetyl phosphotransferase (PTA) was constructed and studied . Results from the metabolic analysis of carbon redistribution show the ackA-pta mutation will reduce acetate level at the expense of the growth rate . In addition, in the ackA-pta deficient strain a much higher lactate formation rate with simultaneously lower formate and ethanol synthesis rates was found . Expression of the B . subtilis ALS in ackA-pta mutants further reduces acetate levels while cell density similar to that of the parent strain is attained.

Rev Latinoam Microbiol, 1997 Jul-Dec, 39(3-4), 129 - 40
Molecular cloning of the gene that codes for the pyruvate kinase of Bacillus subtilis: primary characterization of a strain carrying this gene insertionally inactivated; Munoz ME et al.; We have cloned and characterized the pykA gene from Bacillus subtilis which codes for a pyruvate kinase (PK) enzyme . This gene has been located downstream a putative phosphofructokinase gene, suggesting that they are part of the same operon . The deduced amino acid sequence of this PK showed a strong similarity to other PKs from different sources; however, as it has been found in other bacilli, the B . subtilis pykA enzyme had an extra C-terminal sequence consisting of about 112 amino acid residues . This gene was insertionally inactivated at the chromosomal level, with an antibiotic resistance marker . The analysis of this mutation in wild type and pts- backgrounds, indicated that B . subtilis has no other pyruvate kinase activity capable of complementing the absence of PykA.

Mol Microbiol, 2000 Jun, 36(6), 1415 - 24
BofC negatively regulates SpoIVB-mediated signalling in the Bacillus subtilis sigmaK-checkpoint; Wakeley P et al.; The BofC protein acts negatively on intercompartmental signalling of pro-sigma(K) processing in the sigma(K)-checkpoint of Bacillus subtilis . Signalling is brought about by the SpoIVB protein, which is synthesized in the forespore and initiates proteolytic processing of pro-sigmaK to its mature and active form in the opposed mother cell chamber of the developing cell . We have shown here that BofC, like SpoIVB, is secreted across the inner forespore membrane and, from the analysis of a bofC deletion and insertion mutant, is likely to interact with SpoIVB . In the absence of BofC, the amount of SpoIVB found in sporulating cells is substantially reduced, although SpoIVB is still able to activate proteolysis of pro-sigma(K) . Conversely, in the absence of SpoIVB, the levels of BofC accumulate suggesting that the fate of each molecule is dependent upon their mutual interaction . Our results suggest that BofC could maintain SpoIVB in a stable but inactive form . Supporting this, we have shown that overproduction of BofC inhibits SpoIVB autoproteolysis and leads to a delay in proteolytic cleavage of pro-sigma(K) . Based on our work here, we have proposed a model for BofC's functional role in intercompartmental signalling.

Mol Microbiol, 2000 Jun, 36(6), 1336 - 48
Proteolysis of SpolVB is a critical determinant in signalling of Pro-sigmaK processing in Bacillus subtilis; Wakeley PR et al.; SpoIVB is essential for intercompartmental signalling in the sigma(K)-checkpoint of Bacillus subtilis . SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro-sigma(K) to its mature and active form sigma(K) . We show here that SpoIVB is a serine peptidase of the SA clan . Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self-cleave into at least three discrete products, and in vitro studies have shown cleavage in trans . Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro-sigma(K) processing and a yet, uncharacterized, second function which is essential for the formation of heat-resistant spores . In B . subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis . First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species . This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.

Mol Microbiol, 2000 Jun, 36(6), 1327 - 35
Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E . coli; Andersen PA et al.; The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork-arrest assay plasmid . After transferring these new plasmids into B . subtilis, which could overproduce the E . coli terminator protein Tus, it was shown that the E . coli Tus-TerB complex could cause polar replication fork arrest, albeit at a very low level, in B . subtilis . A new B . subtilis-E . coli shuttle plasmid was designed to allow the insertion of either the Terl (B . subtilis) or TerB (E . coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism . Fork-arrest assays for both terminator-containing plasmids replicating in both organisms which also produced saturating levels of either the B . subtilis terminator protein (RTP) or Tus were performed . The efficiency of the Tus-TerB complex in causing fork arrest was much higher in E . coli than in B . subtilis . The efficiency of the B . subtilis RTP-Terl complex was higher in B . subtilis than in E . coli, but the effect was significantly less . Evidently a specificity feature in E . coli operates to enhance appreciably the fork-arrest efficiency of a Tus-Ter complex . The specificity effect is of less significance for an RTP-Ter complex functioning in B . subtilis.

Mol Microbiol, 2000 Jun, 36(6), 1234 - 49
Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis; Regamey A et al.; Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions . This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains . Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences . Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids . Analysis of junctions involved in the transfer of the trpE26 mutation, i.e . simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination . We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome . The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping' . In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

J Biol Chem, 2000 Nov 10, 275(45), 35311 - 9
Functional analysis of the terminase large subunit, G2P, of Bacillus subtilis bacteriophage SPP1; Gual A et al.; The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging . A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities . H6-G2P introduces a cut with preference at the 5'-RCGG downward arrowCW-3' sequence . Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages . H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i) . H6-G2P interacts with two discrete G1P domains (I and II) . Full-length G1P and G1PDeltaN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P . The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC . In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated . Based on these results, we propose a model that can account for the role of terminase in headful packaging.

Biochem J, 2000 Aug 15, 350 Pt 1, 31 - 9
The influence of secretory-protein charge on late stages of secretion from the Gram-positive bacterium Bacillus subtilis; Stephenson K et al.; Following their secretion across the cytoplasmic membrane, processed secretory proteins of Bacillus subtilis must fold into their native conformation prior to translocation through the cell wall and release into the culture medium . The rate and efficiency of folding are critical in determining the yields of intact secretory proteins . The B . subtilis membrane is surrounded by a thick cell wall comprising a heteropolymeric matrix of peptidoglycan and anionic polymers . The latter confer a high density of negative charge on the wall, endowing it with ion-exchange properties, and secretory proteins destined for the culture medium must traverse the wall as the last stage in the export process . To determine the influence of charge on late stages in the secretion of proteins from this bacterium, we have used sequence data from two related alpha-amylases, to engineer the net charge of AmyL, an alpha-amylase from Bacillus licheniformis that is normally secreted efficiently from B . subtilis . While AmyL has a pI of 7.0, chimaeric enzymes with pI values of 5.0 and 10.0 were produced and characterized . Despite the engineered changes to their physico-chemical properties, the chimaeric enzymes retained many of the enzymic characteristics of AmyL . We show that the positively charged protein interacts with the cell wall in a manner that influences its secretion.

Pharmacol Ther, 2000 Jul, 87(1), 27 - 49
The structure and mechanism of bacterial type I signal peptidases . A novel antibiotic target; Paetzel M et al.; Type I signal peptidases are essential membrane-bound serine proteases that function to cleave the amino-terminal signal peptide extension from proteins that are translocated across biological membranes . The bacterial signal peptidases are unique serine proteases that utilize a Ser/Lys catalytic dyad mechanism in place of the classical Ser/His/Asp catalytic triad mechanism . They represent a potential novel antibiotic target at the bacterial membrane surface . This review will discuss the bacterial signal peptidases that have been characterized to date, as well as putative signal peptidase sequences that have been recognized via bacterial genome sequencing . We review the investigations into the mechanism of Escherichia coli and Bacillus subtilis signal peptidase, and discuss the results in light of the recent crystal structure of the E . coli signal peptidase in complex with a beta-lactam-type inhibitor . The proposed conserved structural features of Type I signal peptidases give additional insight into the mechanism of this unique enzyme.

J Nat Prod, 2000 Jul, 63(7), 984 - 6
New macrolactins from a marine Bacillus sp . Sc026; Jaruchoktaweechai C et al.; Bioassay-guided fractionation of the EtOAc extract of a marine Bacillus sp . Sc026 culture broth has led to the isolation of the known compound, macrolactin F (1), together with two new compounds, 7-O-succinyl macrolactin F (2) and 7-O-succinyl macrolactin A (3) . The chemical structures of 1-3 were elucidated on the basis of spectral data analyses and literature data comparison . Compounds 1-3 exhibited antibacterial activity against Bacillus subtilis and Staphylococcus aureus.

RNA, 2000 Jul, 6(7), 988 - 1003
Selective importation of RNA into isolated mitochondria from Leishmania tarentolae; Rubio MA et al.; All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol . Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection . Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA . The presence of a membrane potential is not required for in vitro importation . A variety of small synthetic RNAs were also found to be efficiently imported in vitro . The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported . The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln) . Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

EMBO J, 2000 Aug 1, 19(15), 4182 - 90
Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7; Meijer WJ et al.; We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence . To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA . On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid . At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane . Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid . Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

J Biochem (Tokyo), 2000 Aug, 128(2), 207 - 11
Glutamate 83 is important for stabilization of domain-domain conformation of Thermus aquaticus glycerol kinase; Huang HS et al.; The gene glpK, encoding glycerol kinase (GlpK) of Thermus aquaticus, has recently been identified . The protein encoded by glpK was found to have an unusually high identity of 81% with the sequence of GlpK from Bacillus subtilis . Three residues (Arg-82, Glu-83, and Asp-244) of T . aquaticus GlpK are conserved in all the known GlpK sequences, including those from various bacteria, yeast and human . The roles that these three residues play in the catalytic mechanism were investigated by using site-directed mutagenesis to produce three mutants: Arg-82-Ala, Glu-83-Ala, and Asp-244-Ala . Replacement of Asp-244 by Ala resulted in a complete loss of activity, thus suggesting that Asp-244 is important for catalysis . Taking k(cat)/K(m) as a simple measure of catalytic efficiency, the mutants Arg-82-Ala and Glu-83-Ala were judged to cause 190- and 37,000-fold decrease, respectively, when compared to the wild-type GlpK . Thus, these three residues play a critical role in the catalytic mechanism . However, only mutant Glu-83-Ala was cleaved by alpha-chymotrypsin, and proteolysis studies showed that the mutant Glu-83-Ala involves a change in the exposure of Tyr-331 at the alpha-chymotrypsin site . This indicates a large domain conformational change, since the residues corresponding to Glu-83 and Tyr-331 in the Escherichia coli GlpK sequence are located in domain IB and domain IIB, respectively . The apparent conformational change caused by replacement of Glu-83 leads us to propose that Glu-83 is an important residue for stabilization of domain conformation.

Can J Microbiol, 2000 Jun, 46(6), 506 - 14
Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis; Cropp TA et al.; Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed . This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors . The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain . The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature . No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S . avermitilis was grown at different temperatures . The principal UFA produced by S . avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli . This observation, and the inability of S . avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis . Incorporation studies with the S . avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.

FEMS Microbiol Lett, 2000 Aug 1, 189(1), 31 - 8
Identification and transcriptional characterization of the gene encoding the stress-response sigma factor sigma(H) in streptomyces coelicolor A3(2); Kormanec J et al.; We cloned a new gene, sigH, encoding an alternative sigma factor in Streptomyces coelicolor A3(2) . The deduced protein of 354 amino acids with an M(r) of 39486 showed greatest similarity to the sporulation sigma factor (sigma(F)) of S . coelicolor, general stress-response sigma(B) of Bacillus subtilis, and stationary-phase stress-response sigma(F) of Mycobacterium tuberculosis . Sequence analysis of the upstream region revealed an ORF encoding a protein (UshX) similar to several anti-sigma factors, and short ORF (UshY) containing zinc-finger DNA binding motif . Transcriptional analysis revealed that all three genes are located on the same polycistronic transcript in order ushY, ushX, and sigH . Expression of the operon was directed by four promoters differentially expressed in the course of differentiation . The first (P1) constitutive promoter was located upstream of ushY . The other three promoters (P2, P3, and P4) were located upstream of ushX, and were differentially induced after various stress conditions . The magnitude of the induction was greatest after osmotic stress and heat shock.

FEMS Microbiol Lett, 2000 Jul 15, 188(2), 203 - 8
Lack of a significant role for the PerR regulator in Bacillus subtilis spore resistance; Casillas-Martinez L et al.; Bacillus subtilis cells lacking the PerR repressor which regulates transcription of genes encoding oxidative stress protective proteins grew at 30-50% the rate of wild-type cells, and perR cultures accumulated rapidly growing suppressor mutants lacking the catalase whose expression is regulated by PerR . However, perR spores which retained the perR regulated catalase were obtained on plates . These perR spores had levels of oxidative stress protective proteins from 7- to 50-fold higher than those in wild-type spores, but perR spore resistance to heat, hydrogen peroxide and t-butyl hydroperoxide was essentially identical to that of wild-type spores, indicating that elevated levels of proteins that protect growing cells from oxidizing agents play no role in dormant spore resistance to these compounds . However, germinated perR spores were much more resistant to alkyl hydroperoxides than were wild-type spores.

FEMS Microbiol Lett, 2000 Jul 15, 188(2), 165 - 9
Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants; Kobayashi G et al.; A recombinant lipase, CWB-LipB, localized on the Bacillus subtilis cell surface and retaining lipase activity was unstable and not accumulated in a high yield . To improve the accumulation, we examined cell wall binding protease (wprA)- and/or sigma D (sigD)-deficient mutants, and also a NprE and AprA protease-deficient mutant as host strains . The nprE aprA mutation did not lead to a significant increase in the CWB-LipB accumulation . The wprA mutant accumulated a greater amount than the wild-type only in the stationary phase, but the sigD mutant accumulated a greater amount in both the exponential and stationary phases . The double mutant exhibited great accumulation of CWB-LipB, the amount being 36% of the total proteins extracted from the cell surface.

Biochemistry, 2000 Jul 25, 39(29), 8617 - 24
The distal heme center in Bacillus subtilis succinate:quinone reductase is crucial for electron transfer to menaquinone; Matsson M et al.; Succinate:quinone reductases are membrane-bound enzymes that catalyze electron transfer from succinate to quinone . Some enzymes in vivo reduce ubiquinone (exergonic reaction) whereas others reduce menaquinone (endergonic reaction) . The succinate:menaquinone reductases all contain two heme groups in the membrane anchor of the enzyme: a proximal heme (heme b(P)) located close to the negative side of the membrane and a distal heme (heme b(D)) located close to the positive side of the membrane . Heme b(D) is a distinctive feature of the succinate:menaquinone reductases, but the role of this heme in electron transfer to quinone has not previously been analyzed . His28 and His113 are the axial ligands to heme b(D) in Bacillus subtilis succinate:menaquinone reductase . We have individually replaced these His residues with Leu and Met, respectively, resulting in assembled membrane-bound enzymes . The H28L mutant enzyme lacks succinate:quinone reductase activity probably due to a defective quinone binding site . The H113M mutant enzyme contains heme b(D) with raised midpoint potential and is impaired in electron transfer to menaquinone . Our combined experimental data show that the heme b(D) center, into which we include a quinone binding site, is crucial for succinate:menaquinone reductase activity . The results support a model in which menaquinone is reduced on the positive side of the membrane and the transmembrane electrochemical potential provides driving force for electron transfer from succinate via heme b(P) and heme b(D) to menaquinone.

J Bacteriol, 2000 Aug, 182(16), 4491 - 9
Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation; Meador-Parton J et al.; A major structural element of bacterial endospores is a peptidoglycan (PG) wall . This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers . The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination . The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination . Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration . Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG . We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores . The germ cell wall PG is synthesized first, followed by the cortex PG . The germ cell wall is relatively highly cross-linked . The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex . Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration . We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration . Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.

J Bacteriol, 2000 Aug, 182(16), 4478 - 90
Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis; Antelmann H et al.; The phosphate starvation response in Bacillus subtilis was analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells . Most of the phosphate starvation-induced proteins are under the control of sigma(B), the activity of which is increased by energy depletion . In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and a phoR mutant . By matrix-assisted laser desorption ionization-time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium . In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS . Transcriptional studies of glpQ and ydhF confirmed the strong PhoPR dependence . Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a sigma(A) promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites . Furthermore, ydhF might be cotranscribed with phoB initiating from the phoB promoter . Only a small group of proteins remained phosphate starvation inducible in both phoR and sigB mutant and did not form a unique regulation group . Among these, YfhM and YjbC were controlled by sigma(B)-dependent and unknown PhoPR-independent mechanisms . Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a sigma(B)-dependent manner and in the sigB mutant probably via sigma(H) . YxiE was induced by phosphate starvation independently of sigma(B) and PhoPR.

J Bacteriol, 2000 Aug, 182(16), 4458 - 65
Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions; Ye RW et al.; Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation . A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale . When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions . These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response . Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function . Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.

J Bacteriol, 2000 Aug, 182(16), 4425 - 9
The chromosomal location of the Bacillus subtilis sporulation gene spoIIR is important for its function; Khvorova A et al.; Formation of the asymmetrically located septum during sporulation of Bacillus subtilis results in enclosure of the origin-proximal 30% of the chromosome in the prespore compartment . The rest of the chromosome is then translocated into the prespore from the mother cell . Transcription of spoIIR is initiated in the prespore by RNA polymerase containing sigma(F) soon after the septum is formed . The SpoIIR protein is required for the activation of the transcription program directed by sigma(E) in the mother cell . The spoIIR locus is located at 324 degrees, near the origin of replication (0/360 degrees ) . We show here that movement of spoIIR to 28 degrees had little effect on sporulation . However, movement to regions not in the origin-proximal part of the chromosome substantially reduced sporulation efficiency . At 283 degrees sporulation was reduced to less than 20% of the level obtained when spoIIR was at its natural location, and movement to 190 degrees reduced sporulation to about 6% of that level . These positional effects were also seen in the transcription of a spoIIR-lacZ fusion . In contrast, movement of other spo-lacZ fusions from 28 degrees to 190 degrees had little effect on their expression . These results suggest that spoIIR is the subject of "positional regulation," in the sense that the chromosomal position of spoIIR is important for its expression and function.

J Bacteriol, 2000 Aug, 182(16), 4414 - 24
Characterization of LrpC DNA-binding properties and regulation of Bacillus subtilis lrpC gene expression; Beloin C et al.; The lrpC gene was identified during the Bacillus subtilis genome sequencing project . Previous experiments suggested that LrpC has a role in sporulation and in the regulation of amino acid metabolism and that it shares features with Escherichia coli Lrp, a transcription regulator (C . Beloin, S . Ayora, R . Exley, L . Hirschbein, N . Ogasawara, Y . Kasahara, J . C . Alonso, and F . Le Hegarat, Mol . Gen . Genet . 256:63-71, 1997) . To characterize the interactions of LrpC with DNA, the protein was overproduced and purified . We show that LrpC binds to multiple sites in the upstream region of its own gene with a stronger affinity for a region encompassing P1, one of the putative promoters identified (P1 and P2) . By analyzing lrpC-lacZ transcriptional fusions, we demonstrated that P1 is the major in vivo promoter and that, unlike many members of the lrp/asnC family, lrpC is not negatively autoregulated but rather slightly positively autoregulated . Production of LrpC in vivo is low in both rich and minimal media (50 to 300 LrpC molecules per cell) . In rich medium, the cellular LrpC content is six- to sevenfold lower during the exponentional phase than during the stationary growth phase . Possible determinants and the biological significance of the regulation of lrpC expression are discussed.

J Bacteriol, 2000 Aug, 182(16), 4394 - 400
Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus; Sebulsky MT et al.; Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine . An S . aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin . Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant . This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis . DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B . subtilis . FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins . Given this, the S . aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates . Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S . aureus . This regulation is attributable to Fur in S . aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions . By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.

J Bacteriol, 2000 Aug, 182(16), 4384 - 93
Transcription of the nfrA-ywcH operon from Bacillus subtilis is specifically induced in response to heat; Moch C et al.; The NfrA protein, an oxidoreductase from the soil bacterium Bacillus subtilis, is synthesized during the stationary phase and in response to heat . Analysis of promoter mutants revealed that the nfrA gene belongs to the class III heat shock genes in B . subtilis . An approximate 10-fold induction at both the transcriptional and the translational levels was found after thermal upshock . This induction resulted from enhanced synthesis of mRNA . Genetic and Northern blot analyses revealed that nfrA and the gene downstream of nfrA are transcribed as a bicistronic transcriptional unit . The unstable full-length transcript is processed into two short transcripts encoding nfrA and ywcH . The nfrA-ywcH operon is not induced by salt stress or by ethanol . According to previously published data, the transcription of class III genes in general is activated in response to the addition of these stressors . However, this conclusion is based on experiments which lacked a valid control . Therefore, it seems possible that the transcription of all class III genes is specifically induced by heat shock.

Protein Expr Purif, 2000 Aug, 19(3), 350 - 4
Rapid purification of His(6)-tagged Bacillus subtilis core RNA polymerase; Anthony LC et al.; Bacillus subtilis core RNA polymerase, containing a His(6)-fusion to the C-terminus of the beta' subunit, was isolated by Ni-NTA, Superdex 200 gel filtration, and Mono Q anion-exchange chromatography . The purified core enzyme was shown to be free of the major sigma factor(A) and the transcription factors NusA and GreA . The purification procedure can be completed within 1 working day, is scalable, and yields highly purified and active core RNA polymerase .

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 9246 - 51
The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis; Hamoen LW et al.; The response regulator DegU is involved in various late-growth developmental processes in Bacillus subtilis, including the production of degradative enzymes and the development of genetic competence . DegU is essential for the expression of the competence transcription factor, encoded by comK . ComK is required for the transcription of genes encoding the DNA uptake and integration machinery, as well as for the transcription of its own gene . We have purified DegU to study its role in the expression of comK, and we demonstrate here that DegU binds specifically to the comK promoter . The binding of the response regulator DegU to a promoter target had not been reported previously . DNase I protection analyses show that the DegU binding site overlaps with the ComK binding site, and gel retardation experiments indicate that DegU strongly stimulates the binding of ComK to the comK promoter . We propose that DegU functions at the initiation of competence development, when ComK concentrations are insufficient to support comK transcription, by facilitating ComK binding to the comK promoter . DegU therefore acts as a priming protein that primes the autostimulatory transcription of comK . Such priming activity adds a function to the class of response regulator proteins.

J Biol Chem, 2000 Oct 6, 275(40), 31407 - 13
Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM; Garinot-Schneider C et al.; ExoM is a beta(1-4)-glucosyltransferase involved in the assembly of the repeat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti . By comparing the sequence of ExoM to those of other members of the Pfam Glyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the three-dimensional structure has been resolved, three potentially important aspartic acid residues of ExoM were identified . Single substitutions of each of the Asp amino acids at positions 44, 96, and 187 with Ala resulted in the loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay . Mutants harboring Glu instead of Asp-44 or Asp-96 possessed no in vitro activity but could restore succinoglycan production in vivo . However, replacement of Asp-187 with Glu completely inactivated ExoM as judged by both the in vitro and in vivo assays . These results indicate that Asp-44, Asp-96, and Asp-187 are essential for the activity of ExoM . Furthermore, these data are consistent with the functions proposed for each of the analogous aspartic acids of SpsA based on the SpsA-UDP structure, namely, that Asp-44 and Asp-96 are involved in UDP substrate binding and that Asp-187 is the catalytic base in the glycosyltransferase reaction.

Adv Microb Physiol, 2000, 42, 47 - 238
Primary metabolism and its control in streptomycetes: a most unusual group of bacteria; Hodgson DA; Streptomycetes are Gram-positive bacteria with a unique capacity for the production of a multitude of varied and complex secondary metabolites . They also have a complex life cycle including differentiation into at least three distinct cell types . Whilst much attention has been paid to the pathways and regulation of secondary metabolism, less has been paid to the pathways and the regulation of primary metabolism, which supplies the precursors . With the imminent completion of the total genome sequence of Streptomyces coelicolor A3(2), we need to understand the pathways of primary metabolism if we are to understand the role of newly discovered genes . This review is written as a contribution to supplying these wants . Streptomycetes inhabit soil, which, because of the high numbers of microbial competitors, is an oligotrophic environment . Soil nutrient levels reflect the fact that plant-derived material is the main nutrient input; i.e . it is carbon-rich and nitrogen- and phosphate-poor . Control of streptomycete primary metabolism reflects the nutrient availability . The variety and multiplicity of carbohydrate catabolic pathways reflects the variety and multiplicity of carbohydrates in the soil . This multiplicity of pathways has led to investment by streptomycetes in pathway-specific and global regulatory networks such as glucose repression . The mechanism of glucose repression is clearly different from that in other bacteria . Streptomycetes feed by secreting complexes of extracellular enzymes that break down plant cell walls to release nutrients . The induction of these enzyme complexes is often coordinated by inducers that bear no structural relation to the substrate or product of any particular enzyme in the complex; e.g . a product of xylan breakdown may induce cellulase production . Control of amino acid catabolism reflects the relative absence of nitrogen catabolites in soil . The cognate amino acid induces about half of the catabolic pathways and half are constitutive . There are reduced instances of global carbon and nitrogen catabolite control of amino acid catabolism, which again presumably reflects the relative rarity of the catabolites . There are few examples of feedback repression of amino acid biosynthesis . Again this is taken as a reflection of the oligotrophic nature of the streptomycete ecological niche . As amino acids are not present in the environment, streptomycetes have rarely invested in feedback repression . Exceptions to this generalization are the arginine and branched-chain amino acid pathways and some parts of the aromatic amino acid pathways which have regulatory systems similar to Escherichia coli and Bacillus subtilis and other copiotrophic bacteria.

Dis Aquat Organ, 2000 May 25, 41(1), 9 - 18
A new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS) caused by virus; Wang YG et al.; This paper describes a new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon . The affected shrimp showed white spots similar to those caused by white spot syndrome virus (WSSV), but the shrimp remained active and grew normally without significant mortalities . The study revealed no evidence of WSSV infection using electron microscopy, histopathology and nested polymerase chain reaction . Electron microscopy indicated bacteria associated with white spot formation, and with degeneration and discoloration of the cuticle as a result of erosion of the epicuticle and underlying cuticular layers . Grossly the white spots in BWSS and WSS look similar but showed different profiles under wet mount microscopy . The bacterial white spots were lichen-like, having perforated centers unlike the melanized dots in WSSV-induced white spots . Bacteriological examination showed that the dominant isolate in the lesions was Bacillus subtilis . The occurrence of BWSS may be associated with the regular use of probiotics containing B . subtilis in shrimp ponds . The externally induced white spot lesions were localized at the integumental tissues, i.e., cuticle and epidermis, and connective tissues . Damage to the deeper tissues was limited . The BWS lesions are non-fatal in the absence of other complications and are usually shed through molting.

J Biol Chem, 2000 Sep 29, 275(39), 30287 - 92
Bacillus subtilis YqkI is a novel malic/Na+-lactate antiporter that enhances growth on malate at low protonmotive force; Wei Y et al.; Bacillus subtilis yheL encodes a Na(+)/H(+) antiporter, whereas its paralogue, yqkI, encodes a novel antiporter that achieves a simultaneous Na(+)/H(+) and malolactate antiport . B . subtilis yufR, a control in some experiments, encodes a Na(+)/malate symporter . YqkI complemented a malate transport mutant of Escherichia coli if Na(+) and lactate were present . YheL conferred Na(+) uptake capacity on everted membrane vesicles from an antiporter-deficient E . coli mutant that was consistent with a secondary Na(+)/H(+) antiport, but YqkI-dependent Na(+) uptake depended on intravesicular malate and extravesicular lactate . YqkI-dependent lactate uptake depended on intravesicular malate and extravesicular Na(+) . YqkI mediated an electroneutral exchange, which is proposed to be a malic(-2)-2H(+) (or fully protonated malate)/Na(+)-lactate(-1) antiport . Because the composite YqkI-mediated exchanges could be driven by gradients of the malate-lactate pair, this transporter could play a role in growth of B . subtilis on malate at low protonmotive force . A mutant with a disruption of yqkI exhibited an abrupt arrest in the mid-logarithmic phase of growth on malate when low concentrations of protonophore were present . Thus growth of B . subtilis to high density on a putatively nonfermentative dicarboxylic acid substrate depends on a malolactate exchange at suboptimal protonmotive force.

DNA Seq, 2000, 11(1-2), 1 - 7
An ORF from Bacillus licheniformis encodes a putative DNA repressor; Naval J et al.; The complete sequence of a reading frame adjacent to the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis is reported . It encodes a putative 171 amino acid residues protein with either, low significant sequence similarity in data banks or the corresponding orthologue in the recently sequenced Bacillus subtilis genome . Computer analyses predict a canonical Helix-Turn-Helix motif characteristic of bacterial repressors/DNA binding proteins . A maxicells assay shows that the encoded polypeptide is expressed . A DNA-protein binding, assay performed by gel electrophoresis shows that the expressed protein specifically binds to Bacillus licheniformis DNA.

Proteins, 2000 Sep 1, 40(4), 613 - 22
Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor; Wen YD et al.; Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor . We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro . Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature . The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B . subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B . subtilis DB1005 . Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface . This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A) . Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.

Arch Biochem Biophys, 2000 Jul 15, 379(2), 292 - 8
Methanococcus jannaschii ORF mj0608 codes for a class C inorganic pyrophosphatase protected by Co(2+) or Mn(2+) ions against fluoride inhibition; Kuhn NJ et al.; Openreading frame mj0608 of the Methanococcus jannaschii genome, recognized by its sequence similarity to that of the gene coding for class C inorganic pyrophosphatase in Bacillus subtilis, was cloned and over-expressed in Escherichia coli . The protein was purified and characterized by SDS-PAGE, M(r), and N-terminal sequence . Under suitable conditions it catalyzed the specific hydrolysis of PPi at about 600 micromol x min(-1) x mg(-1) at 25 degrees C, and at 8000 micromol x min(-1) x mg(-1) at 85 degrees C . Therefore this protein is a specific inorganic pyrophosphatase . The activities of Mg(2+), Mn(2+), Co(2+), and Zn(2+) ions as cofactors for hydrolysis of PPi were compared at pH 7.5 and 9.0 . Unlike the class C pyrophosphatase of B . subtilis, this enzyme required no prior activation by low concentrations of Mn(2+) or Co(2+) ions . However, prior exposure to these ions afforded striking protection against inhibition by sodium fluoride, to which the enzyme was otherwise very sensitive .

Enzyme Microb Technol, 2000 Aug 1, 27(3-5), 248 - 253
Effect of the growth conditions on the synthesis of a recombinant beta-1,4-endoglucanase in continuous and fed-batch culture; Shene C et al.; Continuous culture and fed-batch fermentations were used to test the behavior of the system Bacillus subtilis DN1885(pCH7) that synthesizes a recombinant beta-1,4-endoglucanase . Continuous culture experiments were focused on the study of the instability aspects of the system as well as determination of the biomass growth rate range at which the recombinant enzyme synthesis was improved . Fed-batch fermentations were carried out to study the possibility of enhancing recombinant enzyme synthesis through the control of medium addition . It was found that, in continuous culture fermentations, the culture is less unstable at low dilution rates (dilution rate < 0.1 h(-)(1)) . Also, low dilution rates give a higher specific recombinant enzyme concentration (10 times more than that obtained at high dilution rates) . In fed-batch fermentation, the final recombinant enzyme concentration can be manipulated through the medium addition strategy . To increase the recombinant enzyme concentration, the carbon source has to be fed slowly, otherwise enzyme synthesis is impaired due to catabolite repression . Therefore, an increase in the biomass concentration does not necessarily imply an increase in the recombinant enzyme concentration . Higher recombinant enzyme concentrations were found in fed-batch fermentations compared to those obtained in continuous culture.

Enzyme Microb Technol, 2000 Aug 1, 27(3-5), 227 - 233
Expression of Bacillus circulans Teri-42 xylanase gene in Bacillus subtilis; Qureshy AF et al.; The xylanase gene of Bacillus circulans Teri-42 was cloned in both B . subtilis and Escherichia coli . The enzyme activity was almost 87% higher in B . subtilis (pBA7) than in E . coli (pAQ4) . No cellulase activity was detected in the clones, B . subtilis (pBA7) and E . coli (pAQ4) . Approximately 1120 U (80%) of the xylanase was secreted extracellularly by the clone B . subtilis (pBA7) as compared to 79 U (88%) excreted in E . coli (pAQ4) . In B . subtilis (pBA7) the optimal xylanase activity was at pH 7.0 and 50 degrees C, which was the same as that of the parent B . circulans Teri-42 . The recombinant xylanase in B . subtilis was more stable at higher temperatures than the parent B . circulans Teri-42 . Purification of xylanase from the clone B . subtilis (pBA7) showed a 71 kDa polypeptide similar to that observed in B . circulans Teri-42.

Antimicrob Agents Chemother, 2000 Aug, 44(8), 2154 - 9
Saccharomicins, novel heptadecaglycoside antibiotics produced by Saccharothrix espanaensis: antibacterial and mechanistic activities; Singh MP et al.; Saccharomicins A and B, two new heptadecaglycoside antibiotics, were isolated from the fermentation broth of the rare actinomycete Saccharothrix espanaensis . They represent a novel class of bactericidal antibiotics that are active both in vitro and in vivo against bacteria and yeast (MICs: Staphylococcus aureus, <0.12 to 0 . 5; vancomycin-resistant enterococci, 0.25 to 16; gram-negative bacteria, 0.25 to >128; and yeast, >128 microg/ml), including multiply resistant strains . Saccharomicins protected mice from lethal challenges by staphylococci (subcutaneous 50% effective dose range of 0.06 to 2.6 mg/kg of body weight, depending on the S . aureus strain) . The 50% lethal dose by the subcutaneous route was 16 mg/kg . Mechanistic studies with Escherichia coli imp and Bacillus subtilis suggested complete, nonspecific inhibition of DNA, RNA, and protein biosynthesis within 10 min of drug treatment . Microscopic examination of drug-treated cells also suggested cell lysis . These data are consistent with a strong membrane-disruptive activity . The antibacterial activities of the saccharomicins against gram-positive bacteria were unaffected by the presence of Ca(2+) or Mg(2+), but activity against gram-negative bacteria was substantially reduced.

Bioconjug Chem, 2000 Jul-Aug, 11(4), 469 - 73
Conjugation and modeled structure/function analysis of lysozyme on glycine esterified cotton cellulose-fibers; Edwards JV et al.; The antimicrobial activity of lysozyme covalently bound to glycine-derivatized cotton cellulose was assessed in a 96-well format . Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction . The attachment to cotton fibers was made through both a single glycine and a glycine dipeptide esterified to cotton cellulose . Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples . The antibacterial activity of the lysozyme-conjugated cotton cellulose against Bacillus subtilis was assessed as a suspension of pulverized cotton fibers in microtiter wells . Inhibition of B . subtilis growth was observed to be optimal within a range of 0.14-0.3 mM (equivalent to 4-20 mg of lysozyme-bound cotton/mL) of lysozyme . Enhancement of activity over soluble lysozyme may result from the solid-phase protection afforded by the cellulose linkage of the glycoprotein against proteolytic lysis . Computational models of lysozyme based on its crystal structure attached through aspartate, glutamate, and COOH-terminal residues to cellopentaose-(3) Gly-O-6-glycyl-glycine ester were constructed . The models demonstrate no steric constraints to the active-site cleft from the glycine-conjugated cellulose chain when lysozyme is bound at the carboxylates of Asp-87, Glu-7, Asp-119, Asp-18, and COOH-terminal Leu-129 . The more robust antibacterial activity of the enzyme when bonded to cotton fibers suggests good potential for biologically active enzymes on cotton-based fabrics.

Plant Foods Hum Nutr, 2000, 55(2), 127 - 38
An evaluation of the effect of Bacillus cells and Bacillus spores in association with cowpea granules as starter cultures for the fermentation of African oil bean (Pentaclethra macrophylla Bentham) to 'ugba'; Isu NR et al.; Studies on the improvement of the traditional production of 'ugba', a protein-rich fermented African oil bean seed product, were undertaken, by developing starter cultures of Bacillus subtilis cells and spores in association with cowpea granules . The viability of the cells in association remained stable at 94.5% for 6 months at 30 degrees C and for up to 10 months at 4 degrees C while the viability of the spores in association remained stable at ca . 96% for up to 10 months at both 4 and 30 degrees C . The starter cultures resulted in high increases in protease activity from ca 2.8 mg N/min to about 51.6 +/- 0.4 mg N/min in 48 h and a corresponding increase in amino-nitrogen content of ca 2.0 +/- 0.2 mg N 100 g dry matter (DM) to ca 18.5 +/- 0.3 mg N/100 g (DM) during the same period . Changes in the protease activity of the natural process were gradual and increased from 3.0 mg N/min to 38.0 +/- 0.8 mg N/min after 5 days of fermentation . The maximum amino nitrogen content of 'ugba' produced by the starter cultures (18.5 +/- 0.3 mg N/100 g DM) after 2 days was significantly (p <0.05) higher than the maximum amino nitrogen content (12.5 +/- 0.8 mg N/100 g DM), of 'ugba' obtained by the natural process . 'Ugba' produced by the starter cultures were well accepted and compared favorably with the natural product.

J Bacteriol, 2000 Aug, 182(15), 4352 - 5
Identification of a second region of the Spo0A response regulator of Bacillus subtilis required for transcription activation; Rowe-Magnus DA et al.; Deletion of the 10 C-terminal amino acids of the Bacillus subtilis response regulator Spo0A or valine substitution at D258 and L260 resulted in a sporulation-negative phenotype and loss of in vivo activation of the spoIIG and spoIIA operon promoters . Repression of the abrB promoter was not affected by the mutations . In combination with the previously characterized mutation (A257V), the results identify amino acids at positions 257, 258, and 260 as being required for transcription activation by Spo0A.

J Bacteriol, 2000 Aug, 182(15), 4198 - 206
Reconstitution and partial characterization of phospholipid flippase activity from detergent extracts of the Bacillus subtilis cell membrane; Hrafnsdottir S et al.; In bacteria, phospholipids are synthesized on the inner leaflet of the cytoplasmic membrane and must translocate to the outer leaflet to propagate a bilayer . Transbilayer movement of phospholipids has been shown to be fast and independent of metabolic energy, and it is predicted to be facilitated by membrane proteins (flippases) since transport across protein-free membranes is negligible . However, it remains unclear as to whether proteins are required at all and, if so, whether specific proteins are needed . To determine whether bacteria contain specific proteins capable of translocating phospholipids across the cytoplasmic membrane, we reconstituted a detergent extract of Bacillus subtilis into proteoliposomes and measured import of a water-soluble phospholipid analog . We found that the proteoliposomes were capable of transporting the analog and that transport was inhibited by protease treatment . Active proteoliposome populations were also able to translocate a long-chain phospholipid, as judged by a phospholipase A(2)-based assay . Protein-free liposomes were inactive . We show that manipulation of the reconstitution mixture by prior chromatographic fractionation of the detergent extract, or by varying the protein/phospholipid ratio, results in populations of vesicles with different specific activities . Glycerol gradient analysis showed that the majority of the transport activity sedimented at approximately 4S, correlating with the presence of specific proteins . Recovery of activity in other gradient fractions was low despite the presence of a complex mixture of proteins . We conclude that bacteria contain specific proteins capable of facilitating transbilayer translocation of phospholipids . The reconstitution methodology that we describe provides the basis for purifying a facilitator of transbilayer phospholipid translocation in bacteria.

Biochemistry, 2000 Jul 11, 39(27), 7984 - 9
The role of zinc in Bacillus subtilis cytidine deaminase; Mejlhede N et al.; Cytidine deaminase (CDA) from Bacillus subtilis is a zinc-containing enzyme responsible for the hydrolytic deamination of cytidine to uridine and 2'-deoxycytidine to 2'-deoxyuridine . Titration of the cysteinyl groups of the enzyme with p-hydroxymercuriphenyl sulfonate (PMPS) resulted in release of one zinc ion per subunit . Addition of EDTA to chelate the zinc and dithiothreitol (DTT) to remove PMPS, followed by removal of the low molecular weight compounds by gel filtration, resulted in an apoenzyme with no enzymatic activity . The apoenzyme was almost fully reactivated by addition of zinc chloride, indicating that the zinc ion played a central role in catalysis, in keeping with what has been observed with Escherichia coli CDA {Betts, L., Xiang, S., Short, S . A., Wolfenden, R., and Carter, C . W . J . (1994) J . Mol . Biol . 235, 635-656} . Addition of Cd(2+) or Co(2+) caused partial reactivation of the apoenzyme . Zinc reconstitution of the apoenzyme was strictly dependent on the presence of reducing agents, suggesting that the zinc-ligating cysteines, when unligated, participated in disulfide bond formation . An enzymatically active isoform of the tetrameric CDA protein, containing an extension of 13 amino acids at the C-terminus of each subunit, was used in conjunction with the wild-type CDA in subunit-subunit dissociation studies to show that the zinc ion does not assist in the thermodynamic refolding of the protein . After treatment with PMPS and EDTA, the enzyme existed as unfolded unassociated subunits . Immediately following DTT addition to remove PMPS, the subunits refolded into a tetrameric structure, independent of the presence of zinc.

Biochemistry, 2000 Jul 11, 39(27), 7868 - 77
Crystal structure of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 A resolution; Campobasso N et al.; 4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole (Thz) . This enzyme is a salvage enzyme in the thiamin biosynthetic pathway and enables the cell to use recycled Thz as an alternative to its synthesis from 1-deoxy-D-xylulose-5-phosphate, cysteine, and tyrosine . The structure of ThiK in the rhombohedral crystal form has been determined to 1.5 A resolution and refined to a final R-factor of 21 . 6% (R-free 25.1%) . The structures of the enzyme/Thz complex and the enzyme/Thz-phosphate/ATP complex have also been determined . ThiK is a trimer of identical subunits . Each subunit contains a large nine-stranded central beta-sheet flanked by helices . The overall fold is similar to that of ribokinase and adenosine kinase, although sequence similarity is not immediately apparent . The area of greatest similarity occurs in the ATP-binding site where several key residues are highly conserved . Unlike adenosine kinase and ribokinase, in which the active site is located between two domains within a single subunit, the ThiK active site it formed at the interface between two subunits within the trimer . The structure of the enzyme/ATP/Thz-phosphate complex suggests that phosphate transfer occurs by an inline mechanism . Although this mechanism is similar to that proposed for both ribokinase and adenosine kinase, ThiK lacks an absolutely conserved Asp thought to be important for catalysis in the other two enzymes . Instead, ThiK has a conserved cysteine (Cys198) in this position . When this Cys is mutated to Asp, the enzymatic activity increases 10-fold . Further sequence analysis suggests that another thiamin biosynthetic enzyme (ThiD), which catalyzes the formation of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by two sequential phosphorylation reactions, belongs to the same family of small molecule kinases.

J Agric Food Chem, 2000 Jun, 48(6), 2017 - 22
Endoxylanases in durum wheat semolina processing: solubilization of arabinoxylans, action of endogenous inhibitors, and effects on rheological properties; Ingelbrecht JA et al.; Endoxylanases seriously affect the rheological properties of durum wheat (Triticum durum Desf.) semolina spaghetti doughs prepared with, and as evaluated, by the farinograph . Under the experimental conditions, control doughs (34.9% moisture content) made from two semolinas (semA and semB) yielded a maximal consistency of 525 and 517 farinograph units (FU), with, respectively, 19.4 and 16.4% of the total level of arabinoxylans (TOT-AX) being water-extractable (WE-AX) . When 75.4 Somogyi units/50 g of semolina of the endoxylanases from Trichoderma viride (XTV), rumen microorganisms (XRM), Bacillus subtilis (XBS), and Aspergillus niger (XAN) were used, the maximal consistencies at 34.9% moisture decreased for semA to 467, 436, 448, and 417 FU, respectively . This was accompanied by increased WE-AX contents of 60.8, 71.2, 70.7, and 73.0%, respectively . Similar results were observed for semB . By reducing the total water content of doughs, it was possible to recover the maximal consistency of the original doughs . Both the decrease in maximal consistency and the amount of water to be omitted were significantly related to the decrease in molecular weight (MW) of the WE-AX and the percentage of WE-AX solubilized as a result of the enzymic action . At the same time, it was clear that endogenous endoxylanase inhibitors were present in the durum wheat semolinas and that they inhibited the endoxylanases used to different degrees . Part of the differences in effects between the different endoxylanases (decrease in maximal consistency, amount of AX solubilized, MWs of the WE-AX, and amount of water that could be omitted) could be ascribed to the differences in inhibition of the endoxylanases by endogenous inhibitors.

Yi Chuan Xue Bao, 2000, 27(2), 183 - 8
{Expression and roles of hemoglobin gene in Bacillus subtilis}; Zhang YM et al.; Hemoglobin of the Gram-negative bacterium Vitreoscilla can bind oxygen strongly and reduce the oxygen requirement of the bacterium . A recombinant plasmid pAV was constructed by inserting the hemoglobin gene (Vgb) to the downstream of the promoter of beta-lactase gene of the B . subtilis plasmid pAK4 . The plasmid pAV was transferred into strain DB104, G331 (a subtilisin-producing B . subtilis) and B53 (a xylanase-producing B . subtilis) . The subclones and transfer of the Vgb gene into pAK4 were detected and confirmed by Dot blotting and electrophoresis analysis . The expression of homoglobin was measured by recording CO-difference spectra after bubbling CO into the sample cuvette . The production of subtilisin and xylanase was increased in the fermentation of the Vgb-expressing strains . These results provided a new way to reduce the oxygen requirement and to increase the biomass production in the fermentation industry.

J Microsc, 2000 Jul, 199 ( Pt 1), 56 - 67
Adhesion of microbes using 3-aminopropyl triethoxy silane and specimen stabilisation techniques for analytical transmission electron microscopy; Taylor AP et al.; A variety of adhesive support-films were tested for their ability to adhere various biological specimens for transmission electron microscopy . Support films primed with 3-amino-propyl triethoxy silane (APTES), poly-L-lysine, carbon and ultraviolet-B (UV-B)-irradiated carbon were tested for their ability to adhere a variety of biological specimens including axenic cultures of Bacillus subtilis and Escherichia coli and wild-type magnetotactic bacteria . The effects of UV-B irradiation on the support film in the presence of air and electrostatic charge on primer deposition were tested and the stability of adhered specimens on various surfaces was also compared . APTES-primed UV-B-irradiated Pioloform was consistently the best adhesive, especially for large cells, and when adhered specimens were UV-B irradiated they became remarkably stable under an electron beam . This assisted the acquisition of in situ phase-contrast lattice images from a variety of biominerals in magnetotactic bacteria, in particular metastable greigite magnetosomes . Washing tests indicated that specimens adhering to APTES-primed UV-B-irradiated Pioloform were covalently coupled . The electron beam stability was hypothesised to be the result of mechanical strengthening of the specimen and support film and the reduced electrical resistance in the specimen and support film due to their polymerization and covalent coupling.

Braz J Med Biol Res, 2000 Jul, 33(7), 741 - 7
Structural, functional and immunological studies on a polymeric bacterial protein; Baldi PC et al.; The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research . We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis . The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases . Lumazine is an intermediate product in bacterial riboflavin biosynthesis . The recombinant form of the 18-kDa protein (expressed in E . coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity . Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid . Recombinant lumazine synthase from B . abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures . The macromolecular assembly of the enzyme differs from that of the enzyme from B . subtilis . The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form . Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range . We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Biosci Biotechnol Biochem, 2000 May, 64(5), 1082 - 3
A simple and rapid extraction of high molecular weight chromosomal DNA from Bacillus subtilis protoplasts for cosmid cloning and interspecific transformation; Akamatsu T et al.; After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments . The DNA sample thus prepared was the size of 100-450 kb and could be used for cosmid cloning and interspecific transformation.

Microbiology, 2000 Jul, 146 ( Pt 7), 1573 - 83
The essential two-component regulatory system encoded by yycF and yycG modulates expression of the ftsAZ operon in Bacillus subtilis; Fukuchi K et al.; Essential two-component systems are now being identified in bacteria . The Bacillus subtilis yycF gene encoding a response regulator, and its orthologue in Staphylococcus aureus, were reported recently to be essential for cell growth, although genes under their control have yet to be identified . The essential nature of the yycF regulator gene and its cognate kinase gene, yycG, in B . subtilis was also noted during the course of construction of a knockout mutant bank of newly identified genes in the genome sequence project . It was found that yycG could be deleted in the presence of an active form of the YycF protein, thereby suggesting direct interaction between YycG and YycF . Production of mini-cells and reduction in cell length occurred when the YycF regulator was overproduced in B . subtilis . These observations led to the finding that YycF overproduction up-regulated the expression from the P1 promoter of the cell division operon, ftsAZ . In addition, the YycF protein binds to the P1 promoter region in vitro . These results clearly indicate that the essential two-component regulatory system encoded by yycF and yycG genes has the potential to modulate expression of the ftsAZ operon in B . subtilis.

Appl Environ Microbiol, 2000 Jul, 66(7), 3058 - 64
Introduction of raw starch-binding domains into Bacillus subtilis alpha-amylase by fusion with the starch-binding domain of Bacillus cyclomaltodextrin glucanotransferase; Ohdan K et al.; We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy . Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 alpha-amylase (Ba-S) and the raw starch-binding domain (domain E) of Bacillus A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT) . Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT . Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S) . Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis . Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S . We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy.

World Health Organ Tech Rep Ser, 2000, 891, i - viii, 1-168
Evaluation of certain food additives . Fifty-first report of the Joint FAO/WHO Expert Committee on Food Additives; Bees Scavenge Airborne Bacteria; Microbial Aerosol Research Laboratory LLC, 10975 Doll Rd., Monmouth, OR 97361, USA

An air conditioned wind tunnel system was designed, fabricated, and tested to determine whether tethered bees scavenge microbeads or Bacillus subtilis var . niger spores from aerosols . Tests showed that microbeads and spores were scavenged by bumblebees and honeybees, respectively . Five independent variables and their interactions were used in a stepwise multiple regression . Two of them, the cube root of the electrostatic charge on the honeybee and the dose of the spore aerosol, accounted for most of the statistically significant fit to the model's two dependent variables: the percentage of the dose adsorbed by honeybees and the number of spores adsorbed by the same bees . Both dependent variables increased directly so that an increase in electrostatic charge on the bee (i.e., cube root 32 pC) resulted in an increase (i.e., approximately 1%) in the spore dose adsorbed and the number of spores adsorbed by the bees . It was theorized that the spores were in an adsorption/desorption equilibrium that responded to the concentration "pressure" of the spore aerosol . Further, the charge on the bee affected the adsorption force on the bee's surface, as well as increasing the effective aerosol volume accessible for the bee's scavenging . In short, relating these findings to bees scavenging bacteria from the ambient atmosphere, it appears that the spore exposure (where exposure means the product of the ambient concentration, the time the bee is exposed, and air volume through which the bee flies) controls the number of spores adsorbed by a bee, and the static charge on the bee controls the adsorption/desorption equilibrium and presumably the scavenging volume.

Biotechnol Annu Rev, 2000, 5, 131 - 54
Transcription of the insecticidal crystal protein genes of Bacillus thuringiensis; Komano T et al.; Production of a large amount of insecticidal crystal proteins encoded on large plasmids is largely dependent upon the mother cell, Bacillus thuringiensis (B . thuringiensis, also Bt), specific transcription systems attributable to sporulation . In the middle stages of sporulation, cry4A is most actively transcribed from the promoter cry4A-P1 . The proximal transcriptional start point of cry4A, which is under the control of the promoter P1, is used in Bacillus subtilis (B . subtilis) in the middle stage of sporulation . The nucleotide sequence that determines the cry4A-P1 promoter is homologous to the consensus sequence for the promoter of sigma E-specific genes in B . subtilis, and to those promoters of the insecticidal protein genes that are efficiently transcribed in vitro with the RNA polymerase E sigma 35 isolated from B . thuringiensis . The sigma factor sigma 35 of B . thuringiensis is highly homologous and functionally equivalent to sigma E of B . subtilis . These results suggest that the cry4A transcription from P1 is under the control of sigma E in B . subtilis, and under the control of sigma 35 in B . thuringiensis.

Protein Expr Purif, 2000 Jul, 19(2), 265 - 70
High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases; Changchien LM et al.; Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis . The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells . Basically all that is required to obtain pure enzyme is an induction step from a high-expression vector, followed by a DE-52 column elution . Both enzymes appeared to be fairly stable in that incubation at 43 degrees C for 10 min resulted in the loss of 50% of the E . coli thymidylate synthase activity, while 50 degrees C for 10 min was required to obtain the same effect with the B . subtilis enzyme . In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7 degrees C, which was increased to 9 to 10 degrees C on addition of dihydrofolate . It was shown also that the E . coli thymidylate synthase could be maintained at 4 degrees C for at least 4 months with little or no loss in activity provided that mercaptoethanol was not present . The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cysteine . Addition of dithiothreitol restored the enzyme activity to its original state .

Plasmid, 2000 Jul, 44(1), 100 - 4
A method for demonstrating gene essentiality in Staphylococcus aureus; Jana M et al.; A method for demonstrating whether a gene of Staphylococcus aureus is essential for growth in a rich medium is described . We have used this method to determine whether the murE gene, which encodes the UDP-N-acetylmuramyl tripeptide synthetase required for peptidoglycan synthesis, is essential for growth in S . aureus . In this study, strain CYL368 was constructed from S . aureus RN4220 by placing the murE gene in the chromosome under the control of the spac promoter (a hybrid promoter of the Escherichia coli lac operator and the Bacillus subtilis SPO1 phage promoter) . To regulate the murE gene in CYL368, the E . coli lacI gene was expressed from the B . licheniformis penicillinase gene (pcn) promoter in plasmid pMJ8426 . Strain CYL368(pMJ8426) grew normally in the presence of isopropyl-beta-d-thiogalactopyranoside but could not grow in the absence of the inducer . These results indicate that the murE gene expressed from the spac promoter in CYL368(pMJ8426) is needed for bacterial growth . We concluded that murE is an essential gene of S . aureus .

J Mol Biol, 2000 Jun 23, 299(5), 1217 - 30
Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex; Panaghie G et al.; Formation of an initiation-competent RNA polymerase-promoter complex involves DNA melting over a region of about 12 base-pairs, which includes the start site of transcription, thus enabling the template strand to base-pair with the initiating nucleoside triphosphates . By studying the effects of alanine substitutions, we have investigated the role of the aromatic amino residues in the Escherichia coli sigma(70) conserved region 2.3 in promoter strand separation . The resulting mutants were assessed for their activity in vivo in the context of a sigma(70)/sigma(32) hybrid sigma factor that could be targeted to a specific hybrid promoter in the cell . All substitutions lead to an at least twofold reduction in expression of the hybrid promoter-driven reporter gene . The in vitro assay of single substitutions indicated cold sensitivity similar to that previously observed with analogous substitutions in Bacillus subtilis sigma(A) . Kinetic assays showed that these substitutions slowed the rate of open complex formation at 37 degrees C as well . RNA polymerase reconstituted with a sigma(70) containing multiple alanine substitutions readily binds to promoter DNA, but then proceeds slowly beyond the first intermediate complex on the pathway to formation of the transcription-competent complex . These data demonstrate that together the aromatic residues in region 2.3 of E . coli sigma(70) ensure that DNA strand separation proceeds efficiently, even if no individual residue may be essential for accomplishment of the process .

J Biol Chem, 2000 Sep 1, 275(35), 26696 - 703
D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis; Hyyrylainen HL et al.; The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers . In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins . In this study, a secretion mutant of B . subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ alpha-amylase . One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in the d-alanylation of teichoic acids . The inactivation of dlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins . Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA(+) strain . Furthermore, the dlt mutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s) . Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 8063 - 8
The transcriptional profile of early to middle sporulation in Bacillus subtilis; Fawcett P et al.; Spore formation by Bacillus subtilis is governed by global changes in gene transcription . We used nylon-substrate DNA arrays representing approximately 96% of the predicted open reading frames in the B . subtilis chromosome to compare the pattern of transcripts from wild-type cells with the pattern from cells mutant for the sporulation transcription factors Spo0A or final sigma(F) . We found 520 genes whose transcript levels were at least 3-fold dependent on Spo0A but not on final sigma(F), and an additional 66 genes whose transcript levels were dependent upon both regulatory proteins . Two strategies were used to help assign genes to the direct control of a particular developmental regulatory protein . In one approach, we analyzed the effects on global gene expression of artificially producing a constitutively active form of Spo0A during growth . In a second approach, Hidden Markov models were used to identify promoters likely to be activated by Spo0A, final sigma(F), or a third sporulation transcription factor, final sigma(E) . In addition to detecting known sporulation genes, we identified many genes of unknown function whose patterns of expression and regulation suggest that they could be involved in sporulation . Disruption of two such newly identified genes, yabP and yabQ, blocked sporulation at a late stage.

J Bacteriol, 2000 Jul, 182(14), 4121 - 3
Analysis of the function of a putative 2,3-diphosphoglyceric acid-dependent phosphoglycerate mutase from Bacillus subtilis; Pearson CL et al.; A Bacillus subtilis gene termed yhfR encodes the only B . subtilis protein with significant sequence similarity to 2, 3-diphosphoglycerate-dependent phosphoglycerate mutases (dPGM) . This gene is expressed at a low level during growth and sporulation, but deletion of yhfR had no effect on growth, sporulation, or spore germination and outgrowth . YhfR was expressed in and partially purified from Escherichia coli but had little if any PGM activity and gave no detectable PGM activity in B . subtilis . These data indicate that B . subtilis does not require YhfR and most likely does not require a dPGM.

Int J Pharm, 2000 May 15, 201(1), 29 - 36
The effect of pore formers on the controlled release of cefadroxil from a polyurethane matrix; Kim JE et al.; The effect of various pore formers on the controlled release of an antibacterial agent from a polymeric device was examined in order to develop a novel biomaterial that prevents bacterial adhesion and growth on its surface . Cefadroxil was chosen as the model antibiotic and was incorporated into a polyurethane matrix by the solvent-casting method . Polyethylene glycol (PEG) 1450, D-mannitol, or bovine serum albumin (BSA) was used as a pore former . The amount of cefadroxil released from various formulations at 37 degrees C was measured by HPLC . The morphological change of matrices before and after release studies was investigated by scanning electron microscopy (SEM) . The duration of antimicrobial activities of matrices against Escherichia coli and Bacillus subtilis was evaluated by measuring the diameters of the inhibition zone . Changing the weight fraction and particle size of the pore formers/drug mixtures could control the release of cefadroxil from the matrix . The release rate of cefadroxil increased as the loading dose of the pore former increased (15<20<25%) . Cefadroxil released from these devices exhibited antibacterial activity for 5-6 days . These results imply that an antibiotic-loaded polymeric device that could prevent bacterial infection on its surface can be formulated using appropriate pore formers.

FEMS Microbiol Lett, 2000 Jul 1, 188(1), 103 - 6
The role of the bacitracin ABC transporter in bacitracin resistance and collateral detergent sensitivity; Podlesek Z et al.; The bacitracin resistance of Bacillus licheniformis, a producer of bacitracin, is mediated by the ABC transporter Bcr . Bacillus subtilis cells carrying bcr genes on high-copy number plasmids developed collateral detergent sensitivity, as did human cells with overexpressed multidrug resistance P-glycoprotein . Resistance against bacitracin and sensitivity of resistant cells to detergents were shown to be inseparable phenomena associated with the membrane part of Bcr transporter, namely protein BcrC . A fused protein, consisting of ATP-binding protein BcrA and membrane component BcrC was constructed . It resembled a half molecule of P-glycoprotein and was capable of providing a significant degree of antibiotic resistance and detergent sensitivity.

Res Microbiol, 2000 Mar, 151(2), 129 - 34
Systematic function analysis of Bacillus subtilis genes; Ogasawara N; Information on the complete genome sequence needs to be transformed into information related to function . Toward this goal, research consortia in Japan and Europe have been organized so as to acquire a collection of knockout mutants of genes with no established function . The current status of the Japanese project concerning mutant creation and characterization is summarized.

Transfusion, 2000 Jun, 40(6), 682 - 6
Compodock, a new device for sterile docking; van Der Meer PF et al.; BACKGROUND: A new device for sterile docking, the Compodock (Fresenius NPBI Transfusion Technology), was developed for connecting PVC tubing for medical use while maintaining sterility . STUDY DESIGN AND METHODS: Sterility of the connections was assessed by welding tubing with a heavy exterior contamination with Bacillus subtilis spores and also by welding in an environment contaminated with aerosols of B . subtilis . Tubing was either dry or liquid-filled ("wet") and had various diameters . Bacterial culture medium was flushed through the welded area and subsequently cultured . Tensile strength was measured, and, under semi-routine conditions, Compodock was tested for user friendliness and speed . RESULTS: None of the cultures of welded tubing with exterior contamination showed growth, neither the dry-dry (n = 434) nor the wet-wet connections (n = 622) . Cultures were also negative for welds made in the contaminated environment (dry-dry, 67; wet-wet, 55) . Tensile strength complied fully with ISO 3826 standards (that is, a force of 20 newtons {N} for 15 sec), with a mean maximal strength ranging from 73 to 100 N, depending on diameter and content of the tubing . The semi-routine handling was regarded as good: welds were easily opened; there were clear instructions and error warnings; and the processing time averaged 52 seconds . CONCLUSION: The Compodock is able to maintain a functionally closed system, with maintenance of sterility, despite heavy exterior bacterial contamination; tensile strength conformed to ISO standards . Compodock is suitable for routine implementation in the blood bank.

J Mol Biol, 2000 Jun 30, 300(1), 29 - 40
Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor; Wray LV Jr et al.; The Bacillus subtilis nitrogen regulatory protein TnrA was purified and its interaction with the nrgAB regulatory region examined . The TnrA protein activates transcription from the nrgAB promoter in vitro . DNase I footprinting and methylation protection experiments demonstrated that TnrA binds to an inverted repeat, upstream of the -35 region of the nrgAB promoter . Gel mobility retardation assays were used to determine the affinity of TnrA for its DNA-binding site . The equilibrium dissociation binding constant for the interaction of TnrA with the nrgAB promoter fragment was 7.7 nM under the conditions used here . Mutations in the TnrA consensus sequence that reduce nrgAB expression in vivo were found to reduce significantly the in vitro affinity for TnrA . An A+T rich region located upstream of the TnrA-binding site was found to be necessary for optimal transcriptional activation . A mutant protein, TnrA(HTH), was constructed in which the putative helix-turn-helix DNA-binding motif was altered by exchanging two arginine residues for alanine residues . The TnrA(HTH) protein was unable to activate the in vivo expression of nrgAB and had an in vitro affinity for the nrgAB promoter that was significantly lower than that of the wild-type protein .

J Mol Biol, 2000 Jun 30, 300(1), 17 - 28
The anti-sigma factor SpoIIAB forms a 2:1 complex with sigma(F), contacting multiple conserved regions of the sigma factor; Campbell EA et al.; The developmental regulatory protein sigma(F) of Bacillus subtilis, a member of the sigma(70)-family of bacterial RNA polymerase sigma factors, is negatively regulated by the anti-sigma factor SpoIIAB, which binds to sigma(F), sequestering it in an inactive complex . SpoIIAB binding to sigma(F) is strongly stimulated by ATP . Here, we use a combination of gel filtration chromatography, dynamic light-scattering, analytical ultracentrifugation, limited proteolysis with N-terminal sequencing and electrospray mass spectrometry, and deletion analysis to probe the SpoIIAB-sigma(F) complex . The studies were facilitated by investigating the homologs from Bacillus stearothermophilus as well as co-expression of the proteins in Escherichia coli, allowing purification of large quantities of the in vivo assembled complex . We determined the stoichiometry of the complex to be SpoIIAB(2):sigma(F)(1) . Alone, sigma(F) is rapidly degraded by the protease trypsin . In the complex with SpoIIAB, however, sigma(F) is remarkably resistant to proteolysis . Analysis of the protease cleavage data indicates the anti-sigma binds sigma(F) through contacts with mutliple conserved regions of the sigma factor, supporting previous findings based on genetic data .

Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 196 - 202
Major cold shock proteins, CspA from Escherichia coli and CspB from Bacillus subtilis, interact differently with single-stranded DNA templates; Lopez MM et al.; The family of bacterial major cold shock proteins is characterized by a conserved sequence of 65-75 amino acid residues long which form a three-dimensional structure consisting of five beta-sheets arranged into a beta-barrel topology . CspA from Escherichia coli and CspB from Bacillus subtilis are typical representative members of this class of proteins . The exact biological role of these proteins is still unclear; however, they have been implicated to possess ssDNA-binding activity . In this paper, we report the results of a comparative quantitative analysis of ssDNA-binding activity of CspA and CspB . We show that in spite of high homology on the level of primary structure and very similar three-dimensional structures, CspA and CspB have different ssDNA-binding properties . Both proteins preferentially bind polypyrimidine ssDNA templates, but CspB binds to the T-based templates with one order of magnitude higher affinity than to U- or C-based ssDNA, whereas CspA binds T-, U- or C-based ssDNA with comparable affinity . They also show similarities and differences in their binding to ssDNA at high ionic strength . The results of these findings are related to the chemical structure of DNA bases.

Phytother Res, 2000 Jun, 14(4), 267 - 71
Antimicrobial activity of essential oils and ethanol extract of Phlomis fruticosa L . (Lamiaceae); Ristic MD et al.; The essential oils and an ethanol extract of Phlomis fruticosa L . were evaluated for antibacterial and antifungal activities . Seven bacterial and seven fungal species were used . Among them were human, animal and plant pathogens, food poisoning bacteria and fungi which are known as potential mycotoxin producers . The essential oils showed antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae and Micrococcus luteus . The essential oils extracted from the plants collected from two different localities showed similar antibacterial activities . The antifungal activity of the essential oils was positive against Aspergillus niger, A . ochraceus, Cladosporium cladosporioides, Fusarium tricinctum and Phomopsis helianthi . The ethanol extract showed antibacterial activity against Staphylococcus aureus and Bacillus subtilis and antifungal activity against Aspergillus niger, A . ochraceus, Cladosporium cladosporioides, Fusarium tricinctum and Phomopsis helianthi .

Biotechnol Bioeng, 2000 Jul 20, 69(2), 150 - 9
Effect of variation of Klebsiella pneumoniae acetolactate synthase expression on metabolic flux redistribution in Escherichia coli; Yang YT et al.; Escherichia coli strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene were previously shown to produce less acetate with higher ATP yields . Metabolic flux analysis was used to show that excess pyruvate was channeled into the less inhibitory product, acetoin . To further understand the role of intrinsic enzymatic properties and the effect of variations in enzyme levels in the alternation of metabolic fluxes, we constructed a chromosomal integrant of the Klebsiella pneumoniae ALS gene . The reported in vitro Michaelis-Menten constants (K(m)) for the Bacillus and the Klebsiella ALS are 13.0 mM and 8.0 mM, respectively . Furthermore, expression of the Klebsiella ALS is under the control of an inducible trp promoter system . Shake-flask experiments showed a linear induction response (the ALS activity changes from about 9 to 223 U/mg of protein when the inducer concentration {IAA} varied from 0 to 40 mg/L) . Chemostat experiments showed a similar induction response . Interactions between the branched reactions catalyzed by the PFL, LDH, and the ALS enzymes at the pyruvate node were examined . The results indicate the importance of in vivo enzyme activities in the redistribution of metabolic fluxes .

J Mol Biol, 2000 May 26, 299(1), 181 - 97
The atomic structure of pentameric lumazine synthase from Saccharomyces cerevisiae at 1.85 A resolution reveals the binding mode of a phosphonate intermediate analogue; Meining W et al.; Lumazine synthase of Saccharomyces cerevisiae is a homopentamer with a molecular weight of 90 kDa . Crystals of the recombinant enzyme with a size of up to 1.6 mm were obtained . The space group is P4(1)2(1)2 with lattice dimensions 82.9 A x 82.9 A x 300.2 A . X-ray diffraction data collected under cryogenic conditions were complete to 1.85 A resolution . The structure of the enzyme in complex with the intermediate analogue, 5-(6-D-ribitylamino-2,4-dihydroxypyrimidine-5-yl)-1-pentyl-p hosphonic acid was solved via molecular replacement using the structure of the Bacillus subtilis enzyme as search model and was refined to a final R-factor of 19.8% (Rfree: 22.5%) . The conformation of the active site ligand of the enzyme mimicks that of the Schiff base intermediate of the enzyme-catalyzed reaction . The data enable the reconstruction of the reactant topology during the early steps of the catalytic reaction . Structural determinants, which are likely to be responsible for the inability of the S . cerevisiae enzyme to form icosahedral capsids, will be discussed.

Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 1999, (117), 119 - 22
{A 90-day subchronic oral toxicity study of Bacillus subtilis gum in F344 rats}; Nakamura H et al.; A 90-day subchronic toxicity study of Bacillus subtilis gum was performed in both sexes of F344 rats by feeding of CRF-1 pellet diet containing 0%, 0.18%, 0.55%, 1.66% and 5% . Rats were randomly allocated to 5 groups, each consisting of 10 males and females . No animals died during the administration period and no differences in body weights and food intakes were found among groups of either sex . Kidney weight was significantly increased in both sexes in the groups given concentrations of 1.66% or more . However, the increases of kidney weight were slight in themselves and other data on serum biochemistry and histopathology did not show any apparent toxicological signs including renal toxicity . These findings indicate that the treatment of Bacillus subtilis gum in the diet for 90 days does not exert serious toxicity in rats even at the highest dose.

Antimicrob Agents Chemother, 2000 Jul, 44(7), 1961 - 3
Modulation of erm methyltransferase activity by peptides derived from phage display; Giannattasio RB et al.; Combinatorial peptide display on phage M13 protein pIII was used to discover peptide sequences that selectively bind to ErmC' methyltransferase from Bacillus subtilis . One peptide, Ac-LSGVIAT-NH(2), inhibited methylation in vitro with a 50% inhibitory concentration of 20 microM . Interestingly, the set of six peptides which inhibited ErmC' stimulated ErmSF, a homologous methyltransferase from Streptomyces fradiae . Thus, Ac-LSGVIAT-NH(2) may not act directly at the catalytic center of ErmC', but may modulate its activity by binding at a structurally unrelated, but functionally linked, site.

Appl Microbiol Biotechnol, 2000 May, 53(5), 509 - 16
Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production; Stahmann KP et al.; Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes . These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil . Three microorganisms are currently in use for industrial riboflavin production . The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin . To obtain riboflavin production with the gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase . It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies . This is an important tool for the screening of improved mutants . Antimetabolites like itaconate, which inhibits the isocitrate lyase in A . gossypii, tubercidin, which inhibits purine biosynthesis in C . famata, or roseoflavin, a structural analog of riboflavin used for B . subtilis, have been applied successfully for mutant selections . The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments . Although flux studies in B . subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway . Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity.

J Bacteriol, 2000 Jun, 182(12), 3607 - 11
Septal localization of the membrane-bound division proteins of Bacillus subtilis DivIB and DivIC is codependent only at high temperatures and requires FtsZ; Katis VL et al.; Using immunofluorescence microscopy, we have examined the dependency of localization among three Bacillus subtilis division proteins, FtsZ, DivIB, and DivIC, to the division site . DivIC is required for DivIB localization . However, DivIC localization is dependent on DivIB only at high growth temperatures, at which DivIB is essential for division . FtsZ localization is required for septal recruitment of DivIB and DivIC, but FtsZ can be recruited independently of DivIB . These localization studies suggest a more specific role for DivIB in division, involving interaction with DivIC.

J Bacteriol, 2000 Jun, 182(12), 3446 - 51
Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB); Quisel JD et al.; Two chromosome partitioning proteins, Soj (ParA) and Spo0J (ParB), regulate the initiation of sporulation in Bacillus subtilis . In a spo0J null mutant, sporulation is inhibited by the action of Soj . Soj negatively regulates expression of several sporulation genes by binding to the promoter regions and inhibiting transcription . All of the genes known to be inhibited by Soj are also activated by the phosphorylated form of the transcription factor Spo0A (Spo0A approximately P) . We found that, in a spo0J null mutant, Soj affected sporulation, in part, by decreasing the level of Spo0A protein . Soj negatively regulated transcription of spo0A and associated with the spo0A promoter region in vivo . Expression of spo0A from a heterologous promoter in a spo0J null mutant restored Spo0A levels and partly bypassed the sporulation and gene expression defects . Soj did not appear to significantly affect phosphorylation of Spo0A . Thus, in the absence of Spo0J, Soj inhibits sporulation and sporulation gene expression by inhibiting accumulation of the activator protein Spo0A and by acting downstream of Spo0A to inhibit gene expression directly.

J Food Prot, 2000 Jun, 63(6), 753 - 7
Inactivation of Bacillus subtilis spores on aluminum and polyethylene preformed cartons by UV-excimer laser irradiation; Warriner K et al.; The efficacy of UV KrF-excimer laser light (at 248 nm) to inactivate Bacillus subtilis spores loaded onto preformed cartons was found to be dependent on the interior carton coating and scheme by which the irradiation was applied . When the carton was held static during UV laser treatment, the majority of the dose was delivered to the base of the carton and to a lesser extent to the upper part of the pack . In this arrangement no irradiation of the interior sides of the carton was observed . A more even distribution of dose was achieved, however, by moving the carton within the laser beam during irradiation treatment . The distribution of UV was also found to be dependent on the type of carton interior coating . With aluminum cartons the dose measured was found to be significantly greater (P < 0.01) and more evenly distributed across the interior compared to when polyethylene packs were tested . Under optimized conditions no spore survivors were detected on aluminum cartons preloaded with 9.5 x l0 B . subtilis spores by applying a UV laser output dose of 160 J . In comparison, the same conditions only achieved a significantly lower (P < 0.01) reduction in spore numbers (log count reduction 4.2) when polyethylene cartons were used . This difference in lethality and UV distribution of laser light was associated with the higher internal reflection of photons with aluminum cartons . The suitability of UV-excimer lasers for sterilizing preformed cartons over traditional germicidal lamp-based methods is discussed.

Arch Biochem Biophys, 2000 May 15, 377(2), 315 - 23
A biophysical study of the interaction of the lipopeptide antibiotic iturin A with aqueous phospholipid bilayers; Grau A et al.; Iturin A is a lipopeptide extracted from the culture media of Bacillus subtilis which shows a strong antifungal action . The interaction of iturin A with multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) induced structures which did not sediment during centrifugation . Electron microscopy after negative staining showed that, at 30 mol%, iturin A/DMPC vesicles were visible but smaller than those formed by pure DMPC . Thermograms of DMPC/iturinA obtained after differential scanning calorimetry, at low concentrations of iturin A, were interpreted as indicating the presence of two laterally separated phases, one formed by pure phospholipid and the other by lipopeptide-phospholipid complexes, these two separated phases being already detected even at low concentrations such as 2 mol% . Fluorescence quenching experiments showed that the D-Tyr residue of the lipopeptide was fully accessible to the aqueous medium, indicating that the polar part of iturin A is located outside of the membrane hydrophobic palisade . It was concluded that the membrane barrier properties are likely to be damaged in the area where the lipid complexes are accumulated, due to structural fluctuations, and this may be one of the bases of its biological activity . Iturin-A was also able to greatly destabilize dielaidoylphosphatidylethanolamine (DEPE) membranes in the fluid form, producing a new structure which had a poor correlation in X-ray diffraction, and in 31P NMR spectroscopy gave rise to a spectrum containing a double isotropic signal . Iturin A was shown to induce DEPE to adopt phases other than H(II) inverted hexagonal, underlining that this lipopeptide is capable of modifying the curvature of the membrane, which may also be important in explaining the tendency of iturin A to create small vesicles and which may be another of the bases of its biological activity.

J Bacteriol, 2000 Jul, 182(13), 3870 - 3
The FtsH protein accumulates at the septum of Bacillus subtilis during cell division and sporulation; Wehrl W et al.; The ftsH gene encodes an ATP- and Zn(2+)-dependent metalloprotease which is anchored to the cytoplasmic membrane via two transmembrane segments in such a way that the very short amino- and the long carboxy termini are exposed to the cytoplasm . Deletion of the ftsH gene in Bacillus subtilis results in a pleiotropic phenotype such as filamentous growth . This observation prompted us to ask whether ftsH is involved in cell division . A translational fusion was constructed between the complete coding region of ftsH and gfp(+) the latter carrying five point mutations to obtain enhanced fluorescence . We detected that the FtsH protein accumulates in the midcell septum of dividing cells, and during sporulation first in the asymmetrically located septa of sporulating cells and later in the membrane which engulfs the forespore . These observations revealed a new function of FtsH.

J Bacteriol, 2000 Jul, 182(13), 3717 - 25
Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake; Boncompagni E et al.; The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source . A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S . meliloti . This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism . Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake . These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport . When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2) . Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection . To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 479 - 85
alpha-Amylase production by free and immobilized Bacillus subtilis; Duran-Paramo E et al.; The effect of glucose on the alpha-amylase production by Bacillus subtilis ATCC-21556 was studied . Initial glucose concentrations up to 20 g/L were found to be directly proportional to the specific alpha-amylase production in an immobilized-cell batch system, whereas a free-cell batch system presented an inversely proportional relationship with the initial glucose concentration . This might be owing to the alpha-amylase repression by the glucose present in the culture medium . Three hundred eighty-five percent of the specific alpha-amylase production with the free-cell system was produced by the immobilized-cell batch culture.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 319 - 27
Enhanced alkaline protease production in addition to alpha-amylase via constructing a Bacillus subtilis strain; Zaghloul TI et al.; Bacillus subtilis Bios11 strain was previously isolated and identified . This strain naturally produces a high level of alpha-amylase . The multicopy (pS1) plasmid that carries the complete alkaline protease aprA gene was introduced to this host strain by transformation . The newly constructed strain was found to express the aprA gene and produces a high level of alkaline protease . The level of alpha-amylase production was not affected compared with the parent strain . The pS1 plasmid in the new host was proved to be segregationally and structurally stable, and the multicopy aprA gene was expressed at the stationary phase . This expression did not affect growth rate and sporulation frequency . Moreover, the level of alpha-amylase was maintained . Both alkaline protease and alpha-amylase enzymes were purified using a single-step affinity chromatography column . The use of the newly constructed strain would be valuable to the enzyme industry and would promote recycling of some food-processing wastes.

Eur J Biochem, 2000 Jun, 267(12), 3770 - 83
Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols; Jorasch P et al.; A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis {Jorasch, P., Wolter, F.P., Zahringer, U., and Heinz, E . (1998) Mol . Microbiol . 29, 419-430} . Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S . aureus enzyme shows processivity similar to the B . subtilis enzyme . Both glycosyltransferases characterized in this work show unexpected acceptor specificities . We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S . aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A . thaliana by PCR . After cloning and expression of S . aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa . Thin-layer chromatographic analysis of the lipids extracted from the transformed E . coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls . These lipids were purified from lipid extracts of E . coli cells expressing the S . aureus gene and identified by NMR and mass spectrometry as 1, 2-diacyl-3-{O-beta-D-glucopyranosyl}-sn-glycerol, 1, 2-diacyl-3-{O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyrano-+ ++syl} -sn-glycerol, 1, 2-diacyl-3-{O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-( 1-->6)-O-beta-D-glucopyranosyl}-sn-glycerol, sn-3'-{O-beta-D-glucopyranosyl}-phosphatidylglycerol and sn-3'-{O-(6"'-O-acyl)-beta-D-glucopyranosyl-(1"'-->6")-O-beta-D-gluco pyranosyl}-sn-2'-acyl-phospha-tidylglycerol . A 1, 2-diacyl-3-{O-beta-D-galactopyranosyl}-sn-glycerol was isolated from extracts of E . coli cells expressing the ugt81A1 cDNA from A . thaliana . The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates . Experiments with several of the above described glycolipids as 14C-labeled sugar acceptors and unlabeled UDP-glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP-glucose:1, 2-diacylglycerol-3-beta-D-glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase . As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.

Mol Microbiol, 2000 Jun, 36(5), 1135 - 47
S-adenosylmethionine decarboxylase of Bacillus subtilis is closely related to archaebacterial counterparts; Sekowska A et al.; Bacillus subtilis synthesizes polyamines by decarboxylating arginine to agmatine, which is subsequently hydrolysed to putrescine . Spermidine is synthesized from putrescine and decarboxylated S-adenosylmethionine (dAdoMet) . In Gram-negative bacteria and in eukaryotes, AdoMet is decarboxylated by an unusual 'pyruvoyl' AdoMet decarboxylase (SpeD), the catalytic pyruvoyl moiety of which is generated by serinolysis of an internal serine with self-cleavage of the protein at the upstream peptide bond . Neither the Gram-positive bacterial nor the archaeal counterpart of the Escherichia coli SpeD enzyme were known . We have identified the corresponding B . subtilis speD gene (formely ytcF) . Heterologous expression of the cognate Methanococcus jannaschii protein, MJ0315, demonstrated that it displays the same activity as B . subtilis SpeD, indicating that spermidine biosynthesis in Gram-positive bacteria and in archaea follows a pathway very similar to that of Gram-negatives and eukarya . In B . subtilis, transcription of speD is modulated by spermidine and methionine . Its expression is high under usual growth conditions . In contrast, the SpeD protein self-cleaves slowly in vitro, a noticeable difference with its archaeal counterpart . Under certain growth conditions (minimal medium containing succinate and glutamate as a carbon source), speD is co-transcribed with gapB, the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme required for gluconeogenesis . This observation may couple polyamine metabolism to sulphur and carbon metabolism by a so far unknown mechanism.

Mol Microbiol, 2000 Jun, 36(5), 1037 - 48
Subcellular localization of Dna-initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids; Imai Y et al.; We examined the intracellular distribution of Bacillus subtilis Dna-initiation proteins by immunofluorescence microscopy to visualize the initiation complex of replication in vivo . DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI were always detected as foci during the cell-division cycle . Interaction of DnaI with the DnaC helicase by the yeast two-hybrid assay suggests that DnaI acts as a helicase loader . The number of DnaB and DnaI foci within the cell exceeded that of oriC . Although the foci were not always co-localized with oriC, they seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication . When the replication cycle was synchronized in cells using a temperature-sensitive dnaA mutant, duplication of the oriC region was observed predominantly near an edge of the nucleoid . Before initiation occurred, each one of the DnaB and DnaI foci was frequently observed near there . Furthermore, DnaX-GFP (DnaX is a component of DNA polymerase III) foci were detected near either of the edges of the nucleoids at the onset of replication . These results suggest that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B . subtilis.

Mol Microbiol, 2000 May, 36(4), 846 - 55
The Bacillus subtilis cell division protein FtsL localizes to sites of septation and interacts with DivIC; Sievers J et al.; FtsL is a small bitopic membrane protein required for vegetative cell division and sporulation in Bacillus subtilis . We investigated its localization by fluorescence microscopy using a green fluorescent protein (GFP) fusion . GFP-FtsL was localized at mid-cell in vegetative cells and at the asymmetric septum in sporulating cells . We also show that FtsL forms a ring-like structure at the division site and that it remains localized at mid-cell during the whole septation process . By yeast two-hybrid analysis and non-denaturing polyacrylamide gel electrophoresis (PAGE) with purified proteins, FtsL was found to interact with another membrane-bound division protein, the FtsL-like DivIC protein.

Mol Microbiol, 2000 May, 36(3), 662 - 78
AglU, a protein required for gliding motility and spore maturation of Myxococcus xanthus, is related to WD-repeat proteins; White DJ et al.; The aglU gene of Myxococcus xanthus encodes a protein similar to Het-E1 (vegetative incompatibility) from Podospora anserina, acylaminoacyl-peptidase from Bacillus subtilis, and TolB from Escherichia coli . These proteins all have evenly spaced SPDG repeats that are characteristic of a larger motif called the WD-repeat . The WD-repeat is predicted to form a beta-propeller structure that mediates the assembly of heteromeric protein complexes . AglU has a consensus lipoprotein attachment motif that includes a type II signal sequence followed by a cysteine residue . This suggests that AglU is matured, then attached to the outer membrane via fatty acid acylation at this Cys . Cells carrying a mutation in aglU are blocked in adventurous gliding and can swarm only if cells are in contact with one another . When starved of nutrients, the aglU mutant aggregates and forms multicellular fruiting bodies like the wild-type strain, but is unable to produce heat-resistant spores . This suggests that adventurous gliding motility, per se, is not required for development, but that AglU is essential for a terminal step of spore differentiation.

Mol Microbiol, 2000 May, 36(3), 638 - 50
Genes required for cytochrome c synthesis in Bacillus subtilis; Le Brun NE et al.; Cytochromes of c-type contain covalently bound haem and in bacteria are located on the periplasmic side of the cytoplasmic membrane . More than eight different gene products have been identified as being specifically required for the synthesis of cytochromes c in Gram-negative bacteria . Corresponding genes are not found in the genome sequences of Gram-positive bacteria . Using two random mutagenesis approaches, we have searched for cytochrome c biogenesis genes in the Gram-positive bacterium Bacillus subtilis . Three genes, resB, resC and ccdA, were identified . CcdA has been found previously and is required for a late step in cytochrome c synthesis and also plays a role in spore synthesis . No function has previously been assigned for ResB and ResC but these predicted membrane proteins show sequence similarity to proteins required for cytochrome c synthesis in chloroplasts . Attempts to inactivate resB and resC in B . subtilis have indicated that these genes are essential for growth . We demonstrate that various nonsense mutations in resB or resC can block synthesis of cytochromes c with no effect on other types of cytochromes and little effect on sporulation and growth . The results strongly support the recent proposal that Gram-positive bacteria, cyanobacteria, epsilon-proteobacteria, and chloroplasts have a similar type of machinery for cytochrome c synthesis (System II), which is very different from those of most Gram-negative bacteria (System I) and mitochondria (System III).

J Biol Chem, 2000 Sep 15, 275(37), 28802 - 9
Characterization of YpmQ, an accessory protein required for the expression of cytochrome c oxidase in Bacillus subtilis; Mattatall NR et al.; A search of the Bacillus subtilis genome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast . Sco1 has been found to aid the delivery of copper to cytochrome c oxidase . B . subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, Cu(A) and Cu(B), and a menaquinol oxidase that has only Cu(B) . Deletion of ypmQ in B . subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase . Levels of cytochrome c oxidase recover when copper is added to the growth medium of the DeltaypmQ strain or when ypmQ is expressed from a plasmid . Neither treatment affects the amount or activity of menaquinol oxidase . YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein . We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the Cu(A) site of cytochrome c oxidase.

Appl Environ Microbiol, 2000 Jun, 66(6), 2565 - 71
Biosynthesis of the lantibiotic mersacidin: organization of a type B lantibiotic gene cluster; Altena K et al.; The biosynthetic gene cluster (12.3 kb) of mersacidin, a lanthionine-containing antimicrobial peptide, is located on the chromosome of the producer, Bacillus sp . strain HIL Y-85,54728 in a region that corresponds to 348 degrees on the chromosome of Bacillus subtilis 168 . It consists of 10 open reading frames and contains, in addition to the previously described mersacidin structural gene mrsA (G . Bierbaum, H . Brotz, K.-P . Koller, and H.-G . Sahl, FEMS Microbiol . Lett . 127:121-126, 1995), two genes, mrsM and mrsD, coding for enzymes involved in posttranslational modification of the prepeptide; one gene, mrsT, coding for a transporter with an associated protease domain; and three genes, mrsF, mrsG, and mrsE, encoding a group B ABC transporter that could be involved in producer self-protection . Additionally, three regulatory genes are part of the gene cluster, i.e., mrsR2 and mrsK2, which encode a two-component regulatory system which seems to be necessary for the transcription of the mrsFGE operon, and mrsR1, which encodes a protein with similarity to response regulators . Transcription of mrsA sets in at early stationary phase (between 8 and 16 h of culture).

J Biol Chem, 2000 Aug 25, 275(34), 26404 - 10
Identification of residues within two regions involved in self-association of viral histone-like protein p6 from phage theta29; Abril AM et al.; Protein p6 of Bacillus subtilis phage theta29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA . Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein . Protein p6 is in a monomer-dimer-oligomer equilibrium association . We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation . The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein . Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified . Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of O29 DNA replication . Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate O29 DNA initiation of replication . C-terminal deletion mutants showed an enhanced dimer formation capacity . The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association.

Biochim Biophys Acta, 2000 May 23, 1478(2), 333 - 40
Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2; Cho HY et al.; An additional amylase besides the typical alpha-amylase was detected in the cytoplasm of Bacillus subtilis SUH4-2, an isolate from Korean soil . The corresponding gene encoded a maltogenic amylase, which hydrolyzed cyclodextrin or starch to maltose and glucose; pullulan to panose; acarbose to glucose and acarviosine-glucose . Maltogenic amylase of B . subtilis SUH4-2 transferred sugar molecules to form various branched oligosaccharides upon the hydrolysis of substrates . The enzyme existed in a monomer-dimer equilibrium with a molar ratio of 3:2 in 50 mM KH(2)PO(4)-NaOH buffer (pH 7.0) . The maltogenic amylase is most likely to be associated with carbohydrate metabolism in the cytoplasm, since the nucleotide sequence of the gene was highly homologous to the yvdF gene of B . subtilis 168, which is located in a gene cluster involved in maltose/maltodextrin utilization.

J Biol Chem, 2000 Aug 11, 275(32), 24264 - 72
Selective methylation changes on the Bacillus subtilis chemotaxis receptor McpB promote adaptation; Zimmer MA et al.; The Bacillus subtilis McpB is a class III chemotaxis receptor, from which methanol is released in response to all stimuli . McpB has four putative methylation sites based upon the Escherichia coli consensus sequence . To explore the nature of methanol release from a class III receptor, all combinations of putative methylation sites Gln(371), Gln(595), Glu(630), and Glu(637) were substituted with aspartate, a conservative substitution that effectively eliminates methylation . McpB((Q371D,E630D,E637D)) in a Delta(mcpA mcpB tlpA tlpB)101::cat mcpC4::erm background failed to release methanol in response to either the addition or removal of the McpB-mediated attractant asparagine . In the same background, McpB((E630D,E637D)) produced methanol only upon asparagine addition, whereas McpB((Q371D,E630D)) produced methanol only upon asparagine removal . Thus methanol release from McpB was selective . Mutants unable to methylate site 637 but able to methylate site 630 had high prestimulus biases and were incapable of adapting to asparagine addition . Mutants unable to methylate site 630 but able to methylate site 637 had low prestimulus biases and were impaired in adaptation to asparagine removal . We propose that selective methylation of these two sites represents a method of adaptation novel from E . coli and present a model in which a charged residue rests between them . The placement of this charge would allow for opposing electrostatic effects (and hence opposing receptor conformational changes) . We propose that CheC, a protein not found in enteric systems, has a role in regulating this selective methylation.

Rapid Commun Mass Spectrom, 2000, 14(10), 829 - 33
Application of matrix-assisted laser desorption/ionization to on-line aerosol time-of-flight mass spectrometry
Stowers MA, van Wuijckhuijse AL, Marijnissen JC, Scarlett B, van Baar BL, Kientz CE.
Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained from single biological aerosol particles using an aerosol time-of-flight mass spectrometer (ATOFMS) . The inlet to the ATOFMS was coupled with an evaporation/condensation flow cell that allowed the aerosol to be coated with matrix material as the sampled stream entered the spectrometer . Mass spectra were generated from aerosol composed either of gramicidin-S or erythromycin, two small biological molecules, or from aerosolised spores of Bacillus subtilis var niger . Three different matrices were used: 3-nitrobenzyl alcohol, picolinic acid and sinapinic acid . A spectrum of gramicidin-S was generated from approximately 250 attomoles of material using a molar ratio of 3-nitrobenzyl alcohol to analyte of approximately 20:1 . A single peak, located at 1224 Da, was obtained from the bacterial spores . The washing liquid and extract solution from the spores were analyzed using electrospray mass spectrometry and subsequent MS/MS product ion experiments . This independent analysis suggests that the measured species represents part of the B . subtilis peptidoglycan . The on-line addition of matrix allows quasi-real-time chemical analysis of individual, aerodynamically sized particles, with an overall system residence time of less than 5 seconds . These results suggest that a MALDI-ATOFMS can provide nearly real-time identification of biological aerosols .

Infect Control Hosp Epidemiol, 2000 May, 21(5), 333 - 6
Efficacy of a washer-pasteurizer for disinfection of respiratory-care equipment; Rutala WA et al.; We evaluated the efficacy of a commercial washer-pasteurizer . Carriers were inoculated with 10(4) to 10(6) test organisms and pasteurized at 170 degrees F for 30 minutes . Pasteurization eliminated all test organisms with the exception of Bacillus subtilis spores . Pasteurization appears efficacious for the disinfection of respiratory-care equipment and could result in a cost savings of approximately $30,000 per year.

Photochem Photobiol, 2000 May, 71(5), 514 - 23
Systematic study of parameters influencing the action of Rose Bengal with visible light on bacterial cells: comparison between the biological effect and singlet-oxygen production; Schafer M et al.; As part of a project to study different methods for the disinfection of effluent water, the inactivation of different microorganisms (Escherichia coli, Deinococcus radiodurans and spores of Bacillus subtilis) using a combination of a photosensitizer (Rose Bengal) with simulated sunlight and oxygen was determined under various environmental conditions (temperature, pH index) . In parallel, the singlet-oxygen (1O2) production was also measured under the same conditions . Whereas the vegetative cells could be inactivated much more efficiently at increased temperature and altered index of pH, the production of 1O2 remained essentially the same under these alterations . Additionally, the relations among the sensitivities of different cell types to be killed by our photodynamic treatments (PDT) were opposite to those found after exposure to ionizing radiation . The results of photodynamic experiments do not reflect the cells' capacity to repair DNA strand breaks . Spores of B . subtilis, as a nonvegetative system, could not be inactivated by illuminations up to 100 J cm-2 . Together, these findings indicate that DNA is not the primary target, the inactivation of which leads to the killing of our test organisms . Instead, the cellular envelope appears to be the component being assaulted by our PDT.

Acta Crystallogr D Biol Crystallogr, 2000 Jun, 56 ( Pt 6), 673 - 83
The 1.30 A resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimer; Ladner JE et al.; The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30 A resolution . The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P2(1)2(1)2(1) with unit-cell parameters a = 52.2, b = 83 . 8, c = 86.0 A . The ABC trimer of the monoclinic crystal structure {Chook et al . (1994), J . Mol . Biol . 240, 476-500} was used as the starting model . The final coordinates are composed of three complete polypeptide chains of 127 amino-acid residues . In addition, there are nine sulfate ions, five glycerol molecules and 424 water molecules clearly visible in the structure . This structure was refined with aniosotropic temperature factors, has excellent geometry and a crystallographic R factor of 0.169 with an R(free) of 0.236 . The three active sites of the macromolecule are at the subunit interfaces, with residues from two subunits contributing to each site . This orthorhombic crystal form was grown using ammonium sulfate as the precipitant; glycerol was used as a cryoprotectant during data collection . A glycerol molecule and sulfate ion in each of the active sites was found mimicking a transition-state analog . In this structure, the C-terminal tails of the subunits of the trimer are hydrogen bonded to residues of the active site of neighboring trimers in the crystal and thus cross-link the molecules in the crystal lattice.

Infect Immun, 2000 Jun, 68(6), 3200 - 9
Identification of an immunodominant ABC transporter in methicillin-resistant Staphylococcus aureus infections; Burnie JP et al.; Immunoblotting sera from 26 patients with septicemia due to an epidemic strain of methicillin-resistant Staphylococcus aureus (EMRSA-15), 6 of whom died, revealed an immunodominant EMRSA-15 antigen at 61 kDa . There was a statistically significant correlate (P < 0.001) between survival and immunoglobulin G to the 61-kDa band . The antigen was identified by sequencing positive clones obtained by screening a genomic expression library of EMRSA-15 with pooled sera from patients taken after the septicemic episode . Eluted antibody reacted with the 61-kDa antigen on immunoblots . The amino terminus was obtained by searching the S . aureus NCTC 8325 and MRSA strain COL databases, and the whole protein was expressed in Escherichia coli TOP 10F' . The derived amino acid sequence showed homology with ABC transporters, with paired Walker A and Walker B motifs and 73% homology to YkpA from Bacillus subtilis . Epitope mapping of the derived amino acid sequence with sera from patients who had recovered from EMRSA-15 septicemia delineated seven epitopes . Three of these epitopes, represented by peptides 1 (KIKVYVGNYDFWYQS), 2 (TVIVVSHDRHFLYNNV), and 3 (TETFLRGFLGRMLFS), were synthesized and used to isolate human recombinant antibodies from a phage antibody display library . Recombinant antibodies against peptides 1 and 2 gave logarithmic reductions in organ colony counts, compared with control groups, in a mouse model of the infection . This study suggests the potential role of an ABC transporter as a target for immunotherapy.

Biochem J, 2000 Jun 1, 348 Pt 2, 367 - 73
Interaction of Bacillus subtilis CsaA with SecA and precursor proteins; Muller JP et al.; CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants . CsaA has chaperone-like activities in vivo and in vitro . To examine the role of CsaA in protein export in B . subtilis, expression of the csaA gene was repressed . While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion . CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E . coli and B . subtilis, and binds the B . subtilis preprotein prePhoB . Purified CsaA stimulates the translocation of prePhoB into E . coli membrane vesicles bearing the B . subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E . coli . The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B . subtilis.

Biochem, Eng . J. . 2000 Jun 1, 5(2), 115 - 121
alpha-Amylase production by Bacillus subtilis with dregs in an external-loop airlift bioreactor; Yuguo Z et al.; An external-loop airlift bioreactor, with a low ratio 2.9 of height-to-diameter of the riser and a ratio 6.6 of riser-to-downcomer diameter, was used to produce alpha-amylase from fermentation with dregs by Bacillus subtilis . The effects of gas flow rate and liquid volume on alpha-amylase production were investigated . After a 36-h fermentation time, an average of 432.3U/ml alpha-amylase activity was obtained under the conditions of liquid volume 8.5l and gas flow rate 1.2vvm for the first 12h of fermentation, 1.4vvm from 12 to 27h, and 1.2vvm from 27h to the end . The activity was higher than that obtained in shaking flasks (409.0U/ml) and in a mechanically stirred tank bioreactor (397.2U/ml) under optimized operating conditions . The fermentation cycle of the airlift bioreactor was shorter than the 48h required for the shaking flasks and close to the 36h of the mechanically stirred tank bioreactor . It was demonstrated that the external-loop airlift bioreactor could substitute for the traditional mechanically stirred tank bioreactor to produce alpha-amylase from fermentation by Bacillus subtilis with dregs.

J Biomed Mater Res, 2000 Jul, 51(1), 128 - 35
Effect of gas composition on spore mortality and etching during low-pressure plasma sterilization; Lerouge S et al.; The aim of this work was to investigate possible mechanisms of sterilization by low-temperature gas plasma: spore destruction by plasma is compared with etching of synthetic polymers . Bacillus subtilis spores were inoculated at the bottom of glass vials and subjected to different plasma gas compositions (O(2), O(2)/Ar, O(2)/H(2), CO(2), and O(2)/CF(4)), all known to etch polymers . O(2)/CF(4) plasma exhibited much higher efficacy than all other gases or gas mixtures tested, with a more than 5 log decrease in 7.5 min, compared with a 2 log decrease with pure oxygen . Examination by scanning electron microscopy showed that spores were significantly etched after 30 min of plasma exposure, but not completely . We speculate about their etch resistance compared with that of synthetic polymers on the basis of their morphology and complex coating structure . In contrast to so-called in-house plasma, sterilization by Sterrad(R) tended to increase the observed spores' size; chemical modification (oxidation), rather than etching, is believed to be the sterilization mechanism of Sterrad(R) .

J Bacteriol, 2000 Jun, 182(11), 3305 - 9
Evidence that SpoIVFB is a novel type of membrane metalloprotease governing intercompartmental communication during Bacillus subtilis sporulation; Yu YT et al.; Processing of pro-sigma(K) in the mother cell compartment of sporulating Bacillus subtilis involves SpoIVFB and is governed by a signal from the forespore . SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments . Several conservative single amino acid changes in the HEXXH motif abolished function . However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function . Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence . Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases . A conservative change in this region of SpoIVFB abolished function . Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.

J Bacteriol, 2000 Jun, 182(11), 3274 - 7
Dual control of sbo-alb operon expression by the Spo0 and ResDE systems of signal transduction under anaerobic conditions in Bacillus subtilis; Nakano MM et al.; The Bacillus subtilis sbo-alb operon contains sboA, the structural gene for the bacteriocin subtilosin, and the alb genes required for subtilosin production . Transcription from the sbo-alb promoter is highly induced by oxygen limitation . The transcriptional regulation of the sbo-alb operon is under dual control involving the transition state regulator AbrB and the two-component regulatory proteins ResD and ResE.

J Bacteriol, 2000 Jun, 182(11), 3266 - 73
Mutational analysis of the sbo-alb locus of Bacillus subtilis: identification of genes required for subtilosin production and immunity; Zheng G et al.; The Bacillus subtilis 168 derivative JH642 produces a bacteriocin, subtilosin, which possesses activity against Listeria monocytogenes . Inspection of the amino acid sequence of the presubtilosin polypeptide encoded by the gene sboA and sequence data from analysis of mature subtilosin indicate that the precursor subtilosin peptide undergoes several unique and unusual chemical modifications during its maturation process . The genes of the sbo-alb operon are believed to function in the synthesis and maturation of subtilosin . Nonpolar mutations introduced into each of the alb genes resulted in loss or reduction of subtilosin production . sboA, albA, and albF mutants showed no antilisterial activity, indicating that the products of these genes are critical for the production of active subtilosin . Mutations in albB, -C, and -D resulted in reduction of antilisterial activity and decreased immunity to subtilosin, particularly under anaerobic conditions . A new gene, sboX, encoding another bacteriocin-like product was discovered residing in a sequence overlapping the coding region of sboA . Construction of an sboX-lacZ translational fusion and analysis of its expression indicate that sboX is induced in stationary phase of anaerobic cultures of JH642 . An in-frame deletion of the sboX coding sequence did not affect the antilisterial activity or production of or immunity to subtilosin . The results of this investigation show that the sbo-alb genes are required for the mechanisms of subtilosin synthesis and immunity.

J Bacteriol, 2000 Jun, 182(11), 3259 - 65
The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins; Kruger E et al.; The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis . Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins . Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells . Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions . In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock . This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell . By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.

J Bacteriol, 2000 Jun, 182(11), 3072 - 80
Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression; Cruz Ramos H et al.; Bacillus subtilis grows in the absence of oxygen using nitrate ammonification and various fermentation processes . Lactate, acetate, and 2,3-butanediol were identified in the growth medium as the major anaerobic fermentation products by using high-performance liquid chromatography . Lactate formation was found to be dependent on the lctEP locus, encoding lactate dehydrogenase and a putative lactate permease . Mutation of lctE results in drastically reduced anaerobic growth independent of the presence of alternative electron acceptors, indicating the importance of NADH reoxidation by lactate dehydrogenase for the overall anaerobic energy metabolism . Anaerobic formation of 2,3-butanediol via acetoin involves acetolactate synthase and decarboxylase encoded by the alsSD operon . Mutation of alsSD has no significant effect on anaerobic growth . Anaerobic acetate synthesis from acetyl coenzyme A requires phosphotransacetylase encoded by pta . Similar to the case for lctEP, mutation of pta significantly reduces anaerobic fermentative and respiratory growth . The expression of both lctEP and alsSD is strongly induced under anaerobic conditions . Anaerobic lctEP and alsSD induction was found to be partially dependent on the gene encoding the redox regulator Fnr . The observed fnr dependence might be the result of Fnr-induced arfM (ywiD) transcription and subsequent lctEP and alsSD activation by the regulator ArfM (YwiD) . The two-component regulatory system encoded by resDE is also involved in anaerobic lctEP induction . No direct resDE influence on the redox regulation of alsSD was observed . The alternative electron acceptor nitrate represses anaerobic lctEP and alsSD transcription . Nitrate repression requires resDE- and fnr-dependent expression of narGHJI, encoding respiratory nitrate reductase . The gene alsR, encoding a regulator potentially responding to changes of the intracellular pH and to acetate, is essential for anaerobic lctEP and alsSD expression . In agreement with its known aerobic function, no obvious oxygen- or nitrate-dependent pta regulation was observed . A model for the regulation of the anaerobic fermentation genes in B . subtilis is proposed.

J Bacteriol, 2000 Jun, 182(11), 3055 - 62
Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression; Mirel DB et al.; The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis . Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression . We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression . In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state . In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium . These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity . This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids . However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase . In work by others, CodY has been implicated in the nutritional repression of several genes . Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium . Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B . subtilis.

Gene, 2000 May 2, 248(1-2), 169 - 81
Analysis of the regulation and function of five genes encoding small, acid-soluble spore proteins of Bacillus subtilis; Cabrera-Hernandez A et al.; Four genes {sspI, sspK, sspM, and sspO (originally called cotK)} encoding minor small, acid-soluble proteins (SASP) unique to spores of Bacillus subtilis are expressed only in the forespore compartment of sporulating cells of this organism . The sspI, sspK and sspM genes are monocistronic, while sspO is the first gene in a likely operon with sspP (originally called cotL), which also encodes a putative very small protein . Transcription of these genes is primarily, if not exclusively, by RNA polymerase with the forespore-specific sigma factor, sigma(G) . Sequences centered 10 and 35nt upstream of the 5'-ends of sspI, sspK, sspM and sspO also show homology to the -10 and -35 sequences recognized by sigma(G) . Mutations deleting these genes cause the loss of the appropriate SASP from spores, and the sspK, sspM and sspO (and likely sspP) mutations had no discernable effect on sporulation, spore properties or spore germination . Loss of sspI also had no effect on sporulation, spore properties or spore germination, but DeltasspI spores had a significant defect in spore outgrowth.

Extremophiles, 2000 Apr, 4(2), 99 - 108
Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view; Takami H et al.; Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles . A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments . Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life . To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome . Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far . Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B . subtilis orthologues.

Biosci Biotechnol Biochem, 2000 Mar, 64(3), 652 - 6
Establishment of monoclonal antibodies specific for Bacillus subtilis DB9011; Asano Y et al.; Bacillus subtilis DB9011 is a strain with useful functions for agriculture . To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared . Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B . subtilis DB9011 vegetative cells are recognized by both MAbs . MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella . The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.

Biosci Biotechnol Biochem, 2000 Mar, 64(3), 484 - 91
Role of the DNA sequence downstream of the Bacillus subtilis hut promoter in regulation of the hut operon; Eda S et al.; To identify the role of the downstream region of a hut promoter in regulation of the Bacillus subtilis hut operon, three single-base substitutions (+9G-->A, +14C-->T, and +23T-->G) were introduced into the hut operon . Analysis of expression of the hut operon containing each of these three single-base substitutions and the hut-lacZ fusions with the single-base substitutions at position +14 showed that the position at +14 and probably the position at +23 were required for amino acid repression at the hut promoter, while the position at +14 was not required for catabolite repression at the hut promoter . The position at +9 was required for a histidine-dependent increase of activity of the hut promoter . Analysis of expression of the hut-lacZ fusions and the hut operon in the codY mutant indicated that the position at +14 and probably the position at +23 were involved in CodY-mediated amino acid repression at the hut promoter and that CodY was not required for catabolite repression at the hut promoter.

Appl Microbiol Biotechnol, 2000 Apr, 53(4), 430 - 4
Cloning of the cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp . A2-5a and analysis of the raw starch-binding domain; Ohdan K et al.; The cyclodextrin glucanotransferase (CGTase) gene of alkalophilic Bacillus sp . A2-5a was cloned and expressed in Bacillus subtilis ANA-1 as a host . The DNA region included an open reading frame encoding a 704-amino-acid polypeptide with a typical raw starch-binding motif in its C-terminal region . The CGTase purified from Bacillus sp . A2-5a bound to raw starch as strongly as porcine pancreas alpha-amylase, as expected from the sequence motif . A chromosomal region (a DNA fragment of about 14.1 kbp) including the CGTase gene was also cloned and the nucleotide sequence was determined . Possible cyclodextrinase and putative cyclodextrin-binding protein genes were found in the flanking region of the CGTase gene, which implied that the novel starch-degradation pathway postulated for a gram-negative bacterium {Klebsiella oxytoca; Fiedler et al . (1996) J Mol Biol 256: 279-291} also exists in a gram-positive bacterium i.e . Bacillus.

FEMS Microbiol Lett, 2000 May 15, 186(2), 313 - 7
Surface antigen, SpaA, of erysipelothrix rhusiopathiae binds to Gram-positive bacterial cell surfaces; Makino SI et al.; In a previous study, we isolated the spaA gene encoding the surface protective antigen A, SpaA, of Erysipelothrix rhusiopathiae, and found that the N-terminal region of SpaA was responsible for protective immunity against erysipelas and that the C-terminal region contained eight repeat units consisting of 20 amino acids comprising the binding domain on the Erysipelothrix cell surface . In this study, using recombinant SpaA proteins, we showed that the repeat region bound to the cell surfaces of various Gram-positive bacterial cells, SpaA was a membrane-associated protein, this association depended on the interaction with choline residues in teichoic acid, and SpaA bound to lipoteichoic acid (LTA) of Bacillus subtilis and Staphylococcus aureus . These results showed that LTA was required for the surface association of SpaA in E . rhusiopathiae and that such an association might be common among Gram-positive bacterial cells . We suggested that an LTA-SpaA complex might have an important role in the E . rhusiopathiae infection process.

J Microbiol Methods, 2000 May, 40(3), 241 - 54
Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B . subtilis and B . atrophaeus, closely related species of bacilli; Johnson YA et al.; Assessment of 16S-23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species . Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels . Closely-related species can have ISR PCR products that are similar in size . More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species . Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision . For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp) . Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics . B . subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168) . PCR products amplified from the ISR including the 5' terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS . A 119 or 120 bp PCR product was produced for B . atrophaeus strains . However, strains of B . subtilis subgroups W23 and 168 each produced 114 bp products . In summary, a mass spectrometry method was developed for differentiation of B . subtilis and B . atrophaeus . Also, the genetic similarity of B . subtilis subgroups W23 and 168 was confirmed . Accurate determination of the molecular weight of PCR products from the 16S-23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.

J Biol Chem, 2000 Jul 21, 275(29), 22025 - 30
A role for Asp75 in domain interactions in the Bacillus subtilis response regulator Spo0A; Cervin MA et al.; Spo0A is a two-domain response regulator required for sporulation initiation in Bacillus subtilis . Studies on response regulators have focused on the activity of each domain, but very little is known about the mechanism by which the regulatory domain inhibits the activator domain . In this study, we created a single amino acid substitution in the regulatory domain, D75S, which resulted in a dramatic decrease in sporulation in vivo . In vitro studies with the purified Spo0AD75S protein demonstrated that phosphorylation and DNA binding were comparable with wild type Spo0A . However, the mutant was unable to stimulate transcription by final sigma(A)-RNA polymerase from the Spo0A-dependent spoIIG operon promoter . We suggest that the amino acid Asp(75) and/or the region within which it resides, the alpha3-beta4 loop, are involved in the inhibitory interaction between the regulatory and activator domains of Spo0A.

J Biol Chem, 2000 May 12, 275(19), 14678 - 83
Specific recognition of parental terminal protein by DNA polymerase for initiation of protein-primed DNA replication; Gonzalez-Huici V et al.; The linear genome of Bacillus subtilis phage phi29 has a protein covalently linked to the 5' ends, called parental terminal protein (TP), and is replicated using a free TP as primer . The initiation of phage phi29 DNA replication requires the formation of a DNA polymerase/TP complex that recognizes the replication origins located at the genome ends . The DNA polymerase catalyzes the formation of the initiation complex TP-dAMP, and elongation proceeds coupled to strand displacement . The same mechanism is used by the related phage Nf . However, DNA polymerase and TP from phi29 do not initiate the replication of Nf TP-DNA . To address the question of the specificity of origin recognition, we took advantage of the initiation reaction enhancement in the presence of Mn(2+), allowing us to detect initiation activity in heterologous systems in which DNA polymerase, TP, and template TP-DNA are not from the same phage . Initiation was selectively stimulated when DNA polymerase and TP-DNA were from the same phage, strongly suggesting that specific recognition of origins is brought through an interaction between DNA polymerase and parental TP.

Mol Cell Probes, 2000 Apr, 14(2), 89 - 93
Labelled trinucleotides as quantitative probes to identify Bacillus spp . using fluorescent in situ hybridization; Abella CA et al.; The number of nucleotide triplet repeats in 16 S rRNA sequences can be used for detection and identification of bacteria . Labelled TTT, GGG and ATA triplets were hybridized to the ribonucleic acid of Bacillus subtilis and Bacillus fusiformis whole-cells and the number of such triplets was quantified by synchronous fluorescence spectrometry . Each species was distinctly identified by specific ratios of labelled TTT, GGG and ATA triplets as well as characteristic fluorescence spectra . Notwithstanding the absence of intrinsic specificity, fluorescein-conjugated nucleotide triplet probes appear to be a useful tool for fluorescent spectrometric identification of micro-organisms through the quantitation of trinucleotide repeats .

J Appl Microbiol, 2000 Apr, 88(4), 678 - 85
Inactivation of Bacillus subtilis spores on packaging surfaces by u . v . excimer laser irradiation; Warriner K et al.; Ultraviolet (u.v.) laser irradiation has been used to inactivate Bacillus subtilis spores deposited on to planar aluminium- and polyethylene-coated packaging surfaces . Kill kinetics were found to be diphasic, with an initial rapid inactivation phase followed by tailing . Although no definitive evidence was obtained, it is thought that spores located within packaging crevices/pores were primarily responsible for the observed tailing . Surviving spores were also found on the unexposed underside of cards and, to a lesser extent, within clumps . The log count reduction in B . subtilis was dependent on spore loading and total u.v . dose . In comparison, packaging surface composition, fluence (2-18 Jm-2) and frequency (40-150 Hz) had only a negligible effect . By irradiating boards carrying 106 spores, with a dose of 11.5 J cm-2, a log count reduction >5 was obtained . The mode of spore inactivation was primarily through DNA disruption . This was confirmed by the high sensitivity of spores lacking protective, small, acid-soluble proteins, in addition to the high frequency of auxotrophic and asporogenous mutations found amongst survivors.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 137 - 42
Biochemical and biophysical studies of Bacillus subtilis envelopes under hyperosmotic stress; Lopez CS et al.; The behaviour and state of the envelopes from B . subtilis cultures grown in Luria Bertani (LB) medium with and without 1.5 M NaCl are compared . Under hypertonic conditions, the hydrophobicity of the cultures increases . The phospholipid and fatty acid (FA) compositions show important differences: a higher cardiolipin (CL) content {at the expense of phosphatidylglycerol (PG)}, and a higher unsaturated and straight chain FA content . The fluidity of the membranes, determined with fluorescent probes, indicates an increase in viscosity of the cytoplasmic membrane . The consequences of these variations in membrane permeability and osmotolerance are discussed.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 27 - 31
Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase; Guzzo J et al.; Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation . To survive and grow in such a harsh environment as wine, O . oeni uses several mechanisms of resistance including stress protein synthesis . The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O . oeni of multiple regulation mechanisms as is the case in Bacillus subtilis . One common feature of these genes is that they are expressed under the control of housekeeping promoters . The expression of these genes as a function of growth is significantly different . Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth . In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons . Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.

J Biol Chem, 2000 May 5, 275(18), 13849 - 55
Combined action of two transcription factors regulates genes encoding spore coat proteins of Bacillus subtilis; Ichikawa H et al.; During sporulation of Bacillus subtilis, spore coat proteins encoded by cot genes are expressed in the mother cell and deposited on the forespore . Transcription of the cotB, cotC, and cotX genes by final sigma(K) RNA polymerase is activated by a small, DNA-binding protein called GerE . The promoter region of each of these genes has two GerE binding sites . 5' deletions that eliminated the more upstream GerE site decreased expression of lacZ fused to cotB and cotX by approximately 80% and 60%, respectively but had no effect on cotC-lacZ expression . The cotC-lacZ fusion was expressed later during sporulation than the other two fusions . Primer extension analysis confirmed that cotB mRNA increases first during sporulation, followed by cotX and cotC mRNAs over a 2-h period . In vitro transcription experiments suggest that the differential pattern of cot gene expression results from the combined action of GerE and another transcription factor, SpoIIID . A low concentration of GerE activated cotB transcription by final sigma(K) RNA polymerase, whereas a higher concentration was needed to activate transcription of cotX or cotC . SpoIIID at low concentration repressed cotC transcription, whereas a higher concentration only partially repressed cotX transcription and had little effect on cotB transcription . DNase I footprinting showed that SpoIIID binds strongly to two sites in the cotC promoter region, binds weakly to one site in the cotX promoter, and does not bind specifically to cotB . We propose that late in sporulation the rising level of GerE and the falling level of SpoIIID, together with the position and affinity of binding sites for these transcription factors in cot gene promoters, dictates the timing and level of spore coat protein synthesis, ensuring optimal assembly of the protein shell on the forespore surface.

Appl Environ Microbiol, 2000 May, 66(5), 2243 - 7
Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure; Woo IS et al.; Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions . However, no significant reduction of cell density was observed in either cell suspension . It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed . Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E . coli cells, yet there was no significant change observed in the B . subtilis cells . Microwave-injured E . coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B . subtilis cells were resistant to SDS.

Eur J Biochem, 2000 May, 267(9), 2609 - 16
Biophysical studies of the development of amyloid fibrils from a peptide fragment of cold shock protein B; Wilkins DK et al.; The peptide CspB-1, which represents residues 1-22 of the cold shock protein CspB from Bacillus subtilis, has been shown to form amyloid fibrils when solutions containing this peptide in aqueous (50%) acetonitrile are diluted in water {M . Gross et al . (1999) Protein Science 8, 1350-1357} We established conditions in which reproducible kinetic steps associated with the formation of these fibrils can be observed . Studies combining these conditions with a range of biophysical methods reveal that a variety of distinct events occurs during the process that results in amyloid fibrils . A CD spectrum indicative of beta structure is observed within 1 min of the solvent shift, and its intensity increases on a longer timescale in at least two kinetic phases . The characteristic wavelength shift of the amyloid-binding dye Congo Red is established within 30 min of the initiation of the aggregation process and corresponds to one of the phases observed by CD and to changes in the Fourier transform-infrared spectrum indicative of beta structure . Short fibrillar structures begin to be visible under the electron microscope after these events, and longer, well-defined amyloid fibrils are established on a timescale of hours . NMR spectroscopy shows that there are no significant changes in the concentration of monomeric species in solution during the events leading to fibril formation, but that soluble aggregates too large to be visible in NMR spectra are present throughout the process . A model for amyloid formation by this peptide is presented which is consistent with these kinetic data and with published work on a variety of disease-related systems . These findings support the concept that the ability to form amyloid fibrils is a generic property of polypeptide chains, and that the mechanism of their formation is similar for different peptides and proteins.

Microbiology, 2000 Apr, 146 ( Pt 4), 823 - 8
RT-PCR as a tool for systematic transcriptional analysis of large regions of the Bacillus subtilis genome; Hernandez A et al.; Transcriptional analysis of five different regions of the Bacillus subtilis 168 genome, comprising a total of 175 kb encoding newly identified genes, was carried out using the RT-PCR technique as part of the functional analysis of the whole genome of this bacterium . Amplification of mRNA fragments allowed the detection of both highly and poorly transcribed genes covering 81% of putative ORFs, and also the monitoring of variations in the expression level among genes differentially expressed during particular bacterial growth phases.

Microbiology, 2000 Apr, 146 ( Pt 4), 815 - 21
The Bacillus subtilis cysP gene encodes a novel sulphate permease related to the inorganic phosphate transporter (Pit) family; Mansilla MC et al.; Sulphate permeases in the plasma membrane are responsible for uptake of environmental sulphate used in the sulphate assimilation pathway in bacteria and plants . Here it is reported that the ORF designated cysP, located on the Bacillus subtilis chromosome between cysH and five putative genes involved in sulphate assimilation, encodes a sulphate permease . cysP is able to complement Escherichia coli cysteine auxotrophs with mutations affecting either the membrane or periplasmic components of the sulphate-thiosulphate permease . Transport studies with cell suspensions of a cysA97 E . coli strain transformed with a plasmid expressing the B . subtilis cysP gene indicated that CysP catalyses sulphate uptake . Analysis of the primary sequence showed that CysP (354 amino acids, estimated molecular mass 24 kDa) is a highly hydrophobic protein which has 11 putative transmembrane helices . Sequence comparisons revealed that CysP, together with the phosphate permease of Neurospora crassa, Pho-4, and E . coli PitA, belongs to the family of related transporters, the inorganic phosphate transporter (Pit) family . Among the putative phosphate permeases, CysP shows a similar size and the same domain organization as the archaeal transporters . This is the first report of a sulphate permease in a Gram-positive organism.

Microbiology, 2000 Apr, 146 ( Pt 4), 807 - 14
The yexA gene product is required for phosphoribosylformylglycinamidine synthetase activity in Bacillus subtilis; Saxild HH et al.; The yexA gene encodes an 84 amino acid reading frame; in Bacillus subtilis it is positioned between the purC and purQ genes of the purine biosynthetic operon . Disruption of yexA resulted in a purine-auxotrophic phenotype . When yexA was expressed in trans it was able to complement a yexA mutation . Growth experiments and enzyme analysis of yexA mutant strains revealed a defective phosphoribosylformylglycinamidine synthetase (FGAM synthetase) . In the organisms in which FGAM synthetase has been studied a single polypeptide is responsible for activity . In some organisms two separate genes - in B . subtilis the purL and purQ genes - encode polypeptides with similarity to the N-terminal and the C-terminal region, respectively, of the single-polypeptide FGAM synthetase . Thus, active FGAM synthetase in B . subtilis requires the yexA gene product in addition to the purL and purQ gene products . Open reading frames with sequence similarity to yexA are found in other Gram-positive organisms, in a cyanobacterium and in methanogenic archaea . The designation purS is proposed for this novel function in purine biosynthesis in B . subtilis.

Microbiology, 2000 Apr, 146 ( Pt 4), 797 - 805
Incorporation of {2-3H}glycerol into cell surface components of Bacillus subtilis 168 and thermosensitive mutants affected in wall teichoic acid synthesis: effect of tunicamycin; Pooley HM et al.; A method is described for measuring the synthesis of poly(glycerol phosphate) {poly(groP)}, the major wall teichoic acid (WTA), lipoteichoic acid (LTA) and phospholipid (P-lipid), through fractionation of {2-3H}glycerol ({2-3H}gro)-labelled Bacillus subtilis cells . When cultures of certain temperature-sensitive mutants defective in one of several tag genes, encoding enzymes involved in WTA synthesis, were transferred to the restrictive temperature, the synthesis of WTA underwent a specific, immediate, block, while that of LTA or P-lipid proceeded unimpeded . These results, in addition to confirming the role of tag genes, demonstrated, reciprocally, the specificity of the fractionation procedure used to distinguish label in WTA from that in LTA or P-lipid . Results of analysis of other, less severely affected, tag-deficient mutants, as well as of another genetically unrelated mutant developing comparable morphological phenotypes in non-permissive conditions, are discussed in relation to a possible mechanism generating the latter phenotype . Fractionation of B . subtilis 168 cells labelled either with {2-3H}gro or with {1-14C}N-acetylglucosamine, to which tunicamycin was added at 0.5 microg ml(-1) (the MIC) revealed a specific and marked inhibition of poly(groP) as well as of poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate), the minor WTA . However, for 60 min at least, the syntheses of PG, LTA and P-lipid were barely affected.

Phytochemistry, 2000 Apr, 53(7), 723 - 31
Biosynthesis of riboflavin in plants . The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase; Herz S et al.; A cDNA segment from Arabidopsis thaliana with similarity to the ribA gene of Bacillus subtilis was sequenced . A similar gene was cloned from tomato . The open reading frame of A . thaliana was fused to the malE gene of Escherichia coli and was expressed in a recombinant E . coli strain . The recombinant fusion protein was purified and shown to have GTP cyclohydrolase II activity as well as 3,4-dihydroxy-2-butanone 4-phosphate synthase activity . The cognate gene was amplified by polymerase chain reaction from chromosomal Arabidopsis DNA and was shown to contain six introns . Intron 4 is located in the region connecting the GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase domain of the putative domains catalyzing the two reaction steps . By comparison with the bacterial ribA gene, the Arabidopsis gene contains an additional 5' element specifying about 120 amino acid residues . This segment contains numerous serine and threonine residues and does not show similarity with other known sequences . The N-terminal segment is not required for catalytic activity and is likely to serve as signal sequence for import into chloroplasts.

Farmaco, 2000 Feb, 55(2), 151 - 7
Synthesis and antimicrobial activity of (E)-acetoxystilbenes and alpha,alpha'-dibromoacetoxybibenzyls; Wyrzykiewicz E et al.; The synthesis of 13 new (E)-acetoxystilbenes and alpha,alpha'-dibromoacetoxybibenzyls and their antimicrobial activity are reported . The results of microbiological screening of 17 (E)-stilbenols and (E)-acetoxystilbenes, unknown in the literature, have also been discussed . In particular, coumpounds 1c, 1g, 2a, 3a, 3b, 4a, 6a, 6b showed good antibacterial activity against Staphylococcus aureus and 1c also against Bacillus subtilis.

J Bacteriol, 2000 May, 182(10), 2989 - 91
Control of initiation of sporulation by replication initiation genes in Bacillus subtilis; Lemon KP et al.; Initiation of spore formation in Bacillus subtilis appears to depend on initiation of DNA replication . This regulation was first identified using a temperature-sensitive mutation in dnaB . We found that mutations in the replication initiation genes dnaA and dnaD also inhibit sporulation, indicating that inhibition of sporulation is triggered by general defects in the function of replication initiation proteins.

J Bacteriol, 2000 May, 182(10), 2970 - 2
Complete nucleotide sequence of Tn10; Chalmers R et al.; The complete nucleotide sequence of Tn10 has been determined . The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit . The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis . The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.

J Bacteriol, 2000 May, 182(10), 2919 - 27
A dispensable role for forespore-specific gene expression in engulfment of the forespore during sporulation of Bacillus subtilis; Sun YL et al.; During the stage of engulfment in the Bacillus subtilis spore formation pathway, the larger mother cell engulfs the smaller forespore . We have tested the role of forespore-specific gene expression in engulfment using two separate approaches . First, using an assay that unambiguously detects sporangia that have completed engulfment, we found that a mutant lacking the only forespore-expressed engulfment protein identified thus far, SpoIIQ, is able to efficiently complete engulfment under certain sporulation conditions . However, we have found that the mutant is defective, under all conditions, in the expression of the late-forespore-specific transcription factor sigma(G); thus, SpoIIQ is essential for spore production . Second, to determine if engulfment could proceed in the absence of forespore-specific gene expression, we made use of a strain in which activation of the mother cell-specific sigma factor sigma(E) was uncoupled from forespore-specific gene expression . Remarkably, engulfment occurred in the complete absence of sigma(F)-directed gene expression under the same conditions permissive for engulfment in the absence of SpoIIQ . Our results demonstrate that forespore-specific gene expression is not essential for engulfment, suggesting that the machinery used to move the membranes around the forespore is within the mother cell.

J Bacteriol, 2000 May, 182(10), 2845 - 54
Efficient spore synthesis in Bacillus subtilis depends on the CcdA protein; Schiott T et al.; CcdA is known to be required for the synthesis of c-type cytochromes in Bacillus subtilis, but the exact function of this membrane protein is not known . We show that CcdA also plays a role in spore synthesis . The expression of ccdA and the two downstream genes yneI and yneJ was analyzed . There is a promoter for each gene, but there is only one transcription terminator, located after the yneJ gene . The promoter for ccdA was found to be weak and was active mainly during the transition from exponential growth to stationary phase . The promoters for yneI and yneJ were both active in the exponential growth phase . The levels of the CcdA and YneJ proteins in the membrane were consistent with the observed promoter activities . The ccdA promoter activity was independent of whether the ccdA-yneI-yneJ gene products were absent or overproduced in the cell . It is shown that the four known cytochromes c in B . subtilis and the YneI and YneJ proteins are not required for sporulation . The combined data from analysis of sporulation-specific sigma factor activity, resistance properties of spores, and spore morphology indicate that CcdA deficiency affects stage V in sporulation . We conclude that CcdA, YneI, and YneJ are functionally unrelated proteins and that the role of CcdA in cytochrome c and spore synthesis probably relates to sulfhydryl redox chemistry on the outer surface of the cytoplasmic membrane.

J Bacteriol, 2000 May, 182(10), 2771 - 7
The Bacillus subtilis GTP binding protein obg and regulators of the sigma(B) stress response transcription factor cofractionate with ribosomes; Scott JM et al.; Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of the sigma(B) transcription factor . We investigated Obg's cellular associations by differential centrifugation of crude B . subtilis extracts, using an anti-Obg antibody as a probe to monitor Obg during the fractionation, and by fluorescent microscopy of a B . subtilis strain in which Obg was fused to green fluorescent protein . The results indicated that Obg is part of a large cytoplasmic complex . In subsequent analyses, Obg coeluted with ribosomal subunits during gel filtration of B . subtilis lysates on Sephacryl S-400 and specifically bound to ribosomal protein L13 in an affinity blot assay . Probing the gel filtration fractions with antibodies specific for sigma(B) and its coexpressed regulators (Rsb proteins) revealed coincident elution of the upstream components of the sigma(B) stress activation pathway (RsbR, -S, and -T) with Obg and the ribosomal subunits . The data implicate ribosome function as a possible mediator of the activity of Obg and the stress induction of sigma(B).

Nucleic Acids Symp Ser, 1999, (42), 209 - 10
Search for a selenocysteine tRNA in Bacillus subtilis; Matsugi J et al.; Activity to convert serine to selenocysteine in B . subtilis was studied but no activity was detected . In addition, although we tried to find its selenocysteine tRNA (tRNA(SeCys)) gene from a total genome sequence (1) by the computer search with FASTA against E . coli selC (2), no convincing candidate was found . These results suggest that in B . subtilis, selenium-related system is considerably different from known one like E . coli.

FEMS Microbiol Lett, 2000 May 1, 186(1), 1 - 9
The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences; Studholme DJ et al.; The bacterial transcription factor sigma(N) (sigma-N, sigma-54, RpoN) confers upon RNA polymerase (RNAP) properties distinct from those of the major house-keeping form of RNAP, which contains sigma(70) (sigma-70, RpoD) . Transcription by RNAP containing sigma(N) is subject to enhancer-dependent regulation . Far from being an 'oddity' or 'exception to the rule', the occurrence of sigma(N) in the genome sequences of such diverse bacteria as Aquifex aeolicus, Bacillus subtilis, Chlamydia spp . and Borrelia burgdorferi argues for its biological importance . The availability of complete genome sequences of several (eu)bacteria offers an opportunity to extend our understanding of this special form of transcriptional regulation . By scanning their genome sequences, new functions have been predicted for enhancer-dependent transcription in A . aeolicus, Chlamydia trachomatis, Escherichia coli, Treponema pallidum and B . burgdorferi.

Mikrobiologiia, 2000 Mar-Apr, 69(2), 270 - 5
{The study of heterogeneity of plasmid-bearing and plasmid-f ree populations of Bacillus subtilis under different environmental conditions}; Krylova TIu et al.; The population heterogeneity of recombinant and plasmid-free Bacillus subtilis strains introduced into aquatic microcosms was studied . After introduction, the population of the plasmid-free strain B . subtilis 2335 in microcosms has long been represented by both vegetative cells and spores, whereas, already ten days after introduction, the population of the recombinant strain B . subtilis 2335/105 (Km{symbol: see text}nf+) was represented only by spores . The number of plasmid copies in the spore isolates of the recombinant strain was the same as before introduction, but the plasmid abundance in the vegetative isolates of this strain decreased . The isolates of B . subtilis 2335/105 obtained from microcosms and the variants of this strain obtained by ten successive subcultures on M9 and 0.1 x M9 media with and without kanamycin (Km) differed in the number of plasmid copies, Km resistance, and maximum biomass yield during batch cultivation . Irrespective of the presence of Km, more than 50% of the variants subcultured on M9 medium showed reduced plasmid abundance . At the same time, about 70% of the variants subcultured on 0.1 x M9 medium with Km and 90% of the variants subcultured on the same medium without Km retained the initial number of plasmid copies . The variants subcultured on media with Km retained the initial biomass level . In more than 70% of the variants isolated from media without Km, the biomass yield increased.

Mikrobiologiia, 2000 Mar-Apr, 69(2), 243 - 7
{Factor of salinity and adaptive capacity of recombinant strains of Escherichia coli and Bacillus subtilis}; Boiandin AN et al.; Effect of different concentrations of salts on natural and recombinant strains of Bacillus subtilis and Escherichia coli was studied . The recombinant strain of B . subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains of E . coli . Some salts exerted a bacteriostatic effect on E . coli and B . subtilis . The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells . The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.

Protein Eng, 2000 Mar, 13(3), 197 - 200
Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus; Manco G et al.; The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family . Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad . In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated . The model reveals the topological organization of the fold corroborating our predictions . As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances . The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the {(3)H}diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.

Arch Biochem Biophys, 2000 May 1, 377(1), 22 - 30
Characterization of a structural model of membrane bound cytochrome c-550 from Bacillus subtilis; David PS et al.; A structural model of Bacillus subtilis cytochrome c-550 has been built based upon hydropathy analysis, sequence alignment, homology modeling, and energy minimization . The model has a single transmembrane alpha-helix and a water-soluble domain folded around covalently attached heme C . Physical measurements on purified, recombinant cytochrome c-550 have been made to test aspects of the model . Excitation at either 280 or 295 nm yields fluorescence with an emission maximum at 334 nm and a quantum yield of 25% relative to n-acetyltryptophanamide . The model places one (i.e., W115) of the two tryptophans of cytochrome c-550 in the heme domain and the second (i.e., W3) in the transmembrane domain . The indole ring of W115 is within 5 A of the heme macrocycle and is expected to be highly quenched via resonance energy transfer to the heme . In contrast, W3 is at the start of the putative transmembrane helix and could be located a considerable distance from the heme . Forster theory assigns a distance of 42 A from W3 to the heme . This distance is important in adjusting the relative positions of the membrane-spanning and heme-binding domains . Circular dichroism measurements in the ultraviolet region indicate increased alpha-helical content of B . subtilis cytochrome c compared to mitochondrial cytochrome c in support of an alpha-helical transmembrane domain . The ionic strength dependence of redox kinetics for cytochrome c-550 indicates an overall negative charge that is consistent with a calculated pI of 5.4 . However, the charge distribution specified by the model indicates a surface for electron exchange that is different from the classical front face used by mitochondrial cytochrome c .

Mol Cells, 2000 Feb 29, 10(1), 102 - 7
Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli; Kim SI et al.; To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully . For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E . coli and B . subtilis . Except GST fusion system in E . coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression . The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form . From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment . The purified EF-Tu showed high GDP binding activity . These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B . subtilis EF-Tu.

Infect Immun, 2000 May, 68(5), 2655 - 62
Pathogenic mycobacteria disrupt the macrophage actin filament network; Guerin I et al.; Phagosomes with pathogenic mycobacteria retain fusion and intermingling characteristics of early endosomes indefinitely . The time course of acquisition of newly endocytosed tracers becomes, however, atypical (lag instead of immediate acquisition) starting from day 1 postinfection (p.i.), thereby suggesting that additional factors affect this process . Disruption of the actin filament (F-actin) network by cytochalasin D perturbs the movement of early endosomes and probably fusion events among early endosomes and phagosomes . Here we compare, by immunofluorescence microscopy, the morphology and distribution of F-actin in macrophages infected with virulent Mycobacterium avium, in uninfected macrophages, or in macrophages after phagocytosis of nonpathogenic bacteria (Mycobacterium smegmatis or Bacillus subtilis) or hydrophobic latex particles . In uninfected cells, F-actin appeared as a network of small filaments distributed throughout the cell; about 80% of the cells also displayed one or two small patches of F-actin at the cell periphery . Virulent M . avium caused a marked disorganization of the F-actin network starting from day 1 p.i . The most salient features were the formation of several large patches, the progressive disappearance of the small filaments, and the appearance of large numbers of tiny punctate structures starting from day 2 p.i . With the three other particles, the F-actin network was unmodified compared to that in uninfected cells . The atypical lag in acquisition of newly endocytosed tracers by M . avium-containing phagosomes, therefore, seems to coincide with the disorganization of the F-actin network.

Gene, 2000 Apr 4, 246(1-2), 187 - 96
Characterization of ywhE, which encodes a putative high-molecular-weight class A penicillin-binding protein in Bacillus subtilis; Pedersen LB et al.; The Bacillus subtilis genome sequencing project {Kunst et al., Nature 390 (1997) 249-256} identified ywhE as a gene that potentially encodes a high-molecular-weight class A penicillin-binding protein . Analysis of the expression of a translational ywhE-lacZ fusion showed that ywhE expression is sporulation-specific, and is controlled predominantly by the forespore-specific sigma factor sigma(F), and to a lesser extent by sigma(G) . Primer extension analysis identified two transcription start sites located 26 and 27 nucleotides upstream of the ywhE translational initiation codon . Sequences located in the -10 and -35 regions relative to the transcription start sites showed good homology to the consensus sequences for promoter elements of sigma(F)-dependent genes . An insertional mutation in ywhE had no significant effect on growth, morphology, and sporulation, and ywhE spores had normal heat-resistance, cortex structure, and germination and outgrowth properties . However, overexpression of ywhE in Escherichia coli resulted in cell lysis.

J Mol Biol, 2000 Apr 14, 297(5), 1031 - 6
Divergence in macromolecular assembly: X-ray crystallographic structure analysis of lumazine synthase from Brucella abortus; Braden BC et al.; We have determined the three-dimensional structure of 6, 7-dimethyl-8-ribityllumazine synthase (lumazine synthase) from Brucella abortus, the infectious organism of the disease brucellosis in animals . This enzyme catalyses the formation of 6, 7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboflavin . The three-dimensional X-ray crystal structure of the enzyme from B . abortus has been solved and refined at 2.7 A resolution to a final R-value of 0.18 (R(free)=0.23) . The macromolecular assembly of the enzyme differs from that of the enzyme from Bacillus subtilis, the only other lumazine synthase structure known . While the protein from B . subtilis assembles into a 60 subunit icosahedral capsid built from 12 pentameric units, the enzyme from B . abortus is pentameric in its crystalline form . Nonetheless, the active sites of the two enzymes are virtually identical indicating inhibitors to theses enzymes could be effective pharmaceuticals across a broad species range . Furthermore, we compare the structures of the enzyme from B . subtilis and B . abortus and describe the C teminus structure which accounts for the differences in quaternary structure .

J Bacteriol, 2000 May, 182(9), 2664 - 7
Expression of UGA-containing Mycoplasma genes in Bacillus subtilis; Kannan TR et al.; We used Bacillus subtilis to express UGA-containing Mycoplasma genes encoding the P30 adhesin (one UGA) of Mycoplasma pneumoniae and methionine sulfoxide reductase (two UGAs) of Mycoplasma genitalium . Due to natural UGA suppression, these Mycoplasma genes were expressed as full-length protein products, but at relatively low efficiency, in recombinant wild-type Bacillus . The B . subtilis-expressed Mycoplasma proteins appeared as single bands and not as multiple bands compared to expression in recombinant Escherichia coli . Bacillus mutants carrying mutations in the structural gene (prfB) for release factor 2 markedly enhanced the level of readthrough of UGA-containing Mycoplasma genes.

J Bacteriol, 2000 May, 182(9), 2639 - 42
The yvaJ gene of Bacillus subtilis encodes a 3'-to-5' exoribonuclease and is not essential in a strain lacking polynucleotide phosphorylase; Oussenko IA et al.; Studies of Bacillus subtilis RNases that are involved in mRNA degradation reveal a different pattern from that of Escherichia coli . A strain lacking polynucleotide phosphorylase, the major 3'-to-5' exoribonuclease activity in cell extracts, is viable . Here, we show that the B . subtilis yvaJ gene encodes a second 3'-to-5' exoribonuclease . A strain lacking both of these RNases grows slowly but is viable . The existence of another, as yet unknown, 3'-to-5' exoribonuclease in B . subtilis is suggested.

J Bacteriol, 2000 May, 182(9), 2513 - 9
Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis; Paidhungat M et al.; Dormant Bacillus subtilis spores germinate in the presence of particular nutrients called germinants . The spores are thought to recognize germinants through receptor proteins encoded by the gerA family of operons, which includes gerA, gerB, and gerK . We sought to substantiate this putative function of the GerA family proteins by characterizing spore germination in a mutant strain that contained deletions at all known gerA-like loci . As expected, the mutant spores germinated very poorly in a variety of rich media . In contrast, they germinated like wild-type spores in a chemical germinant, a 1-1 chelate of Ca(2+) and dipicolinic acid (DPA) . These observations showed that proteins encoded by gerA family members are required for nutrient-induced germination but not for chemical-triggered germination, supporting the hypothesis that the GerA family encodes receptors for nutrient germinants . Further characterization of Ca(2+)-DPA-induced germination showed that the effect of Ca(2+)-DPA on spore germination was saturated at 60 mM and had a K(m) of 30 mM . We also found that decoating spores abolished their ability to germinate in Ca(2+)-DPA but not in nutrient germinants, indicating that Ca(2+)-DPA and nutrient germinants probably act through parallel arms of the germination pathway.

J Bacteriol, 2000 May, 182(9), 2411 - 5
Natural genetic competence in Bacillus subtilis natto OK2; Ashikaga S et al.; We isolated a Bacillus subtilis natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using a comG-lacZ fusion at the amyE locus . Although transcription of the late competence genes was not detected in the B . subtilis natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the mecA or the clpC mutation derived from B . subtilis 168 . In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for B . subtilis 168 . Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes . These results clearly indicated that ComK produced in both the mecA and clpC mutants of strain OK2 (ComK(OK2)) could activate the transcription of the whole set of late competence genes and suggested that ComK(OK2) was not activated in strain OK2 during competence development . We therefore sequenced the comS gene of OK2 and compared it with that of 168 . The comS(OK2) had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.

J Bacteriol, 2000 May, 182(9), 2387 - 92
A new IS4 family insertion sequence, IS4Bsu1, responsible for genetic instability of poly-gamma-glutamic acid production in Bacillus subtilis; Nagai T et al.; Certain Bacillus subtilis strains, such as B . subtilis (natto) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-gamma-glutamate (gammaPGA) . In B . subtilis (natto), gammaPGA synthesis is controlled by the ComP-ComA two-component regulatory system and thereby induced at the beginning of the stationary growth phase . We have found a new insertion sequence (IS), designated IS4Bsu1, in the comP gene of a spontaneous gammaPGA-negative mutant of B . subtilis (natto) NAF4 . IS4Bsu1 (1,406 bp), the first IS discovered in B . subtilis, encodes a putative transposase (Tpase) with a predicted M(r) of 34,895 (374 residues) which displays similarity to the Tpases of IS4 family members . Southern blot analyses have identified 6 to 11 copies of IS4Bsu1, among which 6 copies were at the same loci, in the chromosomes of B . subtilis (natto) strains, including NAF4, three commercial starters, and another three gammaPGA-producing B . subtilis (natto) strains . All of the eight spontaneous gammaPGA(-) mutants, which were derived from five independent NAF4 cultures, had a new additional IS4Bsu1 copy in comP at six different positions within 600 bp of the 5'-terminal region . The target sites of IS4Bsu1 were determined to be AT-rich 9-bp sequences by sequencing the flanking regions of IS4Bsu1 in mutant comP genes . These results indicate that IS4Bsu1 transposes by the replicative mechanism, in contrast to other IS4 members that use the conservative mechanism, and that most, if not all, of spontaneous gammaPGA(-) mutants appear to have resulted from the insertion of IS4Bsu1 exclusively into comP . The presence of insertion hot spots in comP, which is essential for gammaPGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous gammaPGA(-) mutation by IS4Bsu1 in B . subtilis (natto).

J Mol Biol, 2000 Apr 21, 298(1), 7 - 20
The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro; Chedin F et al.; The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli . In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences . Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex . During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site . Therefore, we conclude that 5'-AGCGG-3' is the B . subtilis Chi site and it is hereafter referred to as chi(Bs) . After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist . Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process .

Biotechnol Prog, 2000 Mar-Apr, 16(2), 169 - 75
13C NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis; Phalakornkule C et al.; When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2-fold, and the productivity of recombinant protein increases . It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity . To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed . Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label . "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities) . When experiments were performed with 1-(13)C glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario . This evidence further fortifies the prospects for successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities.

Biochemistry, 2000 Apr 11, 39(14), 4037 - 45
Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship; Epperson JD et al.; Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms . This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II) . Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined . We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe . The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms . The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions . The free amine of Ile-1 does not bind Co(II) . Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode . For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical . However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.

EMBO J, 2000 Apr 3, 19(7), 1467 - 75
Direct interaction between the cell division protein FtsZ and the cell differentiation protein SpoIIE; Lucet I et al.; SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate in Bacillus subtilis . First, SpoIIE is needed for the normal formation of the asymmetrically positioned septum that forms early in sporulation and separates the mother cell from the prespore compartment . Secondly, SpoIIE is essential for the activation of the first compartment-specific transcription factor sigma(F) in the prespore . After initiation of sporulation, SpoIIE localizes to the potential asymmetric cell division sites near one or both cell poles . Localization of SpoIIE was shown to be dependent on the essential cell division protein FtsZ . To understand how SpoIIE is targeted to the asymmetric septum we have now analysed its interaction with FtsZ in vitro . Using the yeast two-hybrid system and purified FtsZ, and full-length and truncated SpoIIE proteins, we demonstrate that the two proteins interact directly and that domain II and possibly domain I of SpoIIE are required for the interaction . Moreover, we show that SpoIIE interacts with itself and suggest that this self-interaction plays a role in assembly of SpoIIE into the division machinery.

Microbiology, 2000 Mar, 146 ( Pt 3), 731 - 9
Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells . European Listeria Genome Consortium; Milohanic E et al.; Insertional mutagenesis was performed with Tn1545 in the genetic background of an inIAB deletion mutant to identify new adhesion determinants in Listeria monocytogenes . Four insertion mutants defective in adhesion to eukaryotic cells were identified . Insertion sites were cloned by inverse-PCR and sequenced . The genetic organization of insertion regions was further analysed by screening and sequencing DNA fragments from a HindIII library and by searching databases . Three adhesion-defective mutants each had one copy of Tn1545 inserted into their chromosome . The insertion sites were different in the three mutants: (i) upstream from two ORFs in tandem, similar to dfp and priA of Bacillus subtilis, respectively; (ii) within an ORF encoding a putative 126 amino-acid-polypeptide with no significant similarity to any known protein; (iii) within an ORF similar to a B . subtilis ORF with no known function, just upstream from an operon similar to an ABC (ATP-binding cassette) transporter operon from B . subtilis . The excisants obtained from these mutants using the excision reporter plasmid pTCR9 recovered full adhesion capacity . A fourth mutant was the most severely defective in adhesion . It had five Tn1545 insertions, one of which was upstream from dfp and priA, and another of which was upstream from ami, a gene encoding a surface-exposed autolysin with a C terminus similar to that of InIB . Ami was clearly involved because an ami null mutant constructed in an EGDdeltainIA-F background was adhesion-defective . Thus new regions involved in the adhesion of L . monocytogenes to eukaryotic cells were identified . Further study is required to define more accurately the roles of these regions in the adhesion process itself.

Microbiology, 2000 Mar, 146 ( Pt 3), 659 - 68
Molecular characterization of the ferric-uptake regulator, fur, from Staphylococcus aureus; Xiong A et al.; Iron is an essential nutrient for the survival and pathogenesis of bacteria, but relatively little is known regarding its transport and regulation in staphylococci . Based on the known sequences of ferric-uptake regulatory (fur) genes from several Gram-positive and Gram-negative bacteria, a fragment containing the fur homologue was cloned from a genomic library of Staphylococcus aureus RN450 . Nucleotide sequence analysis of this fragment revealed the presence of a 447 bp ORF that encodes a putative 149 aa polypeptide with an apparent molecular mass of 17 kDa . A putative ferrichrome-uptake (fhu) operon, containing the conserved Fur-binding sequences (Fur box) in the promoter region, was also cloned from the same S . aureus library . To characterize the impact of Fur on the fhu operon, fur was cloned, overexpressed as a His-tagged protein and purified by Ni2+-affinity column chromatography . The recombinant protein was digested with enterokinase to remove the His tag . Electrophoretic mobility-shift assays indicated that Fur binds to the promoter region of the fhu operon in the presence of divalent cations . Fur also interacted with the promoter region of the recently reported sir operon that has been proposed to constitute a siderophore-transport system in S . aureus . The DNase I-protection assay revealed that Fur specifically binds to the Fur box located in the promoter region of the fhu operon . The primer-extension reaction indicated that the transcription-start site of the fhu operon was located inside the Fur box . S . aureus fur partially complemented a fur- mutation in Bacillus subtilis . The data suggest that Fur regulates iron-transport processes in S . aureus.

Microbiology, 2000 Mar, 146 ( Pt 3), 573 - 9
Systematic study of gene expression and transcription organization in the gntZ-ywaA region of the Bacillus subtilis genome; Yoshida K et al.; Within the framework of the international project 'The functional analysis of the Bacillus subtilis genome' in Japan and Europe, the gene expression and transcription organization of the gntZ-ywaA region (160 kb) of the B . subtilis genome has been systematically analysed . First, all unanalysed genes comprising more than 80 amino acids (125 genes) in this region were inactivated through integration of plasmid pMUTIN . No essential gene was found which could not be inactivated . All the integrants grew normally in both nutrient sporulation medium and glucose minimal medium . But an integrant in the yxbG gene exhibited an oligosporogenic phenotype in the nutrient sporulation medium . The synthesis of beta-galactosidase was examined, as a reporter for expression of the inactivated genes, during growth and sporulation in the two media . The results indicated that 36% of the promoters were inactive when cells were grown in at least one of these two media . Furthermore, the transcription of the 119 genes in this region was analysed by Northern blotting, resulting in a transcription map . The results indicate that the gntZ-ywaA region contains at least 24 polycistronic operons, including several published ones . The operons newly found in this work are yxaAB, yxaGH, yxaJKL, yxbBA-yxnB-asnH-yxaM, yxbCD, yxcED, yxdJK, yxeFGH, yxeKLMNOPQ, yxeR-yxxB, hutPHUIGM, bgIPH-yxiE, wapA-yxxG, yxiM-deaD, katB-yxiS, yxjCDEF, yxjJI and yxkF-mmsX.

Analyst, 1999 Nov, 124(11), 1599 - 604
Dipicolinic acid (DPA) assay revisited and appraised for spore detection; Hindle AA et al.; Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores . DPA was assayed by fluorimetric detection of its chelates with lanthanide metals . The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature . The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA . This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA . A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests.

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