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 an