Bacillus subtilis

Microbiol Immunol, 2001, 45(10), 673 - 8
Bacterial arylsulfate sulfotransferase as a reporter system; Yun HJ et al.; To investigate whether the arylsulfate sulfotransferase (ASST) is suitable as a reporter system for monitoring gene expression, a reporter vector carrying the fragments of the astA coding region without the promoter region was constructed and designated as pSY815 . As a test of the ASST reporter system's suitability, the regulatory regions of ermC and lacZ were inserted upstream of the coding region of the reporter gene to generate pSY815-EC and pSY815-LZ, respectively . In the absence of the inserted regulatory regions, the plasmids displayed very low background activities in Bacillus subtilis and Escherichia coli . The ASST activity under the control of the ermC regulatory region was increased 4.4-fold in B . subtilis when induced by 0.1 microgml(-1) of erythromycin . These results were consistent with a lacZ reporter gene assay of the ermC regulatory region . Furthermore, we confirmed that the lacZ promoter in E . coli was strongly induced to a 17.9-fold increase by 0.05 mM of isopropyl-beta-D-thiogalactopyranoside (IPTG) in this reporter system . These results indicate that the ASST is a suitable reporter system . The lack of endogenous activity, the simple detection of enzyme activity in the living cell, the commercially available non-toxic substrates, and the high sensitivity make ASST a useful genetic reporter system for monitoring gene expression and understanding gene regulation.

J Protein Chem, 2001 Aug, 20(6), 501 - 6
Structure of the Bacillus subtilis peptide antibiotic subtilosin A determined by 1H-NMR and matrix assisted laser desorption/ionization time-of-flight mass spectrometry; Marx R et al.; Subtilosin A produced by Bacillus subtilis is a macrocyclic peptide antibiotic which comprises 35 amino acids . Its molecular mass (3399.7 Da), determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and chemical properties gave experimental support for unusual intramolecular linkages . The three-dimensional fold of native subtilosin in dimethylsulfoxide was determined from two-dimensional 1H-NMR spectra recorded at 600 MHz . Based on the backbone conformation, a structure for subtilosin A is presented which is characterized by three inter-residue bridges where two cysteines are linked with two phenylalanine residues, respectively, and a third cysteine is bound to a threonine residue.

Appl Microbiol Biotechnol, 2001 Oct, 57(3), 379 - 84
Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor; Espinosa-de-los-Monteros J et al.; Cultures using nitrate as the terminal electron acceptor were conducted in Schaeffer's medium to evaluate the growth performance and metabolic profiles of Bacillus subtilis, and its potential to express the aprE (subtilisin) gene under anoxic conditions . Nitrate was converted to ammonia through nitrite reduction; and different product profiles were observed during the growth phase when nitrate was added at various concentrations (4-24 mM) to Schaeffer's medium containing glucose (4 g l(-1)) . If nitrate was not limiting, then acetic acid and acetoin were accumulated, suggesting a limitation of reduced cofactors but, if nitrate became limiting, then lactic acid and butanediol were accumulated, suggesting an excess of reduced cofactors . Due to a strong lysis at the onset of the end of the growth phase, sporulation frequency and aprE expression were negligible in anaerobic batch cultures . Fed-batch fermentation allowed the development of a stationary phase through a continuous supply of glucose and nitrate . In this case, sporulation frequency was almost null, but interestingly aprE expression was similar to that found in aerobic cultures.

Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2306 - 10
Antibacterial agents that inhibit histidine protein kinase YycG of Bacillus subtilis; Yamamoto K et al.; We demonstrated in vitro that YycG-YycF of Bacillus subtilis constitutes a two-component system and shows a specificity of the sensor protein for the cognate phosphorylation partner . Based on inhibition of such an autophosphorylation of YycG, we searched imidazole and zerumbone derivatives to identify the antibacterial agents such as NH125, NH126, NH127, and NH0891.

Rapid Commun Mass Spectrom, 2002, 16(2), 103 - 10
Maturation of the lantibiotic subtilin: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor precursors and their proteolytic processing in crude bacterial cultures; Stein T et al.; Bacillus subtilis synthesizes the lanthionine containing 32-amino-acid peptide antibiotic (lanti-biotic) subtilin from a ribosomally generated 56-amino-acid precursor pre-propeptide by extensive posttranslational modifications . Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to monitor the production of matured subtilin within crude samples taken from B . subtilis culture media without prior fractionation . The processing reaction of subtilin was blocked with the serine protease inhibitor phenylmethylsulfonyl fluoride and different subtilin precursor peptides in the molecular mass range up to 6220 were observed . Two of these species were isolated by reversed-phase high-performance liquid chromatography (HPLC) and structurally analyzed by post-source decay MALDI-TOFMS . We provide evidence that the precursor species comprise the posttranslational modified C-terminal part of subtilin to which leader peptide moieties with different chain lengths are attached . These antimicrobial-inactive species could be processed to antibiotic-active subtilin by incubation with culture media of different subtilin-nonproducing B . subtilis strains as indicated by a combination of antimicrobial growth assays and MALDI-TOFMS analyses . These achievements are strong evidence for the sensitivity of MALDI-TOFMS methodology that allows straightforward investigations of analytes even in complex mixtures without time-consuming sample preparations .

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15260 - 3
Evidence that the cell wall of Bacillus subtilis is protonated during respiration; Calamita HG et al.; Several independent experiments suggest that cell walls of Bacillus subtilis are protonated during growth . When cells were grown in the presence of fluorescein-labeled dextran to saturate the cell walls, centrifuged, and suspended in PBS, fluorescence-activated cell sorter analyses revealed the bacteria were only poorly fluorescent . In contrast, when the bacteria were purged with N(2) to dissipate protonmotive force (pmf) fluorescence became intense . Upon reconstitution of the pmf with phenazine methosulfate, glucose, and oxygen, fluorescence declined . Another approach used pH-dependent chemical modification of cell walls . The walls of respiring B . subtilis cells were amenable to carboxylate modification by {(14)C}ethanolamine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide . The carbodiimide activation of carboxylate groups occurs only in acidic conditions . Upon dissipation of pmf the walls were refractory to chemical modification . Ammonium groups can be condensed with FITC in alkaline medium, but the condensation is very slow in acidic buffers . It was found that the derivatization of the walls with FITC could occur in the absence of pmf . The use of pH-dependent fluorophores and pH-dependent chemical modification reactions suggest that cell walls of respiring B . subtilis cells have a relatively low pH environment . This study shows a bacterium has a protonated compartment . Acidification of cell walls during growth may be one means of regulating autolytic enzymes.

Nucleic Acids Res, 2002 Jan 1, 30(1), 62 - 5
SubtiList: the reference database for the Bacillus subtilis genome; Moszer I et al.; SubtiList is the reference database dedicated to the genome of Bacillus subtilis 168, the paradigm of Gram-positive endospore-forming bacteria . Developed in the framework of the B.subtilis genome project, SubtiList provides a curated dataset of DNA and protein sequences, combined with the relevant annotations and functional assignments . Information about gene functions and products is continuously updated by linking relevant bibliographic references . Recently, sequence corrections arising from both systematic verifications and submissions by individual scientists were included in the reference genome sequence . SubtiList is based on a generic relational data schema and a World Wide Web interface developed for the handling of bacterial genomes, called GenoList . The World Wide Web interface was designed to allow users to easily browse through genome data and retrieve information according to common biological queries . SubtiList also provides more elaborate tools, such as pattern searching, which are tightly connected to the overall browsing system . SubtiList is accessible at Similar bacterial databases are accessible at http://genolist.pasteur.fr/.

J Biol Chem, 2002 Mar 1, 277(9), 7567 - 73 Epub 2002 Jan 07.
Bacillus subtilis isocitrate dehydrogenase . A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase; Singh SK et al.; In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine . The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P) . To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities . BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine . Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved . We now expand the comparison to include a number of biochemical properties . Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction . Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity . Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104) . Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively . These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P.

J Bacteriol, 2002 Jan, 184(2), 596 - 9
Epr is transcribed from a final sigma(D) promoter and is involved in swarming of Bacillus subtilis; Dixit M et al.; Epr is a minor extracellular protease secreted by Bacillus subtilis 168 . In this study, we show that epr is transcribed by E sigma(D), the RNA polymerase associated with transcription of genes involved in chemotaxis and motility . Disruption of epr abolished swarming of Bacillus subtilis, suggesting its involvement in motility.

J Bacteriol, 2002 Jan, 184(2), 584 - 7
The products of the spoVA operon are involved in dipicolinic acid uptake into developing spores of Bacillus subtilis; Tovar-Rojo F et al.; Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation . However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation . ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores . These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase . Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores . These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.

J Bacteriol, 2002 Jan, 184(2), 564 - 71
Postexponential regulation of sin operon expression in Bacillus subtilis; Shafikhani SH et al.; The expression of many gene products required during the early stages of Bacillus subtilis sporulation is regulated by sinIR operon proteins . Transcription of sinIR from the P1 promoter is induced at the end of exponential growth . In vivo transcription studies suggest that P1 induction is repressed by the transition-state regulatory protein Hpr and is induced by the phosphorylated form of Spo0A . In vitro DNase I footprinting studies confirmed that Hpr, AbrB, and Spo0A are trans-acting transcriptional factors that bind to the P1 promoter region of sinIR . We have also determined that the P1 promoter is transcribed in vitro by the major vegetative sigma factor, final sigma(A), form of RNA polymerase.

J Bacteriol, 2002 Jan, 184(2), 488 - 93
Localization of UvrA and effect of DNA damage on the chromosome of Bacillus subtilis; Smith BT et al.; We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells . We suggest that the UvrA(2)B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA . We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth . This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response . We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell's genome . Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.

J Bacteriol, 2002 Jan, 184(2), 459 - 67
Bacillus subtilis tolerance of moderate concentrations of rifampin involves the sigma(B)-dependent general and multiple stress response; Bandow JE et al.; Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of Bacillus subtilis led to an instant inhibition of growth . Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment . L-{(35)S}methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin . The sigma(B)-dependent general stress proteins were found to be induced after treatment with the antibiotic . Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA . The target protein of rifampin, the beta subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the sigma(D) regulon were also induced . The rifampin-triggered growth arrest was extended in a sigB mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was . Activity of the RsbP energy-signaling phosphatase in the sigma(B) signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required . The sigB mutant strain was less capable of growing on rifampin-containing agar plates . When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the sigB mutant by a factor of 100 . We conclude that the general stress response of B . subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.

J Bacteriol, 2002 Jan, 184(2), 410 - 9
Characterization of comQ and comX, two genes required for production of ComX pheromone in Bacillus subtilis; Bacon Schneider K et al.; Many microbes use secreted peptide-signaling molecules to stimulate changes in gene expression in response to high population density, a process called quorum sensing . ComX pheromone is a modified 10-amino-acid peptide used by Bacillus subtilis to modulate changes in gene expression in response to crowding . comQ and comX are required for production of ComX pheromone . We found that accumulation of ComX pheromone in culture supernatant paralleled cell growth, indicating that there was no autoinduction of production of ComX pheromone . We overexpressed comQ and comX separately and together and found that overexpression of comX alone was sufficient to cause an increase in production of ComX pheromone and early induction of a quorum-responsive promoter . These results indicate that the extracellular concentration of ComX pheromone plays a major role in determining the timing of the quorum response and that expression of comX is limiting for production of ComX pheromone . We made alanine substitutions in the residues that comprise the peptide backbone of ComX pheromone . Analysis of these mutants highlighted the importance of the modification for ComX pheromone function and identified three residues (T50, G54, and D55) that are unlikely to interact with proteins involved in production of or response to ComX pheromone . We have also identified and mutated a putative isoprenoid binding domain of ComQ . Mutations in this domain eliminated production of ComX pheromone, consistent with the hypothesis that ComQ is involved in modifying ComX pheromone and that the modification is likely to be an isoprenoid.

J Bacteriol, 2002 Jan, 184(2), 381 - 9
Molecular organization of intrinsic restriction and modification genes BsuM of Bacillus subtilis Marburg; Ohshima H et al.; Transcriptional analysis and disruption of five open reading frames (ORFs), ydiO, ydiP, ydiR, ydiS, and ydjA, in the prophage 3 region of the chromosome of Bacillus subtilis Marburg revealed that they are component genes of the intrinsic BsuM restriction and modification system of this organism . The classical mutant strain RM125, which lacks the restriction and modification system of B . subtilis Marburg, lacks the prophage 3 region carrying these five ORFs . These ORFs constitute two operons, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon, both of which are expressed during the logarithmic phase of growth . The predicted gene products YdiO and YdiP are the orthologues of cytosine DNA methyltransferases . The predicted YdiS product is an orthologue of restriction nucleases, while the predicted YdiR and YdjA products have no apparent paralogues and orthologues whose functions are known . Disruption of the ydiR-ydiS-ydjA operon resulted in enhanced transformation by plasmid DNA carrying multiple BsuM target sequences . Disruption of ydiO or ydiP function requires disruption of at least one of the following genes on the chromosome: ydiR, ydiS, and ydjA . The degrees of methylation of the BsuM target sequences on chromosomal DNAs were estimated indirectly by determining the susceptibility to digestion with XhoI (an isoschizomer of BsuM) of DNAs extracted from the disruptant strains . Six XhoI (BsuM) sites were examined . XhoI digested at the XhoI sites in the DNAs from disruptants with disruptions in both operons, while XhoI did not digest at the XhoI sites in the DNAs from the wild-type strain or from the disruptants with disruptions in the ydiR-ydiS-ydjA operon . Therefore, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon are considered operons that are responsible for BsuM modification and BsuM restriction, respectively.

J Bacteriol, 2002 Jan, 184(2), 337 - 43
Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production; Urushibata Y et al.; The genes required for gamma-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans . There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B . subtlis 168 . Northern blot analysis showed that the four genes constitute an operon . Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis . No PGA was produced in Delta ywsC and Delta ywtA strains, indicating that both of these genes are essential for PGA production . To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the Delta ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography . Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene . (14)C-labeled PGA was synthesized by the purified proteins from L-{(14)C}-glutamate in the presence of ATP and MnCl(2), through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.

Bioinformatics, 2001 Dec, 17(12), 1209 - 12
SPiD: a subtilis protein interaction database; Hoebeke M et al.; MOTIVATION: Protein-protein interactions are a potential source of valuable clues in determining the functional role of as yet uncharacterized gene products in metabolic pathways . Graph-like structures emerging from the accumulation of interaction data make it difficult to maintain a consistent and global overview by hand . Bioinformatics tools are needed to perform this graph visualization while maintaining a link to the experimental data . RESULTS: "SPiD" is an online database for exploring networks of interacting proteins in Bacillus subtilis characterized by the two-hybrid system . Graphical displays of interaction networks are created dynamically as users interactively navigate through these networks . Third party applications can interface the database through a Common Object Request Broker Architecture (CORBA) tier . AVAILABILITY: SPiD is available through its web site at and through an Interoperable Object Reference (IOR) and its associated Interface Definition Language (IDL) . CONTACT: hoebeke@versailles.inra.fr

Biochemistry, 2001 Dec 25, 40(51), 15716 - 24
Evolution of enzymatic activities in the enolase superfamily: crystal structures of the L-Ala-D/L-Glu epimerases from Escherichia coli and Bacillus subtilis; Gulick AM et al.; The members of the enolase superfamily catalyze different overall reactions, yet share a partial reaction that involves Mg(2+)-assisted enolization of the substrate carboxylate anion . The fate of the resulting enolate intermediate is determined by the active site of each enzyme . Several members of this superfamily have been structurally characterized to permit an understanding of the evolutionary strategy for using a common structural motif to catalyze different overall reactions . In the preceding paper, two new members of the superfamily were identified that catalyze the epimerization of the glutamate residue in L-Ala-D/L-Glu . These enzymes belong to the muconate lactonizing enzyme subgroup of the enolase superfamily, and their sequences are only 31% identical . The structure of YcjG, the epimerase from Escherichia coli, was determined by MAD phasing using both the SeMet-labeled protein and a heavy atom derivative . The structure of YkfB, the epimerase from Bacillus subtilis, was determined by molecular replacement using the muconate lactonizing enzyme as a search model . In this paper, we report the three-dimensional structures of these enzymes and compare them to the structure of o-succinylbenzoate synthase, another member of the muconate lactonizing enzyme subgroup.

Biochemistry, 2001 Dec 25, 40(51), 15707 - 15
Evolution of enzymatic activities in the enolase superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases; Schmidt DM et al.; The members of the mechanistically diverse enolase superfamily catalyze different overall reactions by using a common catalytic strategy and structural scaffold . In the muconate lactonizing enzyme (MLE) subgroup of the superfamily, abstraction of a proton adjacent to a carboxylate group initiates reactions, including cycloisomerization (MLE), dehydration {o-succinylbenzoate synthase (OSBS)}, and 1,1-proton transfer (catalyzed by an OSBS that also catalyzes a promiscuous N-acylamino acid racemase reaction) . The realization that a member of the MLE subgroup could catalyze a 1,1-proton transfer reaction, albeit poorly, led to a search for other enzymes which might catalyze a 1,1-proton transfer as their physiological reaction . YcjG from Escherichia coli and YkfB from Bacillus subtilis, proteins of previously unknown function, were discovered to be L-Ala-D/L-Glu epimerases, although they also catalyze the epimerization of other dipeptides . The values of k(cat)/K(M) for L-Ala-D/L-Glu for both proteins are approximately 10(4) M(-1) s(-1) . The genomic context and the substrate specificity of both YcjG and YkfB suggest roles in the metabolism of the murein peptide, of which L-Ala-D-Glu is a component . Homologues possessing L-Ala-D/L-Glu epimerase activity have been identified in at least two other organisms.

Biochemistry, 2001 Dec 25, 40(51), 15660 - 8
Copper trafficking: the solution structure of Bacillus subtilis CopZ; Banci L et al.; A sequence with a high homology (39% residue identity) with that of the copper-transport CopZ protein from Enterococcus hirae and with the same MXCXXC metal-binding motif has been identified in the genome of Bacillus subtilis, and the corresponding protein has been expressed . The protein, constituted by 73 amino acids, does bind copper(I) under reducing conditions and fully folded in both copper-bound and copper-free forms under the present experimental conditions . The solution structure of the copper-bound form was determined through NMR spectroscopy on an 15N-labeled sample . A total of 1508 meaningful nuclear Overhauser effects, 38 dihedral phi angles, and 48 dihedral psi angles were used in the structural calculations, which lead to a family of 30 conformers with an average rmsd to the mean structure of 0.32 +/- 0.06 A for the backbone and of 0.85 +/- 0.07 A for the heavy atoms . NMR data on the apoprotein also show that, also in this form, the protein is in a folded state and essentially maintains the complete secondary structure . Some disorder is observed in the loop devoted to copper binding . These results are compared with those reported for CopZ from E . hirae whose structure is well-defined only in the apo form . The different behaviors of copper-loaded E . hirae and B . subtilis are tentatively accounted for on the basis of the presence of dithiothreitol used in the latter case, which would stabilize the monomeric form . The comparison is extended to other similar proteins, with particular attention to the copper-binding loop . The nature and the location of conserved residues around the metal-binding site are discussed with respect to their relevance for the metal-binding process . Proposals for the role of CopZ are also presented.

Biotechnol Bioeng, 2001 Dec 20, 75(6), 630 - 3
Isolation and expression of a Bacillus cereus gene encoding benzil reductase; Maruyama R et al.; Benzil was reduced stereospecifically to (S)-benzoin by Bacillus cereus strain Tim-r01 . To isolate the gene responsible for asymmetric reduction, we constructed a library consisting of Escherichia coli clones that harbored plasmids expressing Bacillus cereus genes . The library was screened using the halo formation assay, and one clone showed benzil reduction to (S)-benzoin . Thus, this clone seemed to carry a plasmid encoding a Bacillus cereus benzil reductase . The deduced amino acid sequence had marked homologies to the Bacillus subtilis yueD protein (41% identity), the yeast open reading frame YIR036C protein (31%), and the mammalian sepiapterin reductases (28% to 30%), suggesting that benzil reductase is a novel short-chain de-hydrogenases/ reductase .

J Biol Chem, 2002 Mar 1, 277(9), 6985 - 93 Epub 2001 Dec 13.
Glycine oxidase from Bacillus subtilis . Characterization of a new flavoprotein; Job V et al.; Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer . GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline) . The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase . GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex . The approximately 300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction . GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K(m) value for d-alanine . A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine . The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis.

J Ethnopharmacol, 2002 Jan, 79(1), 101 - 7
The pharmacological screening of Pentanisia prunelloides and the isolation of the antibacterial compound palmitic acid; Yff BT et al.; The uses of Pentanisia prunelloides in Zulu traditional medicine indicate that the plant is believed to be effective in relieving inflammation, bacterial and viral infections and also stimulating uterine contraction . Aqueous, ethanolic and ethyl acetate extracts of leaves and roots were screened for prostaglandin-synthesis inhibitors and antibacterial and antiviral activity . In the results of the anti-inflammatory assay all the extracts showed cyclooxygenase-1 inhibition . The ethanolic and ethyl acetate extracts showed greater antibacterial activity than the aqueous extracts against Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae) . Both root and leaf extracts were found to inhibit viral replication of the Influenza A virus . The ethyl acetate extract was fractionated by silica vacuum liquid chromatography and anti-inflammatory activity was found to be most pronounced in the more polar fractions . The presence of antibacterial activity was confirmed by running the fractions on a thin layer chromatography (TLC) plate and performing a bioautographic assay . The active fraction was further purified by TLC and the major antibacterial compound in the ethyl acetate root extract was identified by GC/MS as palmitic acid.

J Biol Chem, 2002 Feb 22, 277(8), 6733 - 42 Epub 2001 Dec 10.
The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein; Serna-Rico A et al.; The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain . Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA) . Protein p16.7A was found to be an ssDNA-binding protein . In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates . Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5 . The results obtained show that both proteins have different, non-overlapping functions . The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed . Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.

J Bacteriol, 2002 Jan, 184(1), 241 - 9
Two regions of GerE required for promoter activation in Bacillus subtilis; Crater DL et al.; GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators . Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif . In this report, we sought to define regions of GerE involved in promoter activation . We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface . A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter . However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter . In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter . The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively . A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.

J Bacteriol, 2002 Jan, 184(1), 191 - 9
The Bacillus subtilis signaling protein SpoIVB defines a new family of serine peptidases; Hoa NT et al.; The protein SpoIVB plays a key role in signaling in the final sigma(K) checkpoint of Bacillus subtilis . This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase . SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-final sigma(K) to its active form, final sigma(K), in the outer mother cell chamber of the developing cell . In this work we have characterized the serine peptidase domain of SpoIVB . Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain . We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein . His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad . In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the final sigma(K) checkpoint . The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily.

Protein Eng, 2001 Oct, 14(10), 807 - 13
Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability; Cambi A et al.; We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively . We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C) . The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity . High sequence similarity was also found with the mesophilic CDA(Bsubt) . Both enzymes were found to be homotetramers of approximately 58 kDa . CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald) . After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated . Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable . No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature.

Microbiology, 2001 Dec, 147(Pt 12), 3413 - 9
yveB, Encoding endolevanase LevB, is part of the sacB-yveB-yveA levansucrase tricistronic operon in Bacillus subtilis; Pereira Y et al.; Transcription of sacB, yveB and yveA, three clustered genes on the Bacillus subtilis chromosome, is simultaneously induced by sucrose . Northern blotting analyses with specific probes showed three distinct mRNAs: a monocistronic 1.7 kb sacB mRNA, a bicistronic 3.3 kb sacB-yveB mRNA and a tricistronic 4.9 kb sacB-yveB-yveA mRNA . These results indicate that sacB, encoding levansucrase, is the proximal gene of a sucrose-inducible operon that includes the two other genes . The yield of the full-length transcript is lower than that of the bicistronic transcript, whose yield is itself lower than that of the monocistronic transcript . This suggested that the 3' terminal parts of sacB and yveB genes worked as internal terminator structures . The protein encoded by yveB, which remains anchored to the membrane, displays an endolevanase activity, which, coupled with exolevanase activity of SacB, leads to a complete degradation of levan, a branched fructosyl polymer . It is proposed to rename yveB as levB.

Genome Biol . 2001;2(11):RESEARCH0048 . Epub 2001 Oct 15.
Prediction of co-regulated genes in Bacillus subtilis on the basis of upstream elements conserved across three closely related species; Terai G et al.; BACKGROUND: Identification of co-regulated genes is essential for elucidating transcriptional regulatory networks and the function of uncharacterized genes . Although co-regulated genes should have at least one common sequence element, it is generally difficult to identify these genes from the presence of this element because it is very easily obscured by noise . To overcome this problem, we used conserved information from three closely related species: Bacillus subtilis, B . halodurans and B . stearothermophilus . RESULTS: Even though such species have a limited number of clearly orthologous genes, we obtained 1,884 phylogenetically conserved elements from the upstream intergenic regions of 1,568 B . subtilis genes . Similarity between these elements was used to cluster these genes . No other a priori knowledge on genes and elements was used . We could identify some genes known or suggested to be regulated by a common transcription factor as well as genes regulated by a common attenuation effector . CONCLUSIONS: We confirmed that our method generates relatively few false positives in clusters with higher scores and that general elements such as -35/-10 boxes and Shine-Dalgarno sequence are not major obstacles . Moreover, we identified some plausible additional members of groups of known co-regulated genes . Thus, our approach is promising for exploring potentially co-regulated genes.

Mol Microbiol, 2001 Nov, 42(4), 1107 - 20
Functional analysis of the Bacillus subtilis morphogenetic spore coat protein CotE; Little S et al.; Bacterial spores are surrounded by a multilayered proteinaceous shell called the coat . In Bacillus subtilis, a coat protein called CotE guides the assembly of a major subset of coat proteins . To understand how CotE carries out its role in coat morphogenesis, we subjected its gene to mutagenesis and studied the effects of altered versions of CotE on coat formation . We identified regions within the C-terminal 28 amino acids that direct the deposition of the coat proteins CotA, CotB, CotG, CotSA, CotS and 35 kDa and 49 kDa proteins likely to be the spore proteins CotR (formerly known as YvdO) and YaaH respectively . The timing and genetic dependency of CotR accumulation are consistent with control of its gene by sigmaK and GerE . In addition, we identified a 35-amino-acid internal region involved in targeting of CotE to the forespore . Finally, we found that sequences within this 35-amino-acid region as well as within an 18-amino-acid stretch in the N-terminus of CotE direct the formation of CotE multimers, most probably homooligomers . These results suggest that: (i) most interactions between CotE and the coat proteins assembled under CotE control take place at the CotE C-terminus; (ii) an internal region of CotE connects it with the forespore surface; and (iii) interactions between CotE molecules depend on residues within an 18-amino-acid region in the N-terminal half of CotE.

J Org Chem, 2001 Dec 14, 66(25), 8320 - 7
Design, synthesis, and evaluation of 9-D-ribityl-1,3,7-trihydro-2,6,8-purinetrione, a potent inhibitor of riboflavin synthase and lumazine synthase; Cushman M et al.; Reduction of 5-nitro-6-D-ribitylaminouracil (9) afforded 5-amino-6-D-ribitylaminouracil (1), which reacted with ethyl chloroformate to yield 5-ethylcarbamoyl-6-D-ribitylaminouracil (12) . The latter compound was cyclized to 9-D-ribityl-1,3,7-trihydropurine-2,6,8-trione (13), which was found to be a relatively potent inhibitor of both Escherichia coli riboflavin synthase (K(i) 0.61 microM) and Bacillus subtilis lumazine synthase (K(i) 46 microM) . Molecular modeling of the lumazine synthase-inhibitor complex indicated the possibility for hydrogen bonding between the Lys135 epsilon-amino group of the enzyme and both the 8-keto group and the 4'-hydroxyl group of the ligand . A bisubstrate analogue of the riboflavin synthase-catalyzed reaction, 1,4-bis{1-(9-D-ribityl-1,3,7-trihydropurine-2,6,8-trionyl)}butane (18), was also synthesized using a similar route and was found to be inactive as an inhibitor of both riboflavin synthase and lumazine synthase.

Arch Microbiol, 2001 Dec, 176(6), 459 - 64 Epub 2001 Oct 05.
Fatty-acid-displaced transcriptional repressor, a conserved regulator of cytochrome P450 102 transcription in Bacillus species; Gustafsson MC et al.; Bacillus subtilis strain 168 encodes two flavocytochromes P450, Cyp102A2 and Cyp102A3 . The cyp102A3 gene is preceded by, and organized in an operon with, a gene for a transcriptional regulator, encoded by fatR . The paralogous gene, cyp102A2, is most likely transcribed as a mono-cistronic message . We show that fatR encodes a protein that binds to an operator sequence that is present upstream of its own reading frame, thereby repressing the expression of the fatR-cyp102A3 operon . Unsaturated fatty acids and phytanic acid have the capacity to interact with FatR and to abrogate its binding to the operator sequence.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15251 - 6 Epub 2001 Dec 04.
PAS-A domain of phosphorelay sensor kinase A: a catalytic ATP-binding domain involved in the initiation of development in Bacillus subtilis; Stephenson K et al.; The major sensor kinase controlling the initiation of development in Bacillus subtilis, KinA, functions by activating the phosphorelay signal-transduction system in response to as yet unknown signal ligands . KinA contains, within its amino-terminal signal-sensing region, three PAS domains that, in other proteins, are known to be involved in sensing changes in oxygen concentration and redox potential among other functions . The most amino-terminal PAS domain, PAS-A, was found to bind ATP and catalyze exchange of phosphate between ATP and nucleoside diphosphates . A cysteine-to-alanine mutation in PAS-A increased the affinity for ATP 5-fold, decreased the exchange reaction 2-fold, and stimulated KinA-dependent sporulation . A model for the role of ATP and the exchange reaction in the PAS domain in sensor kinase signal transduction is presented in which the free energy of nucleotide hydrolysis drives the conformational changes that activate or deactivate the sensor kinase in response to signal ligand binding.

Curr Opin Microbiol, 2001 Dec, 4(6), 660 - 6
Septation and chromosome segregation during sporulation in Bacillus subtilis; Errington J; The early stages of sporulation in Bacillus subtilis incorporate a modified, highly asymmetric cell division . It is now clear that most, if not all, of the components of the vegetative division machinery are used also for asymmetric division . However, the machinery for chromosome segregation may differ significantly between vegetative growth and sporulation . Several interesting checkpoint mechanisms couple cell cycle events to gene expression early in sporulation . This review summarises important advances in the understanding of chromosome segregation and cell division at the onset of sporulation in B.subtilis in the past three years.

FEMS Microbiol Lett, 2001 Nov 13, 204(2), 239 - 45
A putative ATP-binding cassette transporter YbdA involved in sporulation of Bacillus subtilis; Isezaki M et al.; Insertional mutagenesis with mini-Tn10 was performed to identify new genes involved in sporulation of Bacillus subtilis . Here, we report on the characterization of the ybdA locus, which encodes a putative ATP-binding cassette transporter . The ybdA gene is the 6th cistron of the putative ybcOPQST-ybdABDE operon . A deletion mutation in ybdA and an insertional mutation in ybdB exhibited highly oligosporogenous phenotypes and led to a decrease in the transcription controlled by Spo0A, which is a key response regulator required for the initiation of sporulation . We further observed that the transcription of this operon was strongly induced after the end of the exponential growth phase in the wild-type strain, but not in a spo0A null mutant . Our data suggest that the YbdA and YbdB proteins are able to affect incorporation of nutrient signals during initiation of sporulation and may act as components of positive feedback systems of Spo0A activation.

Food Chem Toxicol, 2002 Jan, 40(1), 1 - 8
Safety evaluation of a xylanase expressed in Bacillus subtilis; Harbak L et al.; A programme of studies was conducted to establish the safety of a xylanase expressed in a self-cloned strain of Bacillus subtilis to be used as a processing aid in the baking industry . To assess acute and subchronic oral toxicity, rat feeding studies were conducted . In addition, the potential of the enzyme to cause mutagenicity and chromosomal aberrations was assessed in microbial and tissue culture in vitro studies . Acute and subchronic oral toxicity was not detected at the highest dose recommended by OECD guidelines . There was no evidence of mutagenic potential or chromosomal aberrations . Furthermore, the organism used for production of the xylanase is already accepted as safe by several major national regulatory agencies.

FEMS Microbiol Lett, 2001 Nov 27, 205(1), 91 - 7
Rapid orientated cloning in a shuttle vector allowing modulated gene expression in Bacillus subtilis; Joseph P et al.; An expression vector for systematic protein overproduction in Bacillus subtilis has been constructed . It derives from pDG148 and combines the main property of this vector, i.e . conditional expression of the gene in response to isopropylbeta-D-thiogalactopyranoside, with (i) rapid orientated cloning by a ligation independent procedure and (ii) a ribosome binding site of high translational efficiency . When used for overproduction of several proteins in B . subtilis, this vector gave good levels of protein synthesis.

Nucleic Acids Res, 2001 Dec 1, 29(23), 4851 - 65
Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR; Bonner ER et al.; The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation . The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA . PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes . The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay . Titration indicated that PyrR binds RNA in an equimolar ratio . PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site . PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator . The minimal RNA that bound tightly to PyrR was 28 nt long . Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity . Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding . Conserved elements of RNA secondary structure were also required for tight binding . PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments . PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.

Anticancer Res, 2001 Jul-Aug, 21(4A), 2847 - 53
Cytotoxic and DNA damage-inducing activities of low molecular weight phenols from rhubarb; Shi YQ et al.; Six new phenol (anthraquinone or stilbene) glycosides with an acyl group at 6-position of the glucopyranose moiety were isolated from rhubarb (the roots of Rheum palmatum) cultivated in Japan, together with 22 known compounds . Most of these compounds were evaluated for cytotoxic activity against tumor and normal cells and for induction of DNA damage by spore rec-assay . Among them, emodin and aloe-emodin showed higher cytotoxic activities against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines than against normal human gingival fibroblasts (HGF) . Chrysophanol 8-O-beta-(6'-acetyl)glucopyranoside, 4-(4'-hydroxyphenyl)-2-butanone 4'-O-beta-D-(2"-O-galloyl-6"-O-cinnamoyl) glucopyranoside, and 6"-O-(4'''-hydroxybenzoyl) resveratroloside exhibited relatively higher cytotoxic activities against all these cells . The other glycosides of anthraquinone or stilbene showed weaker cytotoxic activity against these tumor cell lines, but may be considered as cancer chemopreventive agents . Spore rec-assay with a recombination deficient mutant of Bacillus subtilis M45 demonstrated the DNA damage-inducing activity of emodin and aloe-emodin 15-O-beta-D-glucopyranoside among, rhubarb phenols.

Chem Pharm Bull (Tokyo), 2001 Nov, 49(11), 1479 - 81
Studies on constituents from Chamaecyparis pisifera and antibacterial activity of diterpenes; Xiao D et al.; In the course of our research for biologically active constituents from coniferous plants, a chromone derivative (1) and an abietane derivative (2) were isolated along with several diterpenes from Chamaecyparis pisifera . Structures of the new compounds were determined to be 5,7-dihydroxy-2-(1-acetyl-2-methoxycarbonylethyl)-chromone and rel-(8R,10R,20S)-8,10,20-trihydroxy-9(10-->20)-abeo-abieta-9,13-dien-12-one by means of spectral methods including two-dimensional NMR experiments . Some of these abietane-type compounds isolated from this plants showed antibacterial activitv against the gram-positive bacteria Staphylococcus aureus and Bacillus subtilis.

Appl Environ Microbiol, 2001 Dec, 67(12), 5830 - 2
Examination of peak power dependence in the UV inactivation of bacterial spores; Rice JK et al.; We examine whether the rate of delivery of photons from a UV radiation source has an effect on the inactivation of spores . We directly compare the output of a high-peak-power UV laser source at 248 nm to a low-power continuous lamp source (254 nm) in the inactivation of Bacillus subtilis spores . The two UV sources differ by a factor of 10(8) in peak power . Contrary to previous reports, no clear differences in spore survival were observed.

Mol Microbiol, 2001 Nov, 42(3), 573 - 85
CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis in Bacillus subtilis; Kirby JR et al.; Chemotaxis by Bacillus subtilis requires the inter-acting chemotaxis proteins CheC and CheD . In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB . We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor . CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two-hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor-CheA complex . Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist . The distinct physiological roles for CheC and CheD during B . subtilis chemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction . We speculate that CheC interacts with flagellar switch components and dissociates upon CheY-P binding and subsequently interacts with the receptor complex to facilitate adaptation.

Eur J Biochem, 2001 Nov, 268(22), 5771 - 5
Directed mutagenesis studies of the C-terminal fingerprint region of Bacillus subtilis pyrophosphatase; Shizawa N et al.; The sequence SRKKQxxP near the C-terminus is conserved in pyrophosphatases of the recently discovered family II and includes a triplet of positively charged residues, two of which (Arg295 and Lys296 in Bacillus subtilis pyrophosphatase) are part of the active site and one (Lys297) is not . The importance of this triplet for catalysis by B . subtilis pyrophosphatase has been estimated by mutational analysis . R295K and K296R substitutions were found to decrease the catalytic constant 650- and 280-fold, respectively, and decrease the pK(a) of the essential acidic group by 1.1 and 0.5, respectively . K297R substitution was found to increase the catalytic constant 4.7-fold and to markedly change the protein circular dichroism spectrum in the range 250-300 nm . These results, together with the results of theoretical modelling of the enzyme-substrate complex, provide support for the direct involvement of Arg295 and Lys296 in substrate binding in family II pyrophosphatases.

Science, 2001 Nov 23, 294(5547), 1716 - 9
Two essential DNA polymerases at the bacterial replication fork; Dervyn E et al.; DNA replication in bacteria is carried out by a multiprotein complex, which is thought to contain only one essential DNA polymerase, specified by the dnaE gene in Escherichia coli and the polC gene in Bacillus subtilis . Bacillus subtilis genome analysis has revealed another DNA polymerase gene, dnaE(BS), which is homologous to dnaE . We show that, in B . subtilis, dnaE(BS) is essential for cell viability and for the elongation step of DNA replication, as is polC, and we conclude that there are two different essential DNA polymerases at the replication fork of B . subtilis, as was previously observed in eukaryotes . dnaE(BS) appears to be involved in the synthesis of the lagging DNA strand and to be associated with the replication factory, which suggests that two different polymerases carry out synthesis of the two DNA strands in B . subtilis and in many other bacteria that contain both polC and dnaE genes.

J Biol Chem, 2002 Feb 1, 277(5), 3268 - 73 Epub 2001 Nov 21.
The twin-arginine signal peptide of PhoD and the TatAd/Cd proteins of Bacillus subtilis form an autonomous Tat translocation system; Pop O et al.; The bacterial twin-arginine translocation (Tat) pathway has been recently described for PhoD of Bacillus subtilis, a phosphodiesterase containing a twin-arginine signal peptide . The expression of phoD is co-regulated with the expression of tatA(d) and tatC(d) genes localized downstream of phoD . To characterize the specificity of PhoD transport further, translocation of PhoD was investigated in Escherichia coli . By using gene fusions, we analyzed the particular role of the signal peptide and the mature region of PhoD in canalizing the transport route . A hybrid protein consisting of the signal peptide of beta-lactamase and mature PhoD was transported in a Sec-dependent manner indicating that the mature part of PhoD does not contain information canalizing the selected translocation route . Pre-PhoD, as well as a fusion protein consisting of the signal peptide of PhoD (SP(PhoD)) and beta-galactosidase (LacZ), remained cytosolic in the E . coli . Thus, SP(PhoD) is not recognized by E . coli transport systems . Co-expression of B . subtilis tatA(d)/C(d) genes resulted in the processing of SP(PhoD)-LacZ and periplasmic localization of LacZ illustrating a close substrate specificity of the TatA(d)/C(d) transport system . While blockage of the Sec-dependent transport did not affect the localization of SP(PhoD)-LacZ, translocation and processing was dependent on the pH gradient of the cytosolic membrane . Thus, the minimal requirement of a functional Tat-dependent protein translocation system consists of a twin-arginine signal peptide-containing Tat substrate, its specific TatA/C proteins, and the pH gradient across the cytosolic membrane.

Cell, 2001 Nov 16, 107(4), 427 - 35
Bacillus subtilis glutamine synthetase controls gene expression through a protein-protein interaction with transcription factor TnrA; Wray LV Jr et al.; Bacillus subtilis TnrA, a global regulator of transcription, responds to nitrogen availability, but the specific signal to which it responds has been elusive . Genetic studies indicate that glutamine synthetase is required for the regulation of TnrA activity in vivo . We report here that the feedback-inhibited form of glutamine synthetase directly interacts with TnrA and blocks the DNA binding activity of TnrA . Mutations in the tnrA gene (tnrA(C)) that allow constitutive high level expression of tnrA-activated genes were isolated and characterized . Feedback-inhibited glutamine synthetase had a significantly reduced ability to block the in vitro DNA binding by three of the TnrA(C) proteins . Thus, glutamine synthetase, an enzyme of central metabolism, directly interacts with and regulates the DNA binding activity of TnrA.

Acta Crystallogr D Biol Crystallogr, 2001 Dec, 57(Pt 12), 1931 - 2 Epub 2001 Nov 21.
Crystallization and preliminary crystallographic studies of antibacterial polypeptide LCI expressed in Escherichia coli; Zhu JP et al.; LCI is a type of novel antibacterial polypeptide secreted by a Bacillus subtilis strain . It consists of 47 residues with a molecular weight of 5468 Da . Using bioengineering, LCI was expressed in Escherichia coli DH5alpha with recombinant plasmid pBVAB16 . It was crystallized using PEG 4000 as a precipitant . The crystal belongs to space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 29.30, c = 187.09 A, and diffracts to 2.44 A . A set of diffraction data to 2.8 A was collected.

J Bacteriol, 2001 Dec, 183(24), 7381 - 6
Complementation of cold shock proteins by translation initiation factor IF1 in vivo; Weber MH et al.; The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins . This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans . For B . subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock . Analysis of the B . subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores . We show here that heterologous expression of the translation initiation factor IF1 from E . coli in a B . subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s) . Two of the possible explanation models are discussed.

J Bacteriol, 2001 Dec, 183(24), 7371 - 80
RNA expression analysis using an antisense Bacillus subtilis genome array; Lee JM et al.; We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in Bacillus subtilis by using Affymetrix technology . Quality control tests of the B . subtilis GeneChip were performed to ascertain the quality of the array . These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes . In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs) . In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine . Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs . Several of these novel transcripts were subsequently correlated to new coding regions.

J Bacteriol, 2001 Dec, 183(24), 7365 - 70
Comprehensive DNA microarray analysis of Bacillus subtilis two-component regulatory systems; Kobayashi K et al.; It has recently been shown through DNA microarray analysis of Bacillus subtilis two-component regulatory systems (DegS-DegU, ComP-ComA, and PhoR-PhoP) that overproduction of a response regulator of the two-component systems in the background of a deficiency of its cognate sensor kinase affects the regulation of genes, including its target ones . The genome-wide effect on gene expression caused by the overproduction was revealed by DNA microarray analysis . In the present work, we newly analyzed 24 two-component systems by means of this strategy, leaving out 8 systems to which it was unlikely to be applicable . This analysis revealed various target gene candidates for these two-component systems . It is especially notable that interesting interactions appeared to take place between several two-component systems . Moreover, the probable functions of some unknown two-component systems were deduced from the list of their target gene candidates . This work is heuristic but provides valuable information for further study toward a comprehensive understanding of the B . subtilis two-component regulatory systems . The DNA microarray data obtained in this work are available at the KEGG Expression Database website .

J Bacteriol, 2001 Dec, 183(24), 7329 - 40
Correlation between Bacillus subtilis scoC phenotype and gene expression determined using microarrays for transcriptome analysis; Caldwell R et al.; The availability of the complete sequence of the Bacillus subtilis chromosome (F . Kunst et al., Nature 390:249-256, 1997) makes possible the construction of genome-wide DNA arrays and the study of this organism on a global scale . Because we have a long-standing interest in the effects of scoC on late-stage developmental phenomena as they relate to aprE expression, we studied the genome-wide effects of a scoC null mutant with the goal of furthering the understanding of the role of scoC in growth and developmental processes . In the present work we compared the expression patterns of isogenic B . subtilis strains, one of which carries a null mutation in the scoC locus (scoC4) . The results obtained indicate that scoC regulates, either directly or indirectly, the expression of at least 560 genes in the B . subtilis genome . ScoC appeared to repress as well as activate gene expression . Changes in expression were observed in genes encoding transport and binding proteins, those involved in amino acid, carbohydrate, and nucleotide and/or nucleoside metabolism, and those associated with motility, sporulation, and adaptation to atypical conditions . Changes in gene expression were also observed for transcriptional regulators, along with sigma factors, regulatory phosphatases and kinases, and members of sensor regulator systems . In this report, we discuss some of the phenotypes associated with the scoC mutant in light of the transcriptome changes observed.

J Bacteriol, 2001 Dec, 183(24), 7318 - 28
Global transcriptional response of Bacillus subtilis to heat shock; Helmann JD et al.; In response to heat stress, Bacillus subtilis activates the transcription of well over 100 different genes . Many of these genes are members of a general stress response regulon controlled by the secondary sigma factor, sigma(B), while others are under control of the HrcA or CtsR heat shock regulators . We have used DNA microarrays to monitor the global transcriptional response to heat shock . We find strong induction of known sigma(B)-dependent genes with a characteristic rapid induction followed by a return to near prestimulus levels . The HrcA and CtsR regulons are also induced, but with somewhat slower kinetics . Analysis of DNA sequences proximal to newly identified heat-induced genes leads us to propose ~70 additional members of the sigma(B) regulon . We have also identified numerous heat-induced genes that are not members of known heat shock regulons . Notably, we observe very strong induction of arginine biosynthesis and transport operons . Induction of several genes was confirmed by quantitative reverse transcriptase PCR . In addition, the transcriptional responses measured by microarray hybridization compare favorably with the numerous previous studies of heat shock in this organism . Since many different conditions elicit both specific and general stress responses, knowledge of the heat-induced general stress response reported here will be helpful for interpreting future microarray studies of other stress responses.

J Bacteriol, 2001 Dec, 183(24), 7308 - 17
Bacillus subtilis metabolism and energetics in carbon-limited and excess-carbon chemostat culture; Dauner M et al.; The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood . We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing Bacillus subtilis . Based on {U-(13)C(6)}glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited B . subtilis is almost exclusively partitioned in maintenance metabolism and biomass formation . In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible . In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present . In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B . subtilis, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose . ATP-dependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.

J Bacteriol, 2001 Dec, 183(24), 7135 - 44
CheR- and CheB-dependent chemosensory adaptation system of Rhodobacter sphaeroides; Martin AC et al.; Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in three major operons and other, unlinked, loci . These include cheA(1) and cheR(1) (che Op(1)) and cheA(2), cheR(2), and cheB(1) (che Op(2)) . In-frame deletions of these cheR and cheB homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays . Under the conditions tested, CheR(2) and CheB(1) were essential for normal chemotaxis, whereas CheR(1) was not . cheR(2) and cheB(1), but not cheR(1), were also able to complement the equivalent E . coli mutants . However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in R . sphaeroides . In E . coli, CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors . This motif is not contained on any R . sphaeroides chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences . This suggests that CheR(2) either interacts with the NWETF motif of E . coli methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs . Methanol release measurements show that R . sphaeroides has an adaptation system that is different from that of Bacillus subtilis and E . coli, with methanol release measurable on the addition of attractant but not on its removal . Intriguingly, CheA(2), but not CheA(1), is able to phosphorylate CheB(1), suggesting that signaling through CheA(1) cannot initiate feedback receptor adaptation via CheB(1)-P.

Antimicrob Agents Chemother, 2001 Dec, 45(12), 3566 - 73
Gene yerP, involved in surfactin self-resistance in Bacillus subtilis; Tsuge K et al.; Surfactin is a cyclic lipopeptide biosurfactant . Transposon mutagenesis was performed in Bacillus subtilis strain 168, and a surfactin-susceptible mutant, strain 801, was isolated . Analysis of the region of insertion revealed that yerP was the determinant of surfactin self-resistance . YerP had homology with the resistance, nodulation, and cell division (RND) family proton motive force-dependent efflux pumps only characterized in gram-negative strains . The yerP-deficient strain 802, in which the internal region of the yerP gene of B . subtilis strain 168 was deleted, showed susceptibility to acriflavine and ethidium bromide . When strain 802 was converted to a surfactin producer by introducing a functional sfp which encodes a 4'-phosphopantetheinyl transferase and is mutated in B . subtilis strain 168, this yerP-deficient strain produced surfactin, although surfactin production was significantly reduced . The expression of yerP was at its maximum at the end of the logarithmic growth phase and was not induced by surfactin . yerP is the first RND-like gene characterized in gram-positive strains and is supposed to be involved in the efflux of surfactin.

J Biol Chem, 2002 Feb 1, 277(5), 3698 - 707 Epub 2001 Nov 09.
Identification, characterization, and crystal structure of Bacillus subtilis nicotinic acid mononucleotide adenylyltransferase; Olland AM et al.; The nadD gene, encoding the enzyme nicotinic acid mononucleotide (NaMN) adenylyltransferase (AT), is essential for the synthesis of NAD and subsequent viability of the cell . The nadD gene in Bacillus subtilis (yqeJ) was identified by sequence homology with other bacterial nadD genes and by biochemical characterization of the gene product . NaMN AT catalyzes the reversible adenylation of both NaMN and the nicotinamide mononucleotide (NMN) but shows specificity for the nicotinate . In contrast to other known NMN ATs, biophysical characterizations reveal it to be a dimer . The NaMN AT crystal structure was determined for both the apo enzyme and product-bound form, to 2.1 and 3.2 A, respectively . The structures reveal a "functional" dimer conserved in both crystal forms and a monomer fold common to members of the nucleotidyl-transferase alpha/beta phosphodiesterase superfamily . A structural comparison with family members suggests a new conserved motif (SXXXX(R/K)) at the N terminus of an alpha-helix, which is not part of the shared fold . Interactions of the nicotinic acid with backbone atoms indicate the structural basis for specificity.

Mol Microbiol, 2001 Oct, 42(2), 383 - 94
Loss-of-function mutations in yjbD result in ClpX- and ClpP-independent competence development of Bacillus subtilis; Nakano MM et al.; Mutations in clpP and clpX have pleiotropic effects on growth and developmentally regulated gene expression in Bacillus subtilis . ClpP and ClpX are needed for expression of comK, encoding the competence transcription factor required for the expression of genes within the competence regulon . ClpP, in combination with the ATPase ClpC, degrades the inhibitor of ComK, MecA . Proteolysis of MecA is stimulated by a small protein, ComS, which interacts with MecA . Suppressor mutations (cxs) were isolated that bypass the requirement for clpX for comK expression . These were found also to overcome the defect in comK expression conferred by a clpP mutation . These mutations were identified as missense mutations (cxs-5, -7 and -12) and a nonsense (UAG) codon substitution (cxs-10) in the yjbD coding sequence in a locus linked to mecA . That a yjbD disruption confers the cxs phenotype, together with its complementation by an ectopically expressed copy of yjbD, indicated that the suppressor alleles bear recessive, loss-of-function mutations of yjbD . ClpP- and ClpX-independent comK expression rendered by inactivation of yjbD was still medium-dependent and required ComS . MecA levels in a clpP-yjbD mutant were lower that those of clpP mutant cells and ComK protein concentration in the clpP mutant was restored to wild-type levels by the yjbD mutation . Consequently, the yjbD mutation bypasses the defect in competence development conferred by clpP and clpX . YjbD protein is barely detectable in wild-type cells, but is present in large amounts in the clpP mutant cells . The results suggest that the role of ClpP in competence development is to degrade YjbD protein so that ComS can productively interact with the MecA-ClpC-ComK complex . Alternatively, the result could suggest that YjbD has a negative effect on regulated proteolysis and that MecA is degraded independently of ClpP when YjbD is absent.

Arch Microbiol, 2001 Nov, 176(5), 377 - 80
Functional analysis of the Streptomyces lividans type I signal peptidases; Geukens N et al.; Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins . In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases . In this work, the different sip genes were cloned and expressed . Subsequently, the Sip proteins were purified to raise antibodies . Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively . Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo . The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.

Microbiology, 2001 Nov, 147(Pt 11), 2933 - 41
Transcriptional responses during outgrowth of Bacillus subtilis endospores; Horsburgh MJ et al.; The Bacillus subtilis 168 genome contains an array of alternative sigma factors, many of which play important roles in reprogramming expression during stress and sporulation . The role of the different sigma factors during outgrowth, when the germinated endospore is converted back to a vegetative cell, is less well characterized . The activity of the alternative sigma factors sigmaB, sigmaD and sigmaH during endospore outgrowth was analysed by Northern blotting and lacZ reporter assays . While sigmaD and sigmaH were transcriptionally active during outgrowth, sigmaB-dependent transcription was not observed until after the first cell division, when growth slowed . Using an IPTG-controllable copy of sigA, an optimal level of expression was required to maintain growth rate at the end of outgrowth . The genes encoding the putative extracytoplasmic function (ECF) sigma factors sigmaI, sigmaV, sigmaW, sigmaZ and YlaC were insertionally inactivated using pMUTIN4 . These strains, together with sigM and sigX mutants, were tested to determine their role and measure their expression during endospore outgrowth . Transcripts or beta-galactosidase activity were observed for each of the ECF sigma factors early after germination . With the exception of MJH003 (sigM), which showed an exacerbated salt stress defect, inactivation of the ECF sigma factor genes did not affect outgrowth in the conditions tested.

Microbiology, 2001 Nov, 147(Pt 11), 2925 - 32
In vivo roles of the germination-specific lytic enzymes of Bacillus subtilis 168; Atrih A et al.; Germination of endospores of Bacillus subtilis involves the activities of several germination-specific lytic enzymes, including glucosaminidase and lytic transglycosylase . Another non-hydrolytic activity, likely to be due to an epimerase, also occurs . The effect of pH on enzyme activities and the overall germination rate was measured . Optimal germination occurred between pH 7-9; however, optimum glucosaminidase and epimerase activities were noted at pH 5 . Conversely, the lytic transglycosylase activity was greatest at pH 8 . Treatment of spores (15 min) with heat (90 degrees C) or NaOH (0.25 M) led to impaired cortex hydrolysis/modification, but with <20% loss in viability . Analysis of muropeptides in the germination exudate revealed a reduction of >85% in glucosaminidase and epimerase products, when compared to untreated spores . Conversely, lytic transglycosylase activity was increased by alkali or heat treatment, which was possibly due to increased substrate availability . FB101 (sleB) spores, which lack lytic transglycosylase activity, showed 90-fold greater loss in viability than the wild-type after 1 h at 90 degrees C . Similarly, 97% of FB101 (sleB) spores were unable to form a colony on nutrient agar after 130 min exposure to 0.25 M NaOH at 4 degrees C, whereas the wild-type was unaffected . This demonstrates the crucial role of the lytic transglycosylase in cortex hydrolysis of damaged spores . The respective targets of heat and alkali in spores and their role during germination are discussed.

Microbiology, 2001 Nov, 147(Pt 11), 2913 - 23
SvpA, a novel surface virulence-associated protein required for intracellular survival of Listeria monocytogenes; Borezee E et al.; A previously unknown protein, designated SvpA (surface virulence-associated protein) and implicated in the virulence of the intracellular pathogen Listeria monocytogenes, was identified . This 64 kDa protein, encoded by svpA, is both secreted in culture supernatants and surface-exposed, as shown by immunogold labelling of whole bacteria with an anti-SvpA antibody . Analysis of the peptide sequence revealed that SvpA contains a leader peptide, a predicted C-terminal transmembrane region and a positively charged tail resembling that of the surface protein ActA, suggesting that SvpA might partially reassociate with the bacterial surface by its C-terminal membrane anchor . An allelic mutant was constructed by disrupting svpA in the wild-type strain LO28 . The virulence of this mutant was strongly attenuated in the mouse, with a 2 log decrease in the LD50 and restricted bacterial growth in organs as compared to the wild-type strain . This reduced virulence was not related either to a loss of adherence or to a lower expression of known virulence factors, which remained unaffected in the svpA mutant . It was caused by a restriction of intracellular growth of mutant bacteria . By following the intracellular behaviour of bacteria within bone-marrow-derived macrophages by confocal and electron microscopy studies, it was found that most svpA mutant bacteria remained confined within phagosomes, in contrast to wild-type bacteria which rapidly escaped to the cytoplasm . The regulation of svpA was independent of PrfA, the transcriptional activator of virulence genes in L . monocytogenes . In fact, SvpA was down-regulated by MecA, ClpC and ClpP, which are highly homologous to proteins of Bacillus subtilis forming a regulatory complex controlling the competence state of this saprophyte . The results indicate that: (i) SvpA is a novel factor involved in the virulence of L . monocytogenes, promoting bacterial escape from phagosomes of macrophages; (ii) SvpA is, at least partially, associated with the surface of bacteria; and (iii) SvpA is PrfA-independent and controlled by a MecA-dependent regulatory network.

Drug Dev Ind Pharm, 2001 Sep, 27(8), 825 - 9
Calcium alginate microspheres of Bacillus subtilis; Lamas MC et al.; Microspheres of Bacillus subtilis were prepared using sodium alginate . Some typical properties of microencapsulated systems, such as microorganism content, particle size, and germination time, were studied . Calcium alginate microspheres were obtained by the emulsification method, dripping into a solution of calcium salt . The conditions of the preparation steps were very soft to produce calcium alginate microspheres containing cells with no apparent changes in general biological properties . The hydrogel matrix provides protection without preventing communication with the surrounding medium.

Extremophiles, 2001 Oct, 5(5), 351 - 4
A globin-coupled oxygen sensor from the facultatively alkaliphilic Bacillus halodurans C-125; Hou S et al.; We have recently discovered heme-containing signal transducers from the archaeon Halobacterium salinarum (HemAT-Hs) and the gram-positive bacterium Bacillus subtilis (HemAT-Bs) . These proteins bind diatomic oxygen and trigger aerotactic responses . We identified that HemAT oxygen-sensing domains contain a globin-coupled sensor (GCS) motif, which exists as a two-domain transducer, having no similarity to the PAS domain (Period circadian protein, Ah receptor nuclear translocator protein, Single-minded protein) superfamily transducers . Using the GCS motif, we predicted that a 439-amino-acid protein annotated as a methyl-accepting chemotaxis protein (MCP) in the facultatively alkaliphilic bacterium Bacillus halodurans is a globin-coupled oxygen sensor . We cloned, expressed, and purified GCS(Bh), and performed its spectral analysis . GCS(Bh), binds heme and shows myoglobin-like spectra . This suggests that GCS(Bh) acts as an oxygen sensor and transmits a conformational signal through a linked signaling domain to trigger an aerotactic response in B . halodurans.

Extremophiles, 2001 Oct, 5(5), 333 - 41
Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake; van Solingen P et al.; Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes . The strains were identified as novel Streptomyces spp . by 16S rDNA sequence analysis . A cellulase gene (cel12A) from Streptomyces sp . strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli . From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids . From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids . The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively . FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD . Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively . The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain . Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene . The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C . The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.

J Bacteriol, 2001 Dec, 183(23), 6965 - 70
Analysis of early promoters of the Bacillus bacteriophage GA-1; Horcajadas JA et al.; Bacteriophage GA-1, which infects Bacillus sp . strain G1R, is evolutionarily related to phage phi29, which infects Bacillus subtilis . We report the characterization of several GA-1 promoters located at either end of its linear genome . Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of phi29 or related phages . These unique promoters are active at early infection times and are repressed at late times . In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter . The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle . The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds.

J Bacteriol, 2001 Dec, 183(23), 6815 - 21
Modulation of anaerobic energy metabolism of Bacillus subtilis by arfM (ywiD); Marino M et al.; Bacillus subtilis grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes . The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation . Mutation of the open reading frame ywiD located upstream of the respiratory nitrate reductase operon narGHJI resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth . Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged . Anaerobic induction of nasDE expression was found to be significantly dependent on intact ywiD, while anaerobic narGHJI expression was ywiD independent . Anaerobic transcription of hmp, encoding a flavohemoglobin-like protein, and of the fermentative operons lctEP and alsSD, responsible for lactate and acetoin formation, was partially dependent on ywiD . Expression of pta, encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced by ywiD . Transcription of the ywiD gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site . Anaerobic induction of ywiD by resDE was found to be indirect via resDE-dependent activation of fnr . The ywiD gene is subject to autorepression and nitrite repression . These results suggest a ResDE --> Fnr --> YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of B . subtilis . Therefore, ywiD was renamed arfM for anaerobic respiration and fermentation modulator.

Adv Space Res, 2001, 27(9), 1571 - 9
Population dynamics of transgenic microorganisms in the different microecosystem conditions; Popova LY et al.; The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems . Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap (r) Lux+) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human alpha 2-interferon Km (r) Inf+), planned for employment as a living vaccine under varying environmental conditions . Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated . The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied . This trend has been shown to be independent of the microcosm type and its complexity . Grant numbers: 99-04-96017, 25, 00-07-9011 . c 2001 . COSPAR . Published by Elsevier Science Ltd . All rights reserved.

J Biol Chem, 2002 Feb 22, 277(8), 5849 - 57 Epub 2001 Nov 02.
Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A(2); Koduri RS et al.; Group IIA secreted phospholipase A(2) (sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo . We have analyzed the bactericidal activity of the full set of recombinant murine and human groups I, II, V, X, and XII sPLA2s on Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli . The rank order potency among human sPLA2s against Gram-positive bacteria is group IIA > X > V > XII > IIE > IB, IIF (for murine sPLA2s: IIA > IID > V > IIE > IIC, X > IB, IIF), and only human group XII displays detectable bactericidal activity against the Gram-negative bacterium E . coli . These studies show that highly basic sPLA2s display potent bactericidal activity with the exception of the ability of the acidic human group X sPLA2 to kill Gram-positive bacteria . By studying the Bacillus subtilis and S . aureus bactericidal potencies of a large panel of human group IIA mutants in which basic residues were mutated to acidic residues, it was found that: 1) the overall positive charge of the sPLA2 is the dominant factor in dictating bactericidal potency; 2) basic residues on the putative membrane binding surface of the sPLA2 are modestly more important for bactericidal activity than are other basic residues; 3) relative bactericidal potency tracks well with the ability of these mutants to degrade phospholipids in the bacterial membrane; and 4) exposure of the bacterial membrane of Gram-positive bacteria by disruption of the cell wall dramatically reduces the negative effect of charge reversal mutagenesis on bactericidal potency.

Biochim Biophys Acta, 2001 Oct 31, 1521(1-3), 81 - 8
Study on construction of a cDNA library corresponding to an amino acid-specific tRNA and influence of the modified nucleotide upon nucleotide misincorporations in reverse transcription; Matsugi J et al.; The construction of a cDNA library corresponding to an amino acid-specific tRNA and the influence of the modified nucleotide in the tRNA upon misincorporation in reverse transcription were investigated . The distinctive feature of the constructive strategy is that the cDNA library was prepared in connection with the charging activity of the tRNA . The aminoacyl-tRNA was captured selectively by using a biotin-avidin system . After hydrolysis of the ester bond, the tRNA was collected as an amino acid-specific tRNA pool, and a poly(A) tail was attached to the CCA terminus for reverse transcription . To the 3'-terminus of the transcribed cDNA, poly (dC) was added by terminal deoxynucleotidyl transferase, and the cDNA was amplified by PCR . The double-stranded cDNA was used for transformation of Escherichia coli JM109 . Sequence analyses of the obtained clones bearing the tRNA genes revealed that a few nucleotide substitutions occurred at the location where the modified nucleotides exist . Among them, it was noteworthy that 1-methyladenosine (m(1)A22) in the D-loop of Bacillus subtilis tRNA(Ser) was recognized as G in the reverse transcription and the result revealed different tendency of the misincorporation, which has been shown in the study of HIV-1 reverse transcription.

Biochemistry, 2001 Nov 6, 40(44), 13331 - 41
Transient-state reduction and steady-state kinetic studies of menaquinol oxidase from Bacillus subtilis, cytochrome aa3-600 nm . Spectroscopic characterization of the steady-state species; Mattatall NR et al.; Cytochrome aa3-600 or menaquinol oxidase, from Bacillus subtilis, is a member of the heme-copper oxidase family . Cytochrome aa3-600 contains cytochrome a, cytochrome a3, and CuB, and each is coordinated via histidine residues to subunit I . Subunit II of cytochrome aa3-600 lacks CuA, which is a common feature of the cytochrome c oxidase family members . Anaerobic reduction of cytochrome aa3-600 by the substrate analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, single-wavelength spectrometry . Global analysis of time-resolved, multiwavelength spectra shows that during these distinct phases cytochromes a and a3 are both reduced . Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a reduction, whereas cytochrome a3-cyanide reduction is slow . The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function of DMN concentration with a Km of 300 microM and a maximal turnover of 63.5 s(-1) . Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a3-CuB, whereas cytochrome a remains oxidized . Electron paramagnetic resonance (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01 . A model of the catalytic cycle for cytochrome aa3-600 proposes initial electron delivery from DMN to cytochrome a, followed by rapid heme to heme electron transfer, and suggests possible origins of the radical signal in the steady-state form of the enzyme.

Pharmazie, 2001 Oct, 56(10), 825 - 7
Eudesmanolides, aromatic derivatives, and other constituents from Carpesium cernuum; Yang C et al.; A new eudesmanolide 13-hydroxy-4 alpha H-eudesman-5,7(11)-dien-12,8 beta-olide (1) and a new aromatic derivative 3-methyl-8-acetoxy-9,10-diisobutanoyloxy-p-cymene (6), along with ten known compounds were isolated from the roots of Carpesium cernuum L . Their structures were elucidated by spectral methods (IR, EIMS, FAB-MS, 1D and 2D NMR) . Compound 2, 3 and compound 10 exhibited moderate antibacterial activity against Bacillus subtilis, Escherichia coli and Staphylococcus aureus.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 55 - 60
Bacillus subtilis contains a cyclodextrin-binding protein which is part of a putative ABC-transporter; Kamionka A et al.; Bacillus subtilis is able to grow on alpha-, beta- and gamma-cyclodextrins as a carbon source via a yet unknown metabolizing system . Sequence analysis of the B . subtilis genome reveals that the putative yvfK-yvfO operon seems to be involved in cyclodextrin utilization, containing the open reading frame yvfK, now termed cycB . The amino acid sequence derived from the DNA sequence bears high similarities to solute-binding proteins from B . subtilis, as well as to cymE from Klebsiella oxytoca and malE from Escherichia coli, both encoding solute-binding proteins able to interact with cyclodextrins . A {His}(6)-tagged variant of CycB from B . subtilis was constructed, overproduced in E . coli and purified . The modified protein has been used to study its substrate specificity by surface plasmon resonance and fluorescence spectroscopy . From these data, CycB can be classified as a cyclodextrin-binding protein which interacts with all three natural cyclodextrins: alpha, beta and gamma, thereby showing the highest affinity to gamma-cyclodextrin.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1718 - 21 Epub 2001 Oct 25.
Preparation and crystallization of a Bacillus subtilis arsenate reductase; Guan Z et al.; Arsenate reductase (AR) in B . subtilis is encoded by the chromosomal arsC gene . Together with arsB and arsR, arsC participates in detoxification processes for the arsenate and arsenite ions . Full-length arsenate reductase without any modification has been expressed in Escherichia coli and purified in a soluble form . The recombinant protein has been crystallized at 277 K using polyethyleneglycol (PEG) or poly(ethyleneglycol) methyl ether (PME) as the main precipitant . At least two forms of crystals large enough for data collection have been obtained from wild-type protein under different conditions . An orthorhombic crystal diffracted to beyond 2.2 A with space group P2(1)2(1)2(1) and unit-cell parameters a = 51.22, b = 91.62, c = 101.93 A . A near-complete data set has been collected to 2.5 A . The application of the flash-annealing technique was crucial for high resolution during the data collection . The SeMet-substituted AR has also been produced and crystallized under very similar conditions as the wild type, but the unit-cell parameters are very different . The crystals of the SeMet protein diffracted to higher resolution than those of the wild type.

Mol Microbiol, 2001 Oct, 42(1), 245 - 55
DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome; Bruand C et al.; Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks . The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA . We have proposed previously that three proteins involved in the initiation of replication at oriC in B . subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium . However, the involvement of these proteins in replication restart has not yet been studied . Here, we describe dnaB mutations that suppress the phenotypes of B . subtilis priA mutants . In a representative mutant, the DnaC helicase is loaded onto single-stranded DNA in a PriA-independent, DnaD- and DnaI-dependent manner . These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B . subtilis . Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B . subtilis.

Mol Microbiol, 2001 Oct, 42(1), 205 - 14
SigB, an RNA polymerase sigma factor required for osmoprotection and proper differentiation of Streptomyces coelicolor; Cho YH et al.; A gene (sigB) encoding an alternative sigma factor sigmaB in Streptomyces coelicolor A3(2) was isolated and characterized . It encodes a polypeptide of 281 amino acids (31 546 Da) and is highly homologous to Bacillus subtilis sigmaB . The sigB coding region is preceded by four open reading frames (ORFs): dpsA, orfA, rsbB and rsbA in sequential order . RNA analyses revealed that rsbB, rsbA and sigB constitute an operon (sigB operon) . Transcripts were produced constitutively from a promoter (sigBp2) upstream of the rsbB coding region, contributing to the basal level expression of sigmaB protein . An inducible promoter (sigBp1) resembling the catB promoter (catBp) was located between the rsbA and sigB coding regions . Transcripts from sigBp1 dramatically increased as cells differentiated on solid media, at the stationary phase in liquid media or by osmotic stresses similar to the behaviour of catBp transcripts . Both catBp and sigBp1 promoters were recognized specifically by sigmaB-containing RNA polymerase in vitro . Disruption of the sigB gene abolished not only the differentiation-associated expression but also the osmotic induction of the catB gene, indicating that catBp is under the control of sigmaB . The sigB mutant exhibited a similar phenotype to the catB mutant, being sensitive to hyperosmolarity, blocked in forming aerial mycelium and with skewed antibiotic production . Therefore, we conclude that sigmaB ensures the proper differentiation and osmoprotection of S . coelicolor cells, primarily via regulation of the expression of catalase B.

Mol Microbiol, 2001 Oct, 42(1), 159 - 66
The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer; Possoz C et al.; pSAM2 is an 11 kb integrative element from Streptomyces ambofaciens that is capable of conjugal transfer . A system based on differential DNA modification by SalI methyltransferase was used to localize pSAM2 in the donor or recipient strain, and thus to determine the various steps associated with transfer . Initiation (i.e . excision and replication of pSAM2 in the donor) occurs a few hours after mating with a recipient strain . pSAM2 replicates in the recipient strain, spreads within the mycelium and then integrates into the chromosome . Transfer generally involves single-stranded DNA . In Streptomyces, only a few genes, such as traSA for pSAM2, are required for conjugal transfer . Using the differential sensitivity to the SalI restriction-modification system of transfers involving single- and double-stranded DNA, we found that pSAM2 was probably transferred to the recipient as double-stranded DNA . This provides the first experimental evidence for the transfer of double-stranded DNA during bacterial conjugation . Thus, TraSA, involved in pSAM2 transfer, and SpoIIIE, which is involved in chromosome partitioning in Bacillus subtilis, display similarities in both sequence and function: both seem to transport double-stranded DNA actively, either from donor to recipient or from mother cell to prespore.

Mol Microbiol, 2001 Oct, 42(1), 133 - 43
A new family of aspartyl phosphate phosphatases targeting the sporulation transcription factor Spo0A of Bacillus subtilis; Perego M; The initiation of the sporulation developmental pathway in Bacillus subtilis is controlled by the phospho-relay, a multicomponent signal transduction system . Multiple positive and negative signals are integrated by the phosphorelay through the opposing activities of histidine protein kinases and aspartyl phosphate phosphatases . Three members of the Rap family of phosphatases (RapA, RapB and RapE) specifically dephosphorylate the Spo0F approximately P response regulator intermediate, while the Spo0A approximately P transcription factor is specifically dephosphorylated by the Spo0E phosphatase and, as shown here, the newly identified YnzD and YisI proteins . The products of the YnzD and YisI genes are highly homologous to Spo0E and define a new family of phosphatases with a distinct signature motif in their amino acid sequence . As negative regulators of the developmental pathway, YnzD and YisI inhibit spore formation if over-expressed, while a chromosomal deletion of their coding sequences results in increased sporulation frequency . Transcription of the ynzD, yisI and spo0E genes is differentially regulated and generally induced by growth conditions antithetical to sporulation . Negative signals interpreted by aspartyl phosphate phosphatases appear to be a common mechanism in Gram-positive spore-forming microorganisms.

Mol Microbiol, 2001 Oct, 42(1), 101 - 9
Variation in 16S-23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates; Shaver YJ et al.; The genome of the Bacillus subtilis 168-type strain contains 10 ribosomal RNA (rRNA) operons . In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnI-H-G and rrnJ-W, lack a trinucleotide signature region . Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168-type strain and B . subtilis 168-like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence . However, PCR of the ISR segment for five other B . subtilis 168 isolates generated only a 114 bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains . Additional genetic variability between the seven B . subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis . The 168-type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern . Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067 . Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B . subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding . These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B . subtilis subgroup 168 . It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.

Biosci Biotechnol Biochem, 2001 Sep, 65(9), 2007 - 15
Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance; Tsukamoto Y et al.; To invent a functional natto promoting bone formation, the construction of a strain with high productivity of vitamin K2 (menaquinone-7: MK-7), which is important in the carboxylation of a kind of bone protein participating in bone formation, osteocalcin, was investigated . To screen for a strain appropriate to making natto (a Japanese traditional fermented soybean food) with high productivity of MK-7, a combination of analog resistance to the compounds on the biosynthetic pathway of menaquinones with mutation was done . Consequently, strain OUV23481, with 2-fold higher productivity (1,719 microg/100 g natto) of MK-7 than that of a commercial strain, was constructed as a mutant with analog resistance to 1-hydroxy-2-naphthoic acid (HNA), p-fluoro-D,L-phenylalanine (pFP), m-fluoro-D,L-phenylalanine (mFP), and beta-2-thienylalanine (betaTA) . This strain was classified as Bacillus subtilis (natto) . The natto made using this strain was evaluated to have a good quality as natto in all the viewpoints of appearance, flavor, taste, texture, and stringiness.

J Bacteriol, 2001 Nov, 183(22), 6688 - 93
Precise deletion of tagD and controlled depletion of its product, glycerol 3-phosphate cytidylyltransferase, leads to irregular morphology and lysis of Bacillus subtilis grown at physiological temperature; Bhavsar AP et al.; Using a previously reported conditional expression system for use in Bacillus subtilis (A . P . Bhavsar, X . Zhao, and E . D . Brown, Appl . Environ . Microbiol . 67:403-410, 2001), we report the first precise deletion of a teichoic acid biosynthesis (tag) gene, tagD, in B . subtilis . This teichoic acid mutant showed a lethal phenotype when characterized at a physiological temperature and in a defined genetic background . This tagD mutant was subject to full phenotypic rescue upon expression of the complementing copy of tagD . Depletion of the tagD gene product (glycerol 3-phosphate cytidylyltransferase) via modulated expression of tagD from the amyE locus revealed structural defects centered on shape, septation, and division . Thickening of the wall and ultimately lysis followed these events.

J Bacteriol, 2001 Nov, 183(22), 6573 - 8
In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis; Quisel JD et al.; The phosphorylated form of the response regulator Spo0A (Spo0A~P) is required for the initiation of sporulation in Bacillus subtilis . Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B . Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood . We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B . We also determined the effect of Spo0E, a Spo0A~P-specific phosphatase, on sporulation of strains containing the spo0A mutations . Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors . In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants . This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.

Sci Total Environ, 2001 Oct 20, 278(1-3), 231 - 7
Storage effects on bacterial concentration: determination of impinger and filter samples; Li CS et al.; Effects of storage on the colony recovery of airborne bacterial samples were evaluated in a laboratory test chamber . Escherichia coli cells and Bacillus subtilis spores were generated by a Collison three-jet nebulizer . Bioaerosol samples were collected by three sampling methods, AGI-30 impingers, Nuclepore filtration and elution methods, and gelatin filters . Effects of storage time was determined by the ratio, Ct/C0, where Ct and C0 were the CFU concentrations of the simultaneously collected samples stored for t and 0 h, respectively . The effect of storage temperature was also studied for AGI-30 samples stored at 25 and 4 degrees C . For impinger samples, it was demonstrated that the bioefficiency of bacterial bioaerosols could survive in the impinger fluid, and even bud more cells at room temperature . In addition, the inhibition effect of refrigerated samples was observed . Therefore, we suggest that samples collected by an impingement method should be refrigerated and processed as soon as possible to avoid the increase of bacterial culturability . Moreover, the effect of storage time on filtration collection for B . subtilis spores was demonstrated to be insignificant . However, E . coli recovery from filters was demonstrated to decrease as storage time increased . It was concluded that the recovery would not decrease during storage if bioefficiencies of the sampling methods were excellent, for example, using filters to collect B . subtilis spores or impingers to collect E . coli cells.

Genetika, 2001 Sep, 37(9), 1300 - 3
{Study of the mechanism of regulating the activity of the ribC gene in Bacillus subtilis}; Kreneva RA et al.; Sequence analysis of several Bacillus subtilis mutants with increased activity of flavokinase/FAD-synthase and the results of Northern hybridization showed that the TTGCCG-17n-TACATT motif localized to the C-end of the truB gene is a regulatory region that controls the ribC gene at the level of transcription.

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12538 - 43 Epub 2001 Oct 16.
Development of a two-part transcription probe to determine the completeness of temporal and spatial compartmentalization of gene expression during bacterial development; Li Z et al.; We have developed a two-part test, using the Bacillus subtilis sacB/SacY transcription antitermination system, to evaluate the completeness of temporal and spatial compartmentalization of gene expression during bacterial cell development . Transcription of sacY(1-55) (encoding a constitutively active form of the antiterminator, SacY) is directed by one promoter, whereas transcription of sacB'-'lacZ (the target of SacY action) is directed by the same or another promoter . To obtain beta-galactosidase activity, SacY(1-55) needs to be present when sacB'-'lacZ is being transcribed . We tested the system by analyzing the spatial compartmentalization of the activities of RNA polymerase final sigma factors, which are tightly regulated during sporulation of B . subtilis: final sigma(F) and then final sigma(G) in the prespore, final sigma(E) and then final sigma(K) in the mother cell . We have confirmed that the activities of final sigma(F) and final sigma(E) are spatially compartmentalized . We have demonstrated that there is also sharp temporal compartmentalization, with little or no overlap in the activities of final sigma(F) and final sigma(G) or of final sigma(E) and final sigma(K) . In contrast, we found no compartmentalization of the activity of the main vegetative factor, final sigma(A), which continued to be active alongside all of the sporulation-specific final sigma factors . We also found no temporal compartmentalization of expression of loci that are activated during the development of competent cells of B . subtilis, a developmental program distinct from spore formation . A possible mechanism to explain the temporal compartmentalization of final sigma(F) and final sigma(G) activities is that the anti-sigma factor SpoIIAB transfers from final sigma(G) to final sigma(F).

EMBO Rep, 2001 Nov, 2(11), 1003 - 6 Epub 2001 Oct 17.
A hybrid bacterial replication origin; Seitz H et al.; We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC . The AT-rich region could be unwound by E . coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble . The results show that species specificity, i.e . which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.

Eur J Biochem, 2001 Oct, 268(20), 5321 - 8
Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis; Ashiuchi M et al.; An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated . The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B . subtilis . We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate . The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer . Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed . The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.

Protein Sci, 2001 Nov, 10(11), 2317 - 24
Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure; Krath BN et al.; Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity . The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors . All of these properties are characteristic for class II PRPP synthases . K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively . Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer . Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved . Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases . Similarly, residues important for oligomerization of the B . subtilis enzyme show little conservation in the spinach enzyme . In contrast, residues of the active site of B . subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.

FEBS Lett, 2001 Oct 12, 506(3), 235 - 8
In vitro transactivation of Bacillus subtilis RNase P RNA; Kim H et al.; Deletion of the 'signature' PL5.1 stem-loop structure of a Type II RNase P RNA diminished its catalytic activity . Addition of PL5.1 in trans increased catalytic efficiency (kcat/KM) rather than kcat . Transactivation was due to the binding of a single PL5.1 species per ribozyme with an apparent Kd near 600 nM . The results are consistent with the role of PL5.1 being to position the substrate near the active site of the ribozyme, and with the hypothesis that ribozymes can evolve by accretion of preformed smaller structures.

J Mol Biol, 2001 Oct 12, 313(1), 111 - 22
The 1.2 A structure of a novel quorum-sensing protein, Bacillus subtilis LuxS; Ruzheinikov SN et al.; In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing . The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione . The crystal structure of Bacillus subtilis LuxS has been determined at 1.2 A resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 and 2.3 A resolution, respectively . These structures show that LuxS is a homodimer with an apparently novel fold based on an eight-stranded beta-barrel, flanked by six alpha-helices . Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits . S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme-bound zinc ion . Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125-129 and those at the N terminus . The structure contains an oxidised cysteine residue in the active site whose role in the biological process of LuxS has not been determined . The autoinducer-2 signalling pathway has been linked to aspects of bacterial virulence and pathogenicity . The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics .

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 816 - 9
A PCR test to identify bacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment; Wattiau P et al.; A PCR test based on the 16S rRNA gene was set up that could identify any of the five species of the 'Bacillus subtilis group' (B . subtilis, B . pumilus, B . atrophaeus, B . lichenijormis and B . amyloliquefaciens) . The test was directly applicable to single colonies and showed excellent specificity . In the mixed population context of wastewater analysis, direct detection of the target Bacillus species by PCR on either crude or purified DNA extracts had poor sensitivity . When assayed on cell suspensions derived from enriched wastewater samples, sensitivity was increased . Using a simple calibration method, it was possible to estimate the proportion of the target organisms . This method was found suitable for easy monitoring of a wastewater bioaugmentation experiment carried out with a mixture of sporulated Bacillus strains.

Nucleic Acids Res, 2001 Oct 15, 29(20), 4125 - 33
Species-specific differences in the operational RNA code for aminoacylation of tRNA(Trp); Xu F et al.; Identity elements play essential roles in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase . An operational RNA code relates amino acids to specific sequences and structural features of tRNA acceptor stems . In this study, a series of tRNA(Trp) variants was prepared by in vitro transcription and their efficiencies of aminoacylation by tryptophan (k(cat)/K(m)) were measured with the aid of Bacillus subtilis and human tryptophanyl-tRNA synthetases (TrpRS) . The identity elements in the operational RNA code of human tRNA(Trp) were found to be: major element, discriminator base A73; minor elements, G1/C72 and U5/G68 . From the cross-species aminoacylation assays, we conclude that the identity elements in tRNA(Trp) from B.subtilis and human all contribute to species-specific aminoacylation by TrpRS . Analyses of 22 TrpRS sequences covering three taxonomic domains (bacteria, eukarya and archaea) reveal that the sequences are divided into two evolutionarily distant groups . The same partition is also observed in the analyses of tRNA(Trp) acceptor stem sequences . Our data suggest that the two TrpRS groups may reflect co-adaptations needed to accommodate changes in the operational RNA code for tryptophan.

Antimicrob Agents Chemother, 2001 Nov, 45(11), 3104 - 8
Bronchopulmonary disposition of the ketolide telithromycin (HMR 3647); Muller-Serieys C et al.; Telithromycin (HMR 3647) is the first member of a new family of antimicrobials, the ketolides, developed specifically for the treatment of community-acquired respiratory tract infections . Telithromycin has proven in vitro activity against both common and atypical respiratory tract pathogens . The penetration of telithromycin into bronchopulmonary tissues and subsequent elimination from these sites were evaluated in four groups (groups A, B, C, and D) of six healthy male subjects who received telithromycin at 800 mg once daily for 5 days . Subjects in groups A, B, C, and D underwent fiberoptic bronchoscopy and bronchoalveolar lavage 2, 8, 24, and 48 h after receipt of the last dose, respectively . The concentration of telithromycin in the alveolar macrophages, epithelial lining fluid (ELF), and plasma was determined by the agar diffusion method with Bacillus subtilis ATCC 6633 as the test organism . The concentration of telithromycin in alveolar macrophages markedly exceeded that in plasma, reaching up to 146 times the concentration in plasma 8 h after dosing (median concentration, 81 mg/liter) . Telithromycin was retained in alveolar macrophages 24 h after dosing (median concentration, 23 mg/liter), and it was still quantifiable 48 h after dosing (median concentration, 2.15 mg/liter) . Telithromycin median concentrations in ELF also markedly exceeded concentrations in plasma (median concentration in ELF, 3.7 mg/liter 8 h after dosing) . Telithromycin achieves high and sustained concentrations in ELF and in alveolar macrophages, while it maintains adequate levels in plasma, providing an ideal pharmacokinetic profile for effective treatment of community-acquired respiratory tract infections caused by either common or atypical, including intracellular, respiratory tract pathogens.

J Infect Dis, 2001 Nov 1, 184(9), 1143 - 51 Epub 2001 Oct 12.
Characteristics of surfactant protein A and D binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria; van de Wetering JK et al.; Infection with gram-positive bacteria is a major cause of pneumonia . Surfactant proteins A (SP-A) and D (SP-D) are thought to play an important role in the innate immunity of the lung . Both proteins can bind to gram-positive bacteria . Until now, it was not known with which surface component(s) of gram-positive bacteria SP-A and SP-D interact . Lipoteichoic acid (LTA) and peptidoglycan (PepG) are components of the cell wall of gram-positive bacteria . By use of a solid phase-based binding assay, LTA of Bacillus subtilis was shown to be bound by SP-D but not by SP-A . Unmodified PepG of Staphylococcus aureus was bound by SP-D . SP-D binding to both LTA and PepG was calcium dependent and carbohydrate inhibitable . These results indicate that SP-D interacts with gram-positive bacteria via binding to the cell wall components LTA and PepG and that the carbohydrate recognition domain is responsible for this binding.

Bioorg Med Chem Lett, 2001 Nov 5, 11(21), 2879 - 81
Cryptoregiochemical analysis of an unusual bacterial desaturation; Fauconnot L et al.; The cryptoregiochemistry of the cold-induced Delta(5) desaturation of long chain fatty acids, as it occurs in Bacillus subtilis ATCC 23857, has been examined by measuring the individual primary deuterium kinetic isotope effects associated with the C-H bond cleavage at C-5 and C-6 . The results point to C-5 as the site of initial oxidation in Delta(5) desaturation.

J Bacteriol, 2001 Nov, 183(21), 6435 - 43
Localization of cold shock proteins to cytosolic spaces surrounding nucleoids in Bacillus subtilis depends on active transcription; Weber MH et al.; Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells Bacillus subtilis CSPs specifically localize to cytosolic regions surrounding the nucleoid . The subcellular localization of CSPs is influenced by the structure of the nucleoid . Decondensed chromosomes in smc mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present . As a control, histone-like protein HBsu localized to the nucleoids, while beta-galactosidase and GFP were detectable throughout the cell . After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells . However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid . Furthermore, we observed that nucleoids are more condensed and frequently abnormal in cspB cspC and cspB cspD double-mutant cells . This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids . In addition, we found that cspB cspD and cspB cspC double mutants are defective in sporulation, with a block at or before stage 0 . Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell . In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.

J Bacteriol, 2001 Nov, 183(21), 6422 - 8
Catalytic function of an alpha/beta hydrolase is required for energy stress activation of the sigma(B) transcription factor in Bacillus subtilis; Brody MS et al.; The general stress response of Bacillus subtilis is controlled by the sigma(B) transcription factor, which is activated in response to diverse energy and environmental stresses . These two classes of stress are transmitted by separate signaling pathways which converge on the direct regulators of sigma(B), the RsbV anti-anti-sigma factor and the RsbW anti-sigma factor . The energy signaling branch involves the RsbP phosphatase, which dephosphorylates RsbV in order to trigger the general stress response . The rsbP structural gene lies downstream from rsbQ in a two-gene operon . Here we identify the RsbQ protein as a required positive regulator inferred to act in concert with the RsbP phosphatase . RsbQ bound RsbP in the yeast two-hybrid system, and a large in-frame deletion in rsbQ had the same phenotype as a null allele of rsbP-an inability to activate sigma(B) in response to energy stress . Genetic complementation studies indicated that this phenotype was not due to a polar effect of the rsbQ alteration on rsbP . The predicted rsbQ product is a hydrolase or acyltransferase of the alpha/beta fold superfamily, members of which catalyze a wide variety of reactions . Notably, substitutions in the presumed catalytic triad of RsbQ also abolished the energy stress response but had no detectable effect on RsbQ structure, synthesis, or stability . We conclude that the catalytic activity of RsbQ is an essential constituent of the energy stress signaling pathway.

J Bacteriol, 2001 Nov, 183(21), 6294 - 301
Surface display of recombinant proteins on Bacillus subtilis spores; Isticato R et al.; We developed a novel surface display system based on the use of bacterial spores . A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC) . Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores . We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies . The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B . subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

J Bacteriol, 2001 Nov, 183(21), 6265 - 73
Cloning, sequencing, and characterization of the iturin A operon; Tsuge K et al.; Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A . Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined . The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC . The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production . The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase . The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively . Mycosubtilin, which is produced by B . subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser) . Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively . Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases . The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B . subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred . When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.

J Bacteriol, 2001 Nov, 183(21), 6175 - 83
Definition of the Bacillus subtilis PurR operator using genetic and bioinformatic tools and expansion of the PurR regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO; Saxild HH et al.; The expression of the pur operon, which encodes enzymes of the purine biosynthetic pathway in Bacillus subtilis, is subject to control by the purR gene product (PurR) and phosphoribosylpyrophosphate . This control is also exerted on the purA and purR genes . A consensus sequence for the binding of PurR, named the PurBox, has been suggested (M . Kilstrup, S . G . Jessing, S . B . Wichmand-Jorgensen, M . Madsen, and D . Nilsson, J . Bacteriol . 180:3900-3906, 1998) . To determine whether the expression of other genes might be regulated by PurR, we performed a search for PurBox sequences in the B . subtilis genome sequence and found several candidate PurBoxes . By the use of transcriptional lacZ fusions, five selected genes or operons (glyA, yumD, yebB, xpt-pbuX, and yqhZ-folD), all having a putative PurBox in their upstream regulatory regions, were found to be regulated by PurR . Using a machine-learning algorithm developed for sequence pattern finding, we found that all of the genes identified as being PurR regulated have two PurBoxes in their upstream control regions . The two boxes are divergently oriented, forming a palindromic sequence with the inverted repeats separated by 16 or 17 nucleotides . A computerized search revealed one additional PurR-regulated gene, ytiP . The significance of the tandem PurBox motifs was demonstrated in vivo by deletion analysis and site-directed mutagenesis of the two PurBox sequences located upstream of glyA . All six genes or operons encode enzymes or transporters playing a role in purine nucleotide metabolism . Functional analysis showed that yebB encodes the previously characterized hypoxanthine-guanine permease PbuG and that ytiP encodes another guanine-hypoxanthine permease and is now named pbuO . yumD encodes a GMP reductase and is now named guaC.

FEBS Lett, 2001 Sep 28, 506(1), 27 - 32
Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function; Ibrahim HR et al.; A catalytically inactive mutant of hen egg white lysozyme was constructed by site-directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram-positive bacteria . The catalytic residue aspartic acid at position 52 of lysozyme was substituted with serine (D52S-Lz) and the mutant cDNA was inserted into a yeast expression vector, pYES-2 . Western blot analysis indicated that the mutation did not affect secretion of the D52S-Lz lysozyme into the medium of the expressing Saccharomyces cerevisiae, INVSC1 . In addition, circular dichroism and fluorescence spectral analysis revealed no change in the structure of D52S-Lz compared to that of wild-type (Wt-Lz) lysozyme . The mutation (D52S) abolished the catalytic activity of lysozyme . Antimicrobial tests against Staphylococcus aureus and Bacillus subtilis revealed that the catalytically inactive D52S-Lz was as bactericidal as the Wt-Lz lysozyme . Heat treatment leading to enzyme inactivation had no effect on the bactericidal activity of either wild-type or the mutant D52S-Lz lysozyme . The binding affinity of D52S-Lz to the isolated peptidoglycan of S . aureus was unaffected . Our results provide the first demonstration of direct genetic evidence that the antimicrobial activity of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors.

Peptides, 2001 Oct, 22(10), 1549 - 53
A free terminal carboxylate group is required for PhrA pentapeptide inhibition of RapA phosphatase; Core LJ et al.; In the Bacillus subtilis phosphorelay signal transduction system for sporulation initiation, signals competing with the differentiation process are interpreted by aspartyl-phosphate phosphatases that specifically dephosphorylate the Spo0F or Spo0A response regulators . The RapA phosphatase is regulated by the PhrA pentapeptide that directly and specifically inhibits its activity . PhrA specificity for RapA inhibition is dependent upon the amino acid sequence of the peptide . Here we show that the pentapeptide affinity for the phosphatase requires a free carboxylate group at the C-terminal amino acid . A free C-terminal carboxylic acid PhrA pentapeptide inhibits RapA phosphatase activity at a 1:1 ratio and it is approximately 200 fold more active than a C-terminal amide peptide . Therefore, coordination of the terminal carboxylate group appears to be critical for peptide binding to RapA.

Peptides, 2001 Oct, 22(10), 1541 - 7
Pentapeptide regulation of aspartyl-phosphate phosphatases; Perego M et al.; Aspartyl-phosphate phosphatases are integral components of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis . The Rap and Spo0E families of protein phosphatases specifically dephosphorylate the sporulation response regulators Spo0F and Spo0A, respectively . The phosphatases interpret regulatory signals antithetical to sporulation and the Rap phosphatases are subject to inactivation by specific pentapeptides generated from an inactive peptide precursor . Additional regulatory signals are brought about by the complex activation circuit that generates the Phr pentapeptide inhibitors of Rap phosphatases . Phr peptide's recognition of the Rap phosphatase targets is remarkably specific . Specificity is dictated by the amino acid sequence of the pentapeptide . The identification of tetratricopeptide repeats in the Rap proteins may explain the mechanism by which Phr peptides bind to and inhibit the activity of Rap phosphatases.

J Biol Chem, 2001 Dec 7, 276(49), 45818 - 25
Early steps of Bacillus subtilis primosome assembly; Marsin S et al.; Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks . Their primary role is to recruit the replicative helicase onto single-stranded DNA . The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks . Binding of the PriA protein to forked DNA triggers its assembly . PriA is conserved in bacteria, but its primosomal partners are not . In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E . coli . Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin . Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly . They are both multimeric and bind individually to DNA . Furthermore, DnaD stimulates DnaB binding activities . DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B . subtilis on several DNA substrates . This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order . The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.

J Biol Chem, 2001 Dec 7, 276(49), 46422 - 8 Epub 2001 Oct 02.
Development and characterization of a series of soluble tetrameric and monomeric streptavidin muteins with differential biotin binding affinities; Qureshi MH et al.; The strong biotin-streptavidin interaction limits the application of streptavidin as a reversible affinity matrix for purification of biotinylated biomolecules . To address this concern, a series of single, double, and triple streptavidin muteins with different affinities to biotin were designed . The strategy involves mutating one to three strategically positioned residues (Ser-45, Thr-90, and Asp-128) that interact with biotin and other framework structure-maintaining residues of streptavidin . The muteins were produced in soluble forms via secretion from Bacillus subtilis . The impact of individual residues on the overall structure of streptavidin is reflected by the formation of monomeric streptavidin to different extents . Of the three targeted residues, Asp-128 has the most dramatic effect (Asp-128 > Thr-90 > Ser-45) . Conversion of all three targeted residues to alanine results in a soluble biotin binding mutein that exists 100% in the monomeric state . Both wild-type and mutated (monomeric and tetrameric) streptavidin proteins were purified, and their kinetic parameters (on- and off-rates) were determined using a BIAcore biosensor with biotin-conjugated bovine serum albumin immobilized to the sensor chip . This series of muteins shows a wide spectrum of affinity toward biotin (K(d) from 10(-6) to 10(-11) m) . Some of them have the potential to serve as reversible biotin binding agents.

J Biol Chem, 2001 Dec 14, 276(50), 47046 - 51 Epub 2001 Oct 02.
Purification and characterization of a Bacillus subtilis 168 nuclease, YokF, involved in chromosomal DNA degradation and cell death caused by thermal shock treatments; Sakamoto JJ et al.; We purified and characterized a 39-kDa Bacillus subtilis 168 nuclease that has been suggested in this laboratory to be involved in chromosomal DNA degradation induced by lethal heat and cold shock treatments in vivo . The nuclease activity was inhibited in vitro by aurintricalboxylic acid but not by Zn(2+) . By the mutant analysis, we identified the 39-kDa nuclease as a product of yokF gene . The yokF gene contained a putative lipoprotein signal peptide motif . After in vivo exposure to lethal heat and cold stresses, the chromosomal DNA fragmentation was reduced in the yokF mutant, which demonstrated about a 2-10-fold higher survival rate than the wild type . The yokF mutant was found to be more sensitive to mitomycin C than the wild type . The transformation efficiency of the yokF mutant was about 10 times higher than that of the wild type . It is suggested that when B . subtilis cells are exposed to a stressful thermal shock resulting in membrane perturbation, YokF nuclease consequently dislocates into the cytoplasm and then attacks DNA.

FEMS Microbiol Lett, 2001 Sep 25, 203(2), 207 - 10
Role of Cys54 in AbrB multimerization and DNA-binding activity; Phillips ZE et al.; The DNA-binding, global regulatory protein AbrB from Bacillus subtilis is homotetrameric in solution . Mutation of the lone cysteine present in the protomers (C54), to either a serine, tyrosine or tryptophan, abolishes DNA-binding activity in vitro and regulatory activity in vivo . The effect of these changes is not due to abrogation of disulfide bond formation since it can be shown biochemically that none of the C54 residues participates in disulfide bond formation . It is unlikely that C54 is involved in direct contact with DNA targets . Rather, it appears that the role of C54 is to provide a nucleophilic center required for proper spatial orientation of the polypeptide subunits.

FEBS Lett, 2001 Aug 3, 502(3), 89 - 92
Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure; Eggert T et al.; Bacillus subtilis secretes the lipolytic enzymes LipA and LipB . We show here that they are differentially expressed depending on the composition of the growth medium: LipA is produced in rich and in minimal medium, whereas LipB is present only in rich medium . A comparison of biochemical characteristics revealed that LipB is thermostable at pH 11 but becomes thermolabile at pH 5 . However, construction of a variant carrying the substitution A76G in the conserved lipase pentapeptide reversed these effects . The atomic coordinates from the LipA crystal structure were used to build a three-dimensional structural model of LipB, which revealed that 43 out of 45 residues different from LipA are surface-located allowing to rationalize the differences observed in the substrate preferences of the two enzymes.

Mol Microbiol, 2001 Sep, 41(6), 1381 - 93
Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY; Tortosa P et al.; The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl beta-glucoside and sucrose utilization respectively . Their RNA-binding activity is carried by the N-terminal domain (CAT), and is regulated by two similar C-terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS) . In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl beta-glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose . LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression . Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT-PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo . The BglP-mediated inhibition process was found to essentially involve His100 of LicT-PRD1, with His159 of the same domain playing a minor role in this regulation . In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by phosphorylated BglP . We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity . Thus, for both antiterminators, the EII-mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.

Phys Rev Lett . 2001 Oct 8;87(15):158102 . Epub 2001 Sep 21.
Swarming ring patterns in bacterial colonies exposed to ultraviolet radiation; Delprato AM et al.; We report a novel morphological transition in a Bacillus subtilis colony initially growing under ambient conditions, after ultraviolet radiation exposure . The bacteria in the central regions of the colonies are observed to migrate towards the colony edge forming a ring during uniform spatial exposure . When the radiation is switched off, the colonies were observed to grow both inward into the evacuated regions as well as outward indicating that the pattern is not formed due to depletion of nutrients at the center of the colony . We also propose a reaction-diffusion model in which waste-limited chemotaxis initiated by the UV radiation leads to the observed phenomenology.

J Biochem (Tokyo), 2001 Oct, 130(4), 569 - 74
Transcriptional regulation of the Bacillus subtilis bscR-CYP102A3 operon by the BscR repressor and differential induction of cytochrome CYP102A3 expression by oleic acid and palmitate; Lee TR et al.; The adjacent yrhI and yrhJ genes were identified by the Bacillus subtilis genome sequencing project . We now report that yrhJ (renamed CYP102A3) encodes a cytochrome P450 and that yrhI (renamed bscR) encodes a repressor that negatively regulates the transcription of the bscR-CYP102A3 operon . The transcriptional initiation site of bscR has been mapped by primer extension analysis . An 18-bp perfect palindromic sequence centered 65.5 bp downstream from the transcriptional initiation site of bscR has been identified as the binding site for BscR by gel mobility shift assays . Base substitutions in the 18-bp inverted repeat resulted in derepression of the bscR-xylE transcriptional fusion in vivo . bscR-xylE fusion studies and Northern blot analysis revealed that oleic acid and palmitate could induce the expression of the bscR-CYP102A3 operon to a considerable extent . However, only oleic acid was capable of preventing the binding of BscR to its operator DNA in vitro, suggesting that the induction of CYP102A3 expression by oleic acid and palmitate in B . subtilis might be mediated through different mechanisms.

Appl Environ Microbiol, 2001 Oct, 67(10), 4458 - 63
beta-Glutamate as a substrate for glutamine synthetase; Robinson P et al.; The conversion of beta-glutamate to beta-glutamine by archaeal and bacterial glutamine synthetase (GS) enzymes has been examined . The GS from Methanohalophilus portucalensis (which was partially purified) is capable of catalyzing the amidation of this substrate with a rate sevenfold less than the rate obtained with alpha-glutamate . Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for alpha-glutamate than beta-glutamate as a substrate . All the archaeal enzymes were much less selective than the two bacterial GS (from Escherichia coli and Bacillus subtilis), whose specific activities towards beta-glutamate were much smaller than rates with the alpha-isomer . These results are discussed in light of the observation that beta-glutamate is accumulated as an osmolyte in many archaea while beta-glutamine (produced by glutamine synthetase) is used as an osmolyte only in M . portucalensis.

Boll Chim Farm, 2001 Jul-Aug, 140(4), 224 - 7
Preparation and antibacterial evaluation of some nitrone derivative and their zirconium (IV) chloride complexes; Khallow KI et al.; Some new complexes of the general formula ZrCl3.L where L = {N-(p-anisole)-alpha-(2-xanthatophenyl)nitrone}, L1, and {N-(p-chlorophenyl)-alpha-(2-xanthatophenyl)nitrone}, L2, have been prepared and characterized by elemental analysis, i.r . and molar conductances . The antibacterial activities of these compounds have been studied against five species of bacteria in vitro at concentration ranging from 1-10 micrograms/ml . A remarkable activity was exhibited by the ligand L2 against Staph . aureus and Bacillus subtilis.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Jul-Aug, (4), 3 - 6
{Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells}; Zhdanova LV et al.; The gene of microbial lysozyme (lyz) of S . aureus 118 and the gene of lysostaphin (lzf) of S . aureus RN 3239 were cloned and their expression in B . subtilis cells was shown . Lysozyme production in B . subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S . aureus wild strain 118 . Lysostaphin production in B . subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S . aureus initial strain RN 3239 . The production of lysozyme and lysostaphin in the cells of B . subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S . aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.

J Bacteriol, 2001 Oct, 183(20), 6046 - 53
Two class A high-molecular-weight penicillin-binding proteins of Bacillus subtilis play redundant roles in sporulation; McPherson DC et al.; The four class A penicillin-binding proteins (PBPs) of Bacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls . The ywhE product was shown to bind penicillin, so the gene and gene product were renamed pbpG and PBP2d, respectively . Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis . A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core . In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles . A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore . The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic delta-lactam and L-alanine side chains and a slight increase in cross-linking . Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG . We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.

J Bacteriol, 2001 Oct, 183(20), 5918 - 26
Expression of the Bacillus subtilis trpEDCFBA operon is influenced by translational coupling and Rho termination factor; Yakhnin H et al.; The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translational control mechanisms . Both mechanisms require binding of tryptophan-activated TRAP to 11 (G/U)AG repeats in the trp leader transcript . trpE translational control involves formation of a TRAP-dependent RNA structure that sequesters the trpE Shine-Dalgarno (SD) sequence (the SD blocking hairpin) . By comparing expression levels from trpE'-'lacZ translational fusions controlled by the wild-type leader or by a leader that cannot form the SD blocking hairpin, we found that translational control requires a tryptophan concentration higher than that required for transcription attenuation . We also found that inhibition of trpE translation by the SD blocking hairpin does not alter the stability of the downstream message . Since the coding sequences for trpE and trpD overlap by 29 nucleotides, we examined expression levels from trpED'-'lacZ translational fusions to determine if these two genes are translationally coupled . We found that introduction of a UAA stop codon in trpE resulted in a substantial reduction in expression . Since expression was partially restored in the presence of a tRNA suppressor, our results indicate that trpE and trpD are translationally coupled . We determined that the coupling mechanism is TRAP independent and that formation of the SD blocking hairpin regulates trpD translation via translational coupling . We also constructed a rho mutation to investigate the role of Rho-dependent termination in trp operon expression . We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causing transcriptional polarity.

J Bacteriol, 2001 Oct, 183(20), 5877 - 84
Involvement of two distinct catabolite-responsive elements in catabolite repression of the Bacillus subtilis myo-inositol (iol) operon; Miwa Y et al.; The Bacillus subtilis inositol operon (iolABCDEFGHIJ) is involved in myo-inositol catabolism . Glucose repression of the iol operon induced by inositol is exerted through catabolite repression mediated by CcpA and the iol induction system mediated by IolR . In this study, we identified two iol catabolite-responsive elements (cre's), to which CcpA complexed with P-Ser-HPr or P-Ser-Crh probably binds . One is located in iolB (cre-iolB, nucleotides +2397 to +2411; +1 is the transcription initiation nucleotide), which was the only cre-iol found in the previous cre search of the B . subtilis genome using a query sequence of WTGNAANCGNWNNCW (W stands for A or T, and N stands for any base) . Deletion and base substitution analysis of the iol region indicated that cre-iolB functions even if it is located far downstream of the iol promoter . Further deletion and base substitution analysis revealed another cre located between the iol promoter and the iolA gene (cre-iiolA, nucleotides +86 to +100); the prefix "i" indicates a location in the intergenic region . Both cre-iiolA and cre-iolB appeared to be recognized to almost the same extent by CcpA complexed with either P-Ser-HPr or P-Ser-Crh . Sequence alignment of the six known cre's, including cre-iiolA, which were not revealed in the previous cre search, exhibited another consensus sequence of WTGAAARCGYTTWWN (R stands for A or G, and Y stands for C or T); the right two thymines (TT) were found to be essential for the function of cre-iiolA by means of base substitution analysis . A cre search with this query sequence led to the finding of 14 additional putative cre's.

J Bacteriol, 2001 Oct, 183(20), 5862 - 9
Bacillus subtilis YxkJ is a secondary transporter of the 2-hydroxycarboxylate transporter family that transports L-malate and citrate; Krom BP et al.; The genome of Bacillus subtilis contains two genes that code for membrane proteins that belong to the 2-hydroxycarboxylate transporter family . Here we report the functional characterization of one of the two, yxkJ, which codes for a transporter protein named CimHbs . The gene was cloned and expressed in Escherichia coli and complemented the citrate-negative phenotype of wild-type E . coli and the malate-negative phenotype of the E . coli strain JRG4008, which is defective in malate uptake . Subsequent uptake studies in whole cells expressing CimHbs clearly demonstrated the citrate and malate transport activity of the protein . Immunoblot analysis showed that CimHbs is a 48-kDa protein that is well expressed in E . coli . Studies with right-side-out membrane vesicles demonstrated that CimHbs is an electroneutral proton-solute symporter . No indications were found for the involvement of Na(+) ions in the transport process . Inhibition of the uptake catalyzed by CimHbs by divalent metal ions, together with the lack of effect on transport by the chelator EDTA, showed that CimHbs translocates the free citrate and malate anions . Among a large set of substrates tested, only malate, citramalate, and citrate competitively inhibited citrate transport catalyzed by CimHbs . The transporter is strictly stereoselective, recognizing only the S enantiomers of malate and citramalate . Remarkably, though citramalate binds to the transporter, it is not translocated.

Electrophoresis, 2001 Aug, 22(14), 2908 - 35
A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis; Buttner K et al.; Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology . This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago . Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified . In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7 . The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control . Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot . In a few selected cases evidence for phosphorylation of these proteins is presented . The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B . subtilis proteome such as alkaline proteins as well as extracellular proteins . A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.

J Protein Chem, 2001 Feb, 20(2), 165 - 9
Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1; Suh HJ et al.; A keratinolytic enzyme produced by Bacillus subtilis KS-1 isolated from poultry waste was purified and characterized using ultrfiltration, DEAE-Sephadex, and Sephadex G-100 chromatographies . The specific activity of the purified protease was 538.2 units/mg . The enzyme was shown to have a relative molecular mass of 25.4 kDa . The enzyme was made completely inactive by PMSF, which indicates a serine-protease . Dithiothreitol enhanced keratinolytic activity by 1.6 times at a concentration of 5.0 mM . These results suggest that the cleavage of the disulfide bonds with reducing agents can occur directly or by excretion of sulfite, which causes the sulfitolysis of the disulfide bonds . The first 10 amino acids of the N-terminal sequence are Ala-Gin-Pro-Val-Glu-Trp-GlyIle-Ser-Gln . The enzyme hydrolyzed casein and feather, but hydrolyzed casein more effectively than it did feather.

J Struct Biol, 2001 Jul, 135(1), 18 - 25
Composition and mass of the bacteriophage phi29 prohead and virion; Peterson C et al.; The protein composition of the Bacillus subtilis bacteriophage phi29 prohead and virion was determined by combustion of gel bands of (3)H-labeled proteins . Copy numbers of individual proteins were calculated relative to the 12 copies of the head-tail connector protein . The mean numbers of copies of the major capsid protein in the prohead and virion were 241 and 218, respectively, approaching the 235 copies determined previously by cryoelectron microscopy . The mean numbers of copies of the dimeric head fiber on the prohead and virion were 24 and 31, respectively, demonstrating partial occupancy of the 55 fiber binding sites . Measured copies of neck and tail proteins in the virion included 11 of the lower collar, 58 of the appendage, and 9 of the tail; if the true copies of these proteins are 12, 60, and 9, respectively, the entire neck and tail of phi29 has quasi-sixfold symmetry . The mass of the fiberless prohead with pRNA was about 14.2 MDa, and the mass of the prohead determined by scanning transmission electron microscopy was consistent with the biochemical data . The mass of the fiberless virion containing the 12.8-MDa DNA genome was about 30.4 MDa . A full complement of dimeric fibers on the prohead or virion would increase the mass of the particle by about 3.2 MDa . The data complement studies relating the structure of phi29 components to dynamic functions in morphogenesis and infection .

Science, 2001 Sep 14, 293(5537), 2057 - 9
Inhibition of the B . subtilis regulatory protein TRAP by the TRAP-inhibitory protein, AT; Valbuzzi A et al.; An anti-TRAP (AT) protein, a factor of previously unknown function, conveys the metabolic signal that the cellular transfer RNA for tryptophan (tRNATrp) is predominantly uncharged . Expression of the operon encoding AT is induced by uncharged tRNATrp . AT associates with TRAP, the trp operon attenuation protein, and inhibits its binding to its target RNA sequences . This relieves TRAP-mediated transcription termination and translation inhibition, increasing the rate of tryptophan biosynthesis . AT binds to TRAP primarily when it is in the tryptophan-activated state . The 53-residue AT polypeptide is homologous to the zinc-binding domain of DnaJ . The mechanisms regulating tryptophan biosynthesis in Bacillus subtilis differ from those used by Escherichia coli.

Nucleic Acids Res, 2001 Sep 15, 29(18), 3848 - 56
Bacterial ribonuclease P holoenzyme crosslinking analysis reveals protein interaction sites on the RNA subunit; Sharkady SM et al.; The structure of the Escherichia coli ribonuclease P (RNase P) holoenzyme was investigated by site-directed attachment of an aryl azide crosslink reagent to specific sites in the protein subunit of the enzyme . The sites of crosslinking to the RNase P RNA subunit were mapped by primer extension to several conserved residues and structural features throughout the RNA . The results suggest rearrangement of current tertiary models of the RNA subunit, particularly in regions poorly constrained by earlier data . Crosslinks to the substrate precursor-tRNA were also detected, consistent with previous crosslinking results in the Bacillus subtilis RNase P holoenzyme.

Nucleic Acids Res, 2001 Sep 15, 29(18), 3804 - 13
DNA microarray analysis of Bacillus subtilis DegU, ComA and PhoP regulons: an approach to comprehensive analysis of B.subtilis two-component regulatory systems; Ogura M et al.; We have analyzed the regulons of the Bacillus subtilis two-component regulators DegU, ComA and PhoP by using whole genome DNA microarrays . For these experiments we took the strategy that the response regulator genes were cloned downstream of an isopropyl-beta-D-thiogalactopyranoside-inducible promoter on a multicopy plasmid and expressed in disruptants of the cognate sensor kinase genes, degS, comP and phoR, respectively . The feasibility of this experimental design to detect target genes was demonstrated by the following two results . First, expression of lacZ fusions of aprE, srfA and ydhF, the target genes of DegU, ComA and PhoP, respectively, was stimulated in their cognate sensor kinase-deficient mutants upon overproduction of the regulators . Secondly, by microarray analysis most of the known target genes for the regulators were detected and, where unknown genes were found, the regulator dependency of several of them was demonstrated . As the mutants used were deficient in the kinase genes, these results show that target candidates can be detected without signal transduction . Using this experimental design, we identified many genes whose dependency on the regulators for expression had not been known . These results suggest the applicability of the strategy to the comprehensive transcription analysis of the B.subtilis two-component systems.

FEMS Microbiol Lett, 2001 Sep 11, 203(1), 125 - 9
The Bacillus subtilis catabolite control protein CcpA exerts all its regulatory functions by DNA-binding; Ludwig H et al.; In Bacillus subtilis, carbon catabolite control is mediated by the regulatory protein CcpA . In addition to loss of catabolite repression, ccpA mutants exhibit a severe growth defect . They are not able to grow with glucose and ammonium as single sources of carbon and nitrogen, respectively . Only ccpA mutant strains carrying either secondary suppressor mutations or that are affected in specific amino acids in the DNA-binding domain of CcpA grow on minimal media . We addressed the importance of DNA-binding by CcpA for both carbon catabolite repression and growth of a mutant strain . A strain specifically deleted for the N-terminal DNA-binding domain of CcpA was constructed . This deletion resulted in complete loss of catabolite repression of beta-xylosidase synthesis and prevented bacteria from growing on minimal media, suggesting that DNA-binding by CcpA is required for both carbon catabolite repression and efficient growth on minimal media.

J Biol Chem, 2001 Nov 16, 276(46), 42901 - 7 Epub 2001 Sep 12.
The Bacillus subtilis competence transcription factor, ComK, overrides LexA-imposed transcriptional inhibition without physically displacing LexA; Hamoen LW et al.; During the development of competence in Bacillus subtilis the recA gene is activated by the competence transcription factor, ComK, which is presumably required to alleviate the transcriptional repression of recA by LexA . To investigate the mechanism by which ComK activates recA transcription we examined the binding of ComK and LexA to the recA promoter in vitro . Using hydroxyl radical protection analyses to establish the location of ComK dimer-binding sites within the recA promoter, we identified four AT-boxes in a configuration unique for ComK-regulated promoters . Gel mobility shift experiments showed that all four ComK dimer-binding sites were occupied at ComK concentrations in the physiological range . In addition, occupation of all ComK-binding sites did not prevent LexA from binding to the recA promoter, despite the fact that the ComK and LexA recognition motifs partially overlap . Although ComK did not replace LexA from the recA promoter, in vitro transcription analyses indicated that the presence of ComK is sufficient to alleviate LexA repression of recA.

Mol Microbiol, 2001 Sep, 41(5), 1223 - 31
New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster; Gaisser S et al.; Using a previously developed expression system based on the erythromycin-producing strain of Saccharopolyspora erythraea, O-methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide macrolactone . The spnI, spnK and spnH methyltransferase genes were expressed individually in the S . erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII . Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S . erythraea SGT2 (spnI) strain only . When 3-O-(2'-O-methylrhamnosyl)-erythronolide B was, in turn, fed to a culture of S . erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was identified in the culture supernatant, whereas S . erythraea SGT2 (spnH) was without effect . These results confirm the identity of the 2'- and 3'-O-methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway . In contrast, 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was found not to be a substrate for the 4'-O-methyltransferase SpnH . Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S . erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway . 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be significantly active against a strain of erythromycin-sensitive Bacillus subtilis.

Bioresour Technol, 2001 Oct, 80(1), 49 - 51
Production of protease by Bacillus subtilis grown on sardinelle heads and viscera flour; Ellouz Y et al.; Fish flours from Sardinelle (Sardinella aurita) were prepared and tested for protease production by Bacillus subtilis . Protease synthesis was strongly induced when cells were grown in media containing only a combined head and viscera preparation . Sardinelle heads and viscera flour enhanced protease production up to 100% more than commercial peptones . The enhancement could have been due to a high lipid content, which might have contained nutritional factors acting as inducers, since defatting fish meal led to a decrease in protease production.

Mycopathologia, 2001, 151(2), 93 - 8
Ochratoxin A in airborne dust and fungal conidia; Skaug MA et al.; Farm workers are often exposed to high concentrations of airborne organic dust and fungal conidia, especially when working with plant materials . The purpose of this investigation was to study the possibility of exposure to the mycotoxin ochratoxin A (OTA) through inhalation of organic dust and conidia . Dust and aerosol samples were collected from three local cowsheds . Aerosol samples for determination of total conidia and dust concentrations were collected by stationary sampling on polycarbonate filters . Total dust was analysed by gravimetry, and conidia were counted using scanning electron microscopy . A method was developed for extraction and determination of OTA in small samples of settled dust . OTA was extracted with a mixture of methanol, chloroform, HCl, and water, purified on immunoaffinity column, and analysed by ion-pair HPLC with fluorescence detection . Recovery of OTA from spiked dust samples (0.9-1.0 microg/kg) was 74% (quantitation limit 0.150 microg/kg) . OTA was found in 6 out of 14 settled dust samples (0.2-70 microg/kg) . The total concentration of airborne conidia ranged from < 1.1 x 10(4) to 3.9 x 15(5) per m3, and the airborne dust concentration ranged from 0.08 to 0.21 mg/m3 . Conidia collected from cultures of Penicillium verrucosum and Aspergillus ochraceus contained 0.4-0.7 and 0.02-0.06 pg OTA per conidium, respectively . Testing of conidial extracts from these fungi in a Bacillus subtilis bioassay indicated the presence of toxic compounds in addition to OTA . The results show that airborne dust and fungal conidia can be sources of OTA . Peak exposures to airborne OTA may be significant, e.g., in agricultural environments.

Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11169 - 74 Epub 2001 Sep 11.
Crystal structure of the quorum-sensing protein LuxS reveals a catalytic metal site; Hilgers MT et al.; The ability of bacteria to regulate gene expression in response to changes in cell density is termed quorum sensing . This behavior involves the synthesis and recognition of extracellular, hormone-like compounds known as autoinducers . Here we report the structure of an autoinducer synthase, LuxS from Bacillus subtilis, at 1.6-A resolution (R(free) = 0.204; R(work) = 0.174) . LuxS is a homodimeric enzyme with a novel fold that incorporates two identical tetrahedral metal-binding sites . This metal center is composed of a Zn(2+) atom coordinated by two histidines, a cysteine, and a solvent molecule, and is reminiscent of active sites found in several peptidases and amidases . Although the nature of the autoinducer synthesized by LuxS cannot be deduced from the crystal structure, features of the putative active site suggest that LuxS might catalyze hydrolytic, but not proteolytic, cleavage of a small substrate . Our analysis represents a test of structure-based functional assignment.

J Biol Chem, 2001 Nov 23, 276(47), 43618 - 26 Epub 2001 Sep 11.
Phosphorylation of the response regulator CheV is required for adaptation to attractants during Bacillus subtilis chemotaxis; Karatan E et al.; In the Gram-positive soil bacterium Bacillus subtilis, the chemoreceptors are coupled to the central two-component kinase CheA via two proteins, CheW and CheV . CheV is a two-domain protein with an N-terminal CheW-like domain and a C-terminal two-component receiver domain . In this study, we show that CheV is phosphorylated in vitro on a conserved aspartate in the presence of phosphorylated CheA (CheA-P) . This reaction is slower compared with the phospho-transfer reaction between CheA-P and one other response regulator of the system, CheB . CheV-P is also highly stable in comparison with CheB-P . Both of these properties are more pronounced in the full-length protein compared with a truncated form composed only of the receiver domain, that is, deletion of the CheW-like domain results in increase in the rate of the phospho-transfer reaction and decrease in stability of the phosphorylated protein . Phosphorylation of CheV is required for adaptation to the addition of the chemoattractant asparagine . In tethered-cell assays, strains expressing an unphosphorylatable point mutant of cheV or a truncated mutant lacking the entire receiver domain are severely impaired in adaptation to the addition of asparagine . Both of these strains, however, show near normal counterclockwise biases, suggesting that in the absence of the attractant the chemoreceptors are efficiently coupled to CheA kinase by the mutant CheV proteins . Inability of the CheW-like domain of CheV to support complete adaptation to the addition of asparagine also suggests that unlike CheW, this domain by itself may lead to the formation of signaling complexes that stay overactive in the presence of the attractant . A possible structural basis for this feature is discussed.

Biochemistry, 2001 Sep 18, 40(37), 11202 - 10
Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA; Qin H et al.; The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme . The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA . The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor . To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants . Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure . tRNA binding selectivity is affected in one but not in two other mutants of this class . Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity . Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity . Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains . These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.

Mol Plant Microbe Interact, 2001 Sep, 14(9), 1043 - 50
Cloning and expression analysis of Phytoplasma protein translocation genes; Kakizawa S et al.; Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium . The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis . Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli . Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants . Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma . The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B . subtilis . These results suggest the presence of a functional Sec system in phytoplasmas . Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm . This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 16 - 20
{Experimental evaluation of the biological safety of gene-engineered bacteria using a model strain Bacillus subtilis interferon-producing strain}; Beliavskaia VA et al.; The in vitro and in vivo evaluation of the biological and ecological safety of genetically modified bacteria (GMB) was carried out on B . subtilis recombinant strain 2335/105, capable of producing human interferon alpha-2, used as experimental model . As shown in this investigation made with the use of bacteriological analysis and polymerase chain reaction, the oral administration of GMB to calves, chickens and white mice produced no disturbances in the microbial ecology of the gastrointestinal tract of warm-blooded animals and did not lead to the appearance of spontaneous transformants . The present work is the first experimental evaluation of the biological safety of genetically modified microorganisms, used as the component of Subalin, a probiotic preparation intended for use in veterinary practice.

Can J Microbiol, 2001 Jul, 47(7), 618 - 25
Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1; Manjula K et al.; Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut . A steady increase in cell numbers of introduced B . subtilis AF 1 was observed in all the formulations at 30 degrees C . The increase in cell numbers was about 5.0 log units . Peat or spent compost inoculated with physiologically active and dormant states of B . subtilis AF 1 showed different time period requirements to attain maximum cell numbers . The presence of chitin or A . niger (in peat) or A . bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B . subtilis AF 1 . When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A . niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea . Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure . Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.

Nat Prod Lett, 2001, 15(1), 35 - 42
Vanillic acid 4-O-beta-D-(6'-O-galloyl) glucopyranoside and other constituents from the bark of Terminalia macroptera Guill.et Perr; Conrad J et al.; A new phenolic glucoside gallate, vanillic acid 4-O-beta-D-(6'-O-galloyl) glucopyranoside (1) was isolated from the bark of Terminalia macroptera Guill.et Perr., together with 3,3',4'-tri-O-methylellagic acid (2) and two triterpene glucopyranosyl esters, 24-deoxysericoside (3) and chebuloside II (4) . Compounds 2-4, not described previously for this plant, showed antimicrobial activities against Bacillus subtilis, while 3 and 4 possessed haemolytic properties . In both assays 1 was found to be inactive.

J Biol Chem, 2001 Nov 23, 276(47), 43627 - 34 Epub 2001 Aug 23.
Oxalate decarboxylase requires manganese and dioxygen for activity . Overexpression and characterization of Bacillus subtilis YvrK and YoaN; Tanner A et al.; The Bacillus subtilis oxalate decarboxylase (EC ), YvrK, converts oxalate to formate and CO(2) . YvrK and the related hypothetical proteins YoaN and YxaG from B . subtilis have been successfully overexpressed in Escherichia coli . Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manganese salts . No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts . YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H(2)O(2); and oxalate-dependent, H(2)O(2)-independent dye oxidations . The oxalate decarboxylase activity of purified YvrK was O(2)-dependent . YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit . EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state . The hyperfine coupling constant (A = 9.5 millitesla) of the main g = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry . The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase . Although YvrK possesses two potential active sites, only one could be fully occupied by manganese . The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed . A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O(2) are cofactors that act together as a two-electron sink during catalysis.

BMC Microbiol . 2001;1(1):15 . Epub 2001 Aug 08.
MtnK, methylthioribose kinase, is a starvation-induced protein in Bacillus subtilis; Sekowska A et al.; BACKGROUND: Methylthioadenosine, the main by-product of spermidine synthesis, is degraded in Bacillus subtilis as adenine and methylthioribose . The latter is an excellent sulfur source and the precursor of quorum-sensing signalling molecules . Nothing was known about methylthioribose recycling in this organism . RESULTS: Using trifluoromethylthioribose as a toxic analog to select for resistant mutants, we demonstrate that methylthioribose is first phosphorylated by MtnK, methylthioribose kinase, the product of gene mtnK (formerly ykrT), expressed as an operon with mtnS (formerly ykrS) in an abundant transcript with a S-box leader sequence . Although participating in methylthioribose recycling, the function of mtnS remained elusive . We also show that MtnK synthesis is boosted under starvation condition, in the following decreasing order: carbon-, sulfur- and nitrogen-starvation . We finally show that this enzyme is part of the family Pfam 01633 (choline kinases) which belongs to a large cluster of orthologs comprizing antibiotic aminoglycoside kinases and protein serine/threonine kinases . CONCLUSIONS: The first step of methylthioribose recycling is phosphorylation by MTR kinase, coded by the mtnK (formerly ykrT) gene . Analysis of the neighbourhood of mtnK demonstrates that genes located in its immediate vicinity (now named mtnUVWXYZ, formerly ykrUVWXYZ) are also required for methylthioribose recycling.

J Bacteriol, 2001 Oct, 183(19), 5772 - 7
Genetic recombination in Bacillus subtilis 168: effect of DeltahelD on DNA repair and homologous recombination; Carrasco B et al.; The B . subtilis DeltahelD allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec(+) strain . The DeltahelD allele was introduced into rec-deficient strains representative of the alpha (recF strain), beta (addA addB), gamma (recH), epsilon (DeltarecU), and zeta (DeltarecS) epistatic groups . The DeltahelD mutation increased the sensitivity to DNA-damaging agents of addAB, DeltarecU, and DeltarecS cells, did not affect the survival of recH cells, and decreased the sensitivity of recF cells . DeltahelD also partially suppressed the DNA repair phenotype of other mutations classified within the alpha epistatic group, namely the recL, DeltarecO, and recR mutations . The DeltahelD allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the alpha, beta, and gamma epistatic groups . Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of recFLOR and render recFLOR, addAB, and recH cells impaired in plasmid transformation.

J Bacteriol, 2001 Oct, 183(19), 5617 - 31
Global analysis of the general stress response of Bacillus subtilis; Petersohn A et al.; Gene arrays containing all currently known open reading frames of Bacillus subtilis were used to examine the general stress response of Bacillus . By proteomics, transcriptional analysis, transposon mutagenesis, and consensus promoter-based screening, 75 genes had previously been described as sigma(B)-dependent general stress genes . The present gene array-based analysis confirmed 62 of these already known general stress genes and detected 63 additional genes subject to control by the stress sigma factor sigma(B) . At least 24 of these 125 sigma(B)-dependent genes seemed to be subject to a second, sigma(B)-independent stress induction mechanism . Therefore, this transcriptional profiling revealed almost four times as many regulon members as the proteomic approach, but failure of confirmation of all known members of the sigma(B) regulon indicates that even this approach has not yet elucidated the entire regulon . Most of the sigma(B)-dependent general stress proteins are probably located in the cytoplasm, but 25 contain at least one membrane-spanning domain, and at least 6 proteins appear to be secreted . The functions of most of the newly described genes are still unknown . However, their classification as sigma(B)-dependent stress genes argues that their products most likely perform functions in stress management and help to provide the nongrowing cell with multiple stress resistance . A comprehensive screening program analyzing the multiple stress resistance of mutants with mutations in single stress genes is in progress . The first results of this program, showing the diminished salt resistance of yjbC and yjbD mutants compared to that of the wild type, are presented . Only a few new sigma(B)-dependent proteins with already known functions were found, among them SodA, encoding a superoxide dismutase . In addition to analysis of the sigma(B)-dependent general stress regulon, a comprehensive list of genes induced by heat, salt, or ethanol stress in a sigma(B)-independent manner is presented . Perhaps the most interesting of the sigma(B)-independent stress phenomena was the induction of the extracytoplasmic function sigma factor sigma(W) and its entire regulon by salt shock.

J Bacteriol, 2001 Oct, 183(19), 5562 - 70
Positive selection of mutations leading to loss or reduction of transcriptional activity of PrfA, the central regulator of Listeria monocytogenes virulence; Herler M et al.; Transcription factor PrfA controls the expression of virulence genes essential for Listeria monocytogenes pathogenesis . To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity . The system is based on the observation that the listerial iap gene, encoding the p60 protein, is lethal if overexpressed in Bacillus subtilis . A plasmid in which the iap gene is placed under the control of the PrfA-dependent hly promoter was constructed and introduced into B . subtilis . This strain was rapidly killed when expression of iap was induced by introduction of a second plasmid carrying prfA . Two classes of B . subtilis survivor mutants were identified: one carried mutations in iap, and the second carried mutations in prfA . Sequence analysis of the defective prfA genes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the beta-roll domain of PrfA; region B, between amino acids 169 and 193, which corresponds to the DNA-binding helix-turn-helix motif; and region C, comprising the 38 C-terminal amino acids of PrfA, which form a leucine zipper-like structure . PrfA proteins with mutations in regions B and C were unable to bind to the PrfA-binding site in the target DNA, while mutations in region A resulted in a protein still binding the target DNA but unable to form a stable complex with RNA polymerase and initiate transcription in vitro.

J Bacteriol, 2001 Oct, 183(19), 5513 - 22
Regulation of transcription of the Bacillus subtilis pyrG gene, encoding cytidine triphosphate synthetase; Meng Q et al.; The B . subtilis pyrG gene, which encodes CTP synthetase, is located far from the pyrimidine biosynthetic operon on the chromosome and is independently regulated . The pyrG promoter and 5' leader were fused to lacZ and integrated into the chromosomes of several B . subtilis strains having mutations in genes of pyrimidine biosynthesis and salvage . These mutations allowed the intracellular pools of cytidine and uridine nucleotides to be manipulated by the composition of the growth medium . These experiments indicated that pyrG expression is repressed by cytidine nucleotides but is largely independent of uridine nucleotides . The start of pyrG transcription was mapped by primer extension to a position 178 nucleotides upstream of the translation initiation codon . A factor-independent termination hairpin lying between the pyrG promoter and its coding region is essential for regulation of pyrG expression . Primer-extended transcripts were equally abundant in repressed and derepressed cells when the primer bound upstream of the terminator, but they were much less abundant in repressed cells when the primer bound downstream of the terminator . Furthermore, deletion of the terminator from pyrG-lacZ fusions integrated into the chromosome yielded elevated levels of expression that was not repressible by cytidine . We suggest that cytidine repression of pyrG expression is mediated by an antitermination mechanism in which antitermination by a putative trans-acting protein is reduced by elevated levels of cytidine nucleotides . Conservation of sequences and secondary structural elements in the pyrG 5' leaders of several other gram-positive bacteria indicates that their pyrG genes are regulated by a similar mechanism.

Icarus, 2000 Jun, 145(2), 391 - 427
Natural transfer of viable microbes in space; Mileikowsky C et al.; The possibility and probability of natural transfer of viable microbes from Mars to Earth and Earth to Mars traveling in meteoroids during the first 0.5 Ga and the following 4 Ga are investigated, including: --radiation protection against the galactic cosmic ray nuclei and the solar rays, dose rates as a function of the meteorite's radial column mass (radius x density), combined with dose rates generated by natural radioactivity within the meteorite; and survival curves for some bacterial species using NASA's HZETRN transport code --other factors affecting microbe survival: vacuum; central meteorite temperatures at launch, orbiting, and arrival; pressure and acceleration at launch; spontaneous DNA decay; metal ion migration --mean sizes and numbers of unshocked meteorites ejected and percentage falling on Earth, using current semiempirical results --viable flight times for the microbe species Bacillus subtilis and Deinococcus radiodurans R1 --the approximate fraction of microbes (with properties like the two species studied) viably arriving on Earth out of those ejected from Mars during the period 4 Ga BP to the present time, and during the 700 Ma from 4.5 to 3.8 Ga . Similarly, from Earth to Mars . The conclusion is that if microbes existed or exist on Mars, viable transfer to Earth is not only possible but also highly probable, due to microbes' impressive resistance to the dangers of space transfer and to the dense traffic of billions of martian meteorites which have fallen on Earth since the dawn of our planetary system . Earth-to-Mars transfer is also possible but at a much lower frequency.

Biol Sci Space, 2000 Mar, 14(1), 14 - 21
{Survival conditions of microorganisms under extremely severe environment}; Sera I et al.; The survival conditions of microorganisms under extremely severe environment are of interest in various areas of biology, sterilization, and space engineering, especially where resistance to microorganisms is concerned . Despite the interest, the resistance to microorganisms under extremely severe environment such as space environment or other planetary environment is not known well . In order to investigate survival conditions of microorganisms under extremely severe environment, surviving fractions for spores and vegetative cells of Bacillus subtilis were surveyed in various chemical species of atmosphere at various pressures and various temperatures, and the dependence on time for surviving fractions was examined . The results show: (i) Surviving fractions depend on chemical species of atmosphere . (ii) At high pressure and high temperature, surviving fractions are low and the resistance of spores is stronger than that of vegetative cells . (iii) Surviving fractions decrease as first-order reaction along with time elapsed.

Adv Space Res, 1983, 3(8), 85 - 94
Inactivation probability of heavy ion-irradiated Bacillus subtilis spores as a function of the radial distance to the particle's {correction of paricle's} trajectory; Facius R et al.; The understanding of the radiobiological action of heavy ions requires the knowledge of the dependence of the inactivation probability on the distance between the particle's trajectory and the biological test organism (the impact parameter) . Spores of Bacillus subtilis with a cytoplasmic core of about 0.22 micrometer cross section are suitable test objects for the study of this radial inactivation probability in its microscopic details . The spores are irradiated at low fluences of some 10(6) ions/cm2 with very heavy ions at different specific energies up to 10 MeV per atomic mass unit u while in fixed contact with visual nuclear track detectors . The methods are described by which the biological response of individual cells can be evaluated and the impact parameter be determined with an accuracy typically better than 0.2 micrometer . The results demonstrate that the common characteristics of inactivation, e.g., an effective range of inactivation extending to at least 3 micrometers, a nonmonotonic dependence of the inactivation probabilities on the radial distance, and the fact that the inactivation probability even for direct central hits on the cytoplasmic core is substantially below one, are nearly independent of the particle energy and type . The results are incompatible with the assumption that the radiobiological effectiveness can be attributed to the dose of secondary electrons as currently understood . They also demonstrate that the widely held notion of an "overkill" at low impact parameters does not apply for the spores even with the most densely ionizing ions.

Adv Space Res, 1983, 3(8), 79 - 84
Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions; Horneck G et al.; Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology . Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g . Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects . For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve . The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast . Lethal damage produced at LET values < 2000 MeV cm2/g is reparable . Recombination repair is more effective than excision repair . At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied . In addition, at these high LET values, the frequency of induced mutations was drastically decreased . The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.

Adv Space Res, 1995, 16(8), 119 - 29
ERA-experiment "Space Biochemistry"; Dose K et al.; The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light . Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods . The decrease in viability of the microorganisms was as expected from simulation experiments in the laboratory . Accordingly, it could be correlated with the increase in DNA damages . The purple membranes, amino acids and urea were not measurably effected by the dehydrating condition of open space (in the dark) . Plasmid DNA, however, suffered a significant amount of strand breaks under these conditions . The response of these biomolecules to high fluences of short wavelength solar UV-light is very complex . Only a brief survey can be given in this paper . The data on the relatively good survival of some of the microorganisms call for strict observance of COSPAR Planetary Protection Regulations during interplanetary space missions.

Adv Space Res, 1995, 16(8), 105 - 18
Biological responses to space: results of the experiment "Exobiological Unit" of ERA on EURECA I; Horneck G et al.; Spores of different strains of Bacillus subtilis and the Escherichia coli plasmid pUC19 were exposed to selected conditions of space (space vacuum and/or defined wavebands and intensities of solar ultraviolet radiation) in the experiment ER 161 "Exobiological Unit" of the Exobiology Radiation Assembly (ERA) on board of the European Retrievable Carrier (EURECA) . After the approximately 11 months lasting mission, their responses were studied in terms of survival, mutagenesis in the his (B . subtilis) or lac locus (pUC19), induction of DNA strand breaks, efficiency of DNA repair systems, and the role of external protective agents . The data were compared with those of a simultaneously running ground control experiment . The survival of spores treated with the vacuum of space, however shielded against solar radiation, is substantially increased, if they are exposed in multilayers and/or in the presence of glucose as protective, whereas all spores in "artificial meteorites", i.e . embedded in clays or simulated Martian soil, are killed . Vacuum treatment leads to an increase of mutation frequency in spores, but not in plasmid DNA . Extraterrestrial solar ultraviolet radiation is mutagenic, induces strand breaks in the DNA and reduces survival substantially; however, even at the highest fluences, i.e . 3 x 10(8) J m-2, a small but significant fraction of spores survives the insolation . Action spectroscopy confirms results of previous space experiments of a synergistic action of space vacuum and solar UV radiation with DNA being the critical target.

Adv Space Res, 1981, 1(14), 39 - 48
Survival of microorganisms in space: a review; Horneck G; Spores of Bacillus subtilis were exposed to selected factors of space (vacuum, solar UV radiation, heavy ions of cosmic radiation), and their response was studied after recovery . These investigations were supplemented by ground-based studies under simulated space conditions . The vacuum of space did not inactivate the spores . However, vacuum-induced structural changes in the DNA, and probably in the proteins, caused a supersensitivity to solar UV radiation . This phenomenon is caused by the production of specific photoproducts in DNA and protein, which cannot be removed by normal cellular repair processes . In vegetative bacterial cells, exposed to vacuum, cell dehydration led to damage of the cell membrane, which could be partly repaired during subsequent incubation . The high local effectiveness of the cosmic heavy ions further decreases the chance that spores can survive for any length of time in space . Nonetheless, a spore travelling through space and protected from ultraviolet radiation could possibly survive an interplanetary journey . Such a situation favors panspermia as a possible explanation for the origin of life.

J Aerosol Sci, 1997 Apr, 28(3), 471 - 82
Design of an instrument for real-time detection of bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic fluorescence; Hairston PP et al.; A prototype instrument has been constructed to measure individual airborne particles based on their aerodynamic size and their intrinsic fluorescence at selected excitation and emission wavelength bands . The instrument combines features of an aerodynamic particle sizing device with capabilities similar to those of a liquid flow cytometer . The goal of the instrument is to provide real-time data indicative of particle characteristics, and it is especially targeted to respond to bioaerosols from 0.5 to 10 micrometers (aerodynamic diameter) with intrinsic fluorescence exited at a wavelength of 325 nm and emitting from 420 to 580 nm . This size range covers individual airborne bacteria and bacteria clusters, and the fluorescence sensitivity is selected for biological molecules commonly found in cellular systems, for example, reduced nicotinamide adenine dinucleotide phosphate {NAD(P)H} and riboflavin . Initial tests with nebulised Bacillus subtilis var . niger (BG, ATCC 9372) spores have shown that, for both individual spores and spore clumps, a low level of fluorescence is detected from 17% of the particles . This detection percentage is on the same order as previous experiments that have measured viability of about 12% for mechanically dispersed BG spores (Ho and Fisher (1993) Defense Research Establishment Suffield Memorandum 1421) and suggests a need for further investigation into the possible relationship between the detected fluorescence and viability of bacterial spores.

Biol Sci Space, 1996 Dec, 10(4), 283 - 8
{Warning! A crucial period of searching for life on Mars--why international criterion for space quarantine is now required}; Koike J; In connection with quarantine for interplanetary mission, we have examined the survivalities of terrestrial microorganisms under simulated Mars condition . In this study, the Mars conditions were simulated by ultraviolet and proton irradiation under similar low temperature, high vacuum and gaseous conditions by using cryostat vehicle . After exposure to the simulated Mars conditions, the survivabilities of the organisms were examined . From the results, the spores of Bacillus subtilis, the spores of Aspergillus niger, some anaerobic bacteria and algae showed considerable high survivalities even after UV and proton irradiations corresponding to 200 years on Mars . This subject is not restricted to academic curiosity but concerns problems involving the contamination of Mars with terrestrial organisms carried by space-probes . If there is a possibility that the terrestrial organisms carried from Earth to Mars can live for a significant period on Mars, a contamination of the Mars should be prevented for the purpose of life-detection-experiments in future.

Adv Space Res, 1994 Oct, 14(10), 41 - 5
Long-term survival of bacterial spores in space; Horneck G et al.; On board of the NASA Long Duration Exposure Facility (LDEF), spores of Bacillus subtilis in monolayers (10(6)/sample) or multilayers (10(8)/sample) were exposed to the space environment for nearly six years and their survival was analyzed after retrieval . The response to space parameters, such as vacuum (10(-6) Pa), solar electromagnetic radiation up to the highly energetic vacuum-ultraviolet range (10(9) J/m2) and/or cosmic radiation (4.8 Gy), was studied and compared to the results of a simultaneously running ground control experiment . If shielded against solar ultraviolet (UV)-radiation, up to 80 % of spores in multilayers survive in space . Solar UV-radiation, being the most deleterious parameter of space, reduces survival by 4 orders of magnitude or more . However, up to 10(4) viable spores were still recovered, even in completely unprotected samples . Substances, such as glucose or buffer salts serve as chemical protectants . With this 6 year study in space, experimental data are provided to the discussion on the likelihood of "Panspermia".

Adv Space Res, 1994 Oct, 14(10), 207 - 11
Double strand breaks in the DNA of Bacillus subtilis cells irradiated by heavy ions; Micke U et al.; Cells of Bacillus subtilis strain TKJ 8431 in stationary phase were irradiated with X-rays (150 kV at DLR) or heavy ions (Ne, Ar, Pb with residual energies between 3 and 15 MeV/u at GSI) . The action cross section for the formation of double strand breaks in the DNA of the irradiated cells follows a similar dependence on mass and energy of the ions as has been found for various biological endpoints, e.g . inactivation, mutagenesis and repair efficacy.

Adv Space Res, 1994 Oct, 14(10), 1039 - 46
Inactivation of individual Bacillus subtilis spores in dependence on their distance to single accelerated heavy ions; Schafer M et al.; In order to understand radiation mechanisms of heavy ions in detail, it is necessary to study effects of single ions on individual biological test objects . Spores of Bacillus subtilis have been used as a suitable small biological test system to measure the inactivation in dependence on the radial distance to the tracks of charged particles . Accelerator experiments have been performed using a modified Biostack technique--biological objects sandwiched between nuclear track detectors . Results of these experiments using ions differing in their energy and atomic number will be discussed under following aspects: (i) methodological differences between the experiments and their possible influences on the results, (ii) common features which are independent on the particle type and energy, (iii) theoretical expectations and problems to find solid theoretical concepts which explain the results.

Adv Space Res, 1994 Oct, 14(10), 1027 - 38
Inactivation of individual Bacillus subtilis spores in dependence on their distance to single cosmic heavy ions; Facius R et al.; For radiobiological experiments in space, designed to investigate biological effects of the heavy ions of the cosmic radiation field, a mandatory requirement is the possibility to spatially correlate the observed biological response of individual test organisms to the passage of single heavy ions . Among several undertakings towards this goal, the BIOSTACK experiments in the Apollo missions achieved the highest precision and therefore the most detailed information on this question . Spores of Bacillus subtilis as a highly radiation resistant and microscopically small test organism yielded these quantitative results . This paper will focus on experimental and procedural details, which must be included for an interpretation and a discussion of these findings in comparison to control experiments with accelerated heavy ions.

Adv Space Res, 1984, 4(10), 19 - 27
Microorganisms and biomolecules in space environment experiment ES 029 on Spacelab-1; Horneck G et al.; Bacterial spores are proper test organisms for studying problems of space biology and exobiology . During the Spacelab 1 mission, studies on the limiting factors for survival of Bacillus subtilis spores in free space have been performed . An exposure tray on the pallet of Spacelab 1 accomodated 316 samples of dry spores for treatment with space vacuum and/or the following selected wavelengths of solar UV: > 170 nm, 220 nm, 240nm, 260nm and 280 nm . After recovery, inactivation, mutation induction, reparability, and photochemical damages in DNA and protein have been studied . The results contribute to the understanding of the mechanisms of increased UV sensitivity of bacterial spores in vacuo and to a better assessment of the chance of survival of resistant forms in space and of interplanetary transfer of life.

Math Comput Simul, 1996 Apr, 40(3-4), 359 - 70
Consumption, supply and transport: self-organization without direct communication; Kessler JO; Swimming bacteria of the species Bacillus subtilis require and consume oxygen . In static liquid cultures the cells' swimming behaviour leads them to accumulate up oxygen concentration gradients generated by consumption and supply . Since the density of bacterial cells exceeds that of the fluid in which they live, fluid regions where cells have accumulated are denser than depleted regions . These density variations cause convection . The fluid motion is dynamically maintained by the swimming of the cells toward regions of attraction: the air-fluid interface and the fluctuating advecting attractors, gradients of oxygen concentration that are embedded in the convecting fluid . Because of the fluid dynamical conservation laws, these complex physical and biological factors generate patterns ordered over distances > 10000 bacterial cell diameters . The convection enhances long-range transport and mixing of oxygen, cells and extracellular products by orders of magnitude . Thus, through the interplay of physical and biological factors, a population of undifferentiated selfish cells creates functional dynamic patterns . Populations of bacteria that have organised themselves into regularly patterned regions of vigorous convection and varying cell concentration interact with their environment as if they were one purposeful, coherent multicellular individual . The mathematical and experimental ingredients of these remarkable phenomena are presented here.

Adv Space Res, 1996, 18(1-2), 339 - 44
Fundamental studies concerning planetary quarantine in space; Koike J et al.; If there is a possibility that the organisms carried from Earth to space can live for a significant period on planets, the contamination of planets should be prevented for the purpose of future life-detection experiments . In connection with quarantine for interplanetary missions, we have examined the survivabilities of terrestrial microorganisms under simulated space conditions . In this study, examined the survivabilities of terrestrial organisms under simulated Mars conditions . The Mars conditions were simulated by ultraviolet (UV) and proton irradiation under low temperature, high vacuum, and simulated gaseous conditions . After exposure to the simulated Mars condition, the survivabilities of the organisms were examined . The spores of Bacillus subtilis and Aspergillus niger, some anaerobic bacterias and algaes, showed considerably high survivabilities even after UV and proton irradiation corresponding to 200 years on Mars . This subject is not restricted to academic curiosity but concerns problems involving the contamination of Mars with terrestrial organisms carried by space-probes.

Syst Appl Microbiol, 1991, 14(3), 266 - 9
Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies; Rossler D et al.; Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B . laterosporus, B . macerans, B . macquariensis, B . polymyxa and B . stearothermophilus revealed the phylogenetic diversity of the genus Bacillus . Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B . stearothermophilus, a "B . brevis cluster" including B . laterosporus, a "B . alvei cluster" including B . macerans, B . maquariensis and B . polymyxa and a "B . cycloheptanicus branch".

Adv Space Res, 1992, 12(4), 275 - 9
Thymine photoproduct formation and inactivation of intact spores of Bacillus subtilis irradiated with short wavelength UV (200-300nm) at atmospheric pressure and in vacuo; Lindberg C et al.; Vacuum exposure renders the survival of spores of Bacillus subtilis approximately five times more sensitive to ultraviolet light irradiation than exposure under atmospheric conditions . The photoproduct formation in spores irradiated under ultrahigh vacuum (UHV) conditions is compared to the photoproduct formation in spores irradiated at atmospheric pressure . Compared to irradiation at atmospheric pressure, where only the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine (TDHT) can be detected, two additional photoproducts, known as the c,s and t,s isomers of thymine dimer (T<>T) are produced in vacuo . The spectral efficiencies for photoproduct formation in spores under atmospheric and vacuum conditions are compared . Since there is no increased formation of TDHT after irradiation in vacuum, TDHT cannot be made responsible for the observed vacuum effect . "Vacuum specific" photoproducts may cause a synergistic response of spores to the simultaneous action of ultraviolet light (UV) and UHV . Three different mechanisms are discussed for the enhanced sensitivity of B . subtilis spores to UV radiation in vacuum . The experiments described contribute valuable research information on the chance for survival of microorganisms in outer space.

Adv Space Res, 1992, 12(4), 265 - 70
Extreme dryness and DNA-protein cross-links; Bieger-Dose A et al.; Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links . In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins . In fungal conidia these cross-links are detected after selective iodination (125 J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography . Another approach is the labelling of DNA with 32P by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.

Adv Space Res, 1992, 12(4), 249 - 53
The effects of vacuum-UV radiation (50-190nm) on microorganisms and DNA; Ito T; Early difficulties with the light source in the study of vacuum-UV effects on biological materials were overcome largely by the introduction of the synchrotron radiation source in 1977 . Highly monochromatic vacuum-UV radiation has been used to obtain action spectra of various biological targets in the range 50-190 nm . Bacillus subtilis spores were particularly suitable because of their inherent tolerance to vacuum, and action spectra for inactivation were measured down to 50 nm . Action spectra for DNA strand breaks were also measured in the same range with isolated plasmid DNA . Studies are in progress of vacuum-UV induced molecular changes in nucleic acids and their model compounds, especially of chain scission and base modification.

Radiat Meas, 1994 Jan, 23(1), 5 - 8
Single track effects, Biostack and risk assessment; Curtis SB; The scientific career of Prof . Bucker has spanned a very exciting period in the fledgling science of Space Radiation Biology . The capability for placing biological objects in space was developed, and the methods for properly packaging, retrieving and analyzing them were worked out . Meaningful results on the effects of radiation were obtained for the first time . In fact, many of the successful techniques and methodologies for handling biological samples were developed in Prof . Bucker's laboratories, as attested by the extensive Biostack program . He was the first to suggest and successfully carry out experiments in space directly aimed at measuring effects of single tracks of high-energy heavy galactic cosmic rays by specifically identifying whether or not the object had been hit by a heavy particle track . Because the "hit" frequencies of heavy galactic cosmic rays to cell nuclei in the bodies of space travelers will be low, it is expected that any effects to humans on the cellular level will be dominated by single-track cell traversals . This includes the most important generally recognized late effect of space radiation exposure: radiation-induced cancer . This paper addresses the single-track nature of the space radiation environment, and points out the importance of single "hits" in the evaluation of radiation risk for long-term missions occurring outside the earth's magnetic field . A short review is made of biological objects found to show increased effects when "hit" by a single heavy charged-particle in space . A brief discussion is given of the most provocative results from the bacterial spore Bacillus subtilis: experimental evidence that tracks can affect biological systems at much larger distances from the trajectory than previously suspected, and that the resultant inactivation cross section in space calculated for this system is very large . When taken at face value, the implication of these results, when compared to those from experiments performed at ground-based accelerators with beams at low energies in the same LET range, is that high-energy particles can exert their influence a surprising distance from their trajectory and the inactivation cross sections are some 20 times larger than expected . Clearly, beams from high-energy heavy-ion accelerators should be used to confirm these results . For those end points that can also be caused by low-LET beams such as high-energy protons, it is important to measure their action cross sections as well . The ratio of the cross sections for a high-LET beam to that of a low-LET beam is an interesting experimental ratio and, we suggest, of more intrinsic interest than the RBE (Relative Biological Effectiveness) . It is a measure of the "biological" importance of one particle type relative to another particle type . This ratio will be introduced and given the name RPPE (Relative Per Particle Effectiveness) . Values of RPPE have appeared in the literature and will be discussed . A rather well-known value of this quantity (13,520) has been suggested for the RPPE of high-energy iron ions to high-energy protons . This value was suggested by Letaw et al . Nature 330, 709-710 (1987)} we will call it the Letaw limit . It will be discussed in terms of the importance of the heavy-ion component vs light-ion component of the galactic cosmic rays . It is also pointed out, however, that there may be unique effects from single tracks of heavy ions that do not occur from light-ion tracks . For such effects, the concepts of both RBE and RPPE lose their meaning.

Adv Space Res, 1986, 6(11), 109 - 15
Genetic response of bacterial spores to very heavy ions; Baltschukat K et al.; Using spores of two Bacillus subtilis strains differing in repair capacity, we have studied repair and mutation induction in the spores after irradiation with very heavy ions up to uranium with specific particle energies up to 18.6 MeV/u . The results indicate that repair and mutation induction after heavy ion irradiation are closely related to each other and that both phenomena strongly depend on the atomic number and specific energy of the ions . The effects are discussed in comparison with results obtained after X-irradiation.

Adv Space Res, 1992, 12(2-3), 59 - 63
Heavy ion induced double strand breaks in bacteria and bacteriophages; Micke U et al.; DNA damage induced by heavy ions in bacterial cells and bacteriophages such as Bacillus subtilis, E . coli and Bacteriophage T1 were investigated by analyzing the double strand breaks in the chromosomal DNA . This kind of lesion is considered as one of the main reasons for lethal events . To analyze double strand breaks in long molecules of DNA--up to some Mbp in length--the technique of pulse field agarose gel electrophoresis has been used . This allows the detection of one double strand break per genome . Cell lysis and DNA isolation were performed in small agarose blocks directly . This procedure secured minimum DNA destruction by shearing forces . After running a gel, the DNA was stained with ethidium bromide . The light intensity of ethidium bromide fluorescence for both the outcoming (running) DNA and the remaining intact DNA were measured by scanning . The mean number of double strand breaks was calculated by determining the quotient of these intensities . Strand break induction after heavy ion and X-ray irradiation was compared.

Microbiology, 2001 Sep, 147(Pt 9), 2417 - 24
Sigma A recognition sites in the Bacillus subtilis genome; Jarmer H et al.; A hidden Markov model of sigma(A) RNA polymerase cofactor recognition sites in Bacillus subtilis, containing either the common or the extended -10 motifs, has been constructed based on experimentally verified sigma(A) recognition sites . This work suggests that more information exists at the initiation site of transcription in both types of promoters than previously thought . When tested on the entire B . subtilis genome, the model predicts that approximately half of the sigma(A) recognition sites are of the extended type . Some of the response-regulator aspartate phosphatases were among the predictions of promoters containing extended sites . The expression of rapA and rapB was confirmed by site-directed mutagenesis to depend on the extended -10 region.

Mol Microbiol, 2001 Aug, 41(4), 849 - 59
Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA; Herbig AF et al.; The inducible response to H(2)O(2) stress in Bacillus subtilis is under the control of PerR, one of three Fur homologues in this organism . PerR was purified in both an inactive, metal-dependent form and an active, metal-containing form as determined using DNA-binding assays . Active PerR contains both zinc and iron and is designated PerR:Zn,Fe . Added manganous ion competes for binding to the iron site and can restore DNA-binding activity to the metal-dependent form of PerR, presumably generating PerR:Zn,Mn . The DNA-binding activity of PerR:Zn,Fe is eliminated by exposure to H(2)O(2) whereas PerR:Zn,Mn is comparatively resistant . DNA-binding activity can be restored by a thiol-reducing agent, suggesting that redox-active cysteines are involved in peroxide sensing . Experiments using reporter fusions demonstrate that elevated levels of manganese repress PerR regulon genes and prevent their full induction by H(2)O(2) . In contrast, in cells grown with iron supplementation, a PerR-repressed gene is completely derepressed by H(2)O(2) . These results are consistent with the idea that the intracellular form of the PerR metalloprotein, and therefore its hydrogen peroxide sensitivity, can be altered by growth conditions.

Mol Microbiol, 2001 Aug, 41(4), 757 - 74
Genome-wide analysis of the general stress response in Bacillus subtilis; Price CW et al.; Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins . In Bacillus subtilis and related Gram-positive pathogens, this response is governed by the sigma(B) transcription factor . To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce sigma(B) activity . Macroarrays representing more than 3900 annotated reading frames of the B . subtilis genome were hybridized to (33)P-labelled cDNA populations derived from (i) wild-type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which sigma(B) expression was controlled by an inducible promoter . On the basis of their significant sigma(B)-dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the sigma(B) regulon . Of these genes, 30 were known previously or inferred to be sigma(B) dependent by other means . To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible sigma(B) recognition sequences preceding 21 of them . To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the sigma(B)-dependent messages for seven representative genes . For all seven, the 5' end of the message lay near typical sigma(B) recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples . Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins . Notably, products with a direct protective function were in the minority . Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover.

Mol Microbiol, 2001 Aug, 41(3), 743 - 55
Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein; Autret S et al.; Spo0J and Soj belong to the ParB/ParA family of proteins involved in chromosome and plasmid segregation in bacteria . In Bacillus subtilis, Spo0J protein binds to several specific sites, parS, located on both sides of the origin of DNA replication, oriC, and apparently self-associates to form large discrete foci visible by fluorescence microscopy . Soj protein forms large 'patches' probably associated with the nucleoid, which can undergo dynamic, co-operative jumping from nucleoid to nucleoid in the presence of Spo0J . Patches of Soj protein somehow help to bring about the condensation of Spo0J foci . Soj is also a negative regulator of transcription . In the absence of Spo0J, Soj is statically distributed on each of the nucleoids in the cell and blocks the transcription of several sporulation genes . To analyse the functional interaction between Spo0J and Soj further, we have constructed and studied a collection of spo0J mutants . Most of the mutants completely prevent Spo0J from interacting with DNA . One mutation impairs the formation of compact Spo0J foci and simultaneously results in loss of Soj movement . We also isolated one spo0J mutant, in which the frequency of Soj internucleoid oscillation is highly increased . Both mutations affecting the interaction with Soj lie in the N-terminal coding part of spo0J, whereas the substitutions affecting DNA binding lie in the mid- to C-terminal coding region.

Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1324 - 5 Epub 2001 Aug 23.
Cloning, purification, crystallization and preliminary crystallographic analysis of Bacillus subtilis LuxS; Das SK et al.; LuxS of Bacillus subtilis is a member of a novel family of proteins with a potential role in quorum sensing, controlling important aspects of cellular physiology in a range of microbial species . B . subtilis luxS was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant . The crystals belong to one of the enantiomorphic space groups P6(1)22 or P6(5)22, with approximate unit-cell parameters a = b = 63.6, c = 151.5 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.5 A(3) Da(-1) . The crystals diffract X-rays to at least 1.55 A resolution on a synchrotron-radiation source . Determination of the structure will provide insights into the key determinants of function of this class of proteins, for which no structures are currently available.

J Biol Chem, 2001 Nov 9, 276(45), 41991 - 7 Epub 2001 Aug 28.
Definitive identification of mammalian 5-hydroxymethyluracil DNA N-glycosylase activity as SMUG1; Boorstein RJ et al.; Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken . Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection . Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra . Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa . Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry . Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1) . The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20x the specific activity of the most purified bovine fraction . We conclude that hSMUG1 and HMUDG are the same protein.

Appl Environ Microbiol, 2001 Sep, 67(9), 3819 - 23
Fate and dissemination of Bacillus subtilis spores in a murine model; Hoa TT et al.; Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action . As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice . Our results have shown that spores do not appear to disseminate across the mucosal surfaces . However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum . This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again . This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.

FEMS Microbiol Rev, 2001 Aug, 25(4), 437 - 54
Translocation of proteins across the cell envelope of Gram-positive bacteria; van Wely KH et al.; In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment . For this reason, Gram-positive bacteria (e.g . various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium . The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms . Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria . The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway . In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.

J Biotechnol, 2001 Sep 13, 91(1), 1 - 34
The bacterial ParA-ParB partitioning proteins; Bignell C et al.; A pair of genes designated parA and parB are encoded by many low copy number plasmids and bacterial chromosomes . They work with one or more cis-acting sites termed centromere-like sequences to ensure better than random predivisional partitioning of the DNA molecule that encodes them . The centromere-like sequences nucleate binding of ParB and titrate sufficient protein to create foci, which are easily visible by immuno-fluorescence microscopy . These foci normally follow the plasmid or the chromosomal replication oriC complexes . ParA is a membrane-associated ATPase that is essential for this symmetric movement of the ParB foci . In Bacillus subtilis ParA oscillates from end to end of the cell as does MinD of E . coli, a relative of the ParA family . ParA may facilitate ParB movement along the inner surface of the cytoplasmic membrane to encounter and become tethered to the next replication zone . The ATP-bound form of ParA appears to adopt the conformation needed to drive partition . Hydrolysis to create ParA-ADP or free ParA appears to favour a form that is not located at the pole and binds to DNA rather than the partition complex . Definition of the protein domains needed for interaction with membranes and the conformational changes that occur on interaction with ATP/ADP will provide insights into the partitioning mechanism and possible targets for inhibitors of partitioning.

Insect Mol Biol, 2001 Aug, 10(4), 293 - 302
Purification, characterization and gene expression of a glycine and proline-rich antibacterial protein family from larvae of a beetle, Allomyrina dichotoma; Sagisaka A et al.; Two structurally related antibacterial proteins were isolated from larvae of a beetle, Allomyrina dichotoma, immunized with Escherichia coli . The two proteins were designated A . dichotoma (A . d.) coleoptericin A and B . The mature portion of A . d . coleoptericins deduced from nucleotide sequences of the cDNAs consists of seventy-two amino acids without cysteine residues and is rich in glycine (11.1%) and proline (11.1%) . Comparison of the amino acid sequences of the A . d . coleoptericins revealed that these antibacterial proteins have 94%, 75%, 50% and 43% similarity to rhinocerosin, holotricin 2, coleoptericin and acaloleptin A1 . Recombinant A . d . coleoptericin A and B showed strong antibacterial activity against Staphylococcus aureus, methicillin resistant S . aureus (MRSA) and Bacillus subtilis . Recombinant A . d . coleoptericin A and B were shown to not form pores through bacterial membranes of E . coli, but to hamper cell division . Results of Northern blotting showed that A . d . coleoptericin genes are inducible by bacteria and are expressed strongly in the fat bodies and haemocytes, and weakly in the Malpighian tubules . Analysis of the evolutionary relationship of amino acid sequences among A . d . coleoptericins and other antibacterial proteins suggests that A . d . coleoptericins, rhinocerosin and holotricin 2 are closely related and form a gene family.

Chem Pharm Bull (Tokyo), 2001 Aug, 49(8), 1047 - 9
Protease-catalyzed monoacylation of 2-O-alpha-D-Glucopyranosyl-L-ascorbic acid in pyridine; Tai A et al.; 2-O-alpha-D-Glucopyranosyl-6-O-octanoyl-L-ascorbic acid was enzymatically synthesized from 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and vinyl octanoate with a protease from Bacillus subtilis in pyridine . Furthermore, with various linear saturated fatty acid vinylesters as acyl donors, AA-2G was also converted to their corresponding 6-O-acyl AA-2G in the same manner . The reactivities of transacylation decreased with increasing length of the acyl groups . Thus, short chain acyl groups were transferred to AA-2G by this protease more efficiently than were long chain acyl groups . This enzymatic method is recommended for the synthesis of 6-Acyl-AA-2G with short or medium length chain acyl groups.

J Bacteriol, 2001 Sep, 183(18), 5449 - 52
Polymer stability plays an important role in the positional regulation of FtsZ; Levin PA et al.; We conducted a series of experiments examining the effect of polymer stability on FtsZ localization dynamics in Bacillus subtilis . A loss-of-function mutation in ezrA, a putative polymer-destabilizing factor, suppresses the defects in FtsZ polymer stability associated with minCD overexpression . In addition, a mutation that is predicted to stabilize the FtsZ polymer leads to the formation of polar FtsZ rings . These data support the hypothesis that carefully balanced polymer stability is important for the assembly and localization of FtsZ during the bacterial cell cycle.

J Bacteriol, 2001 Sep, 183(18), 5426 - 30
CotA of Bacillus subtilis is a copper-dependent laccase; Hullo MF et al.; The spore coat protein CotA of Bacillus subtilis displays similarities with multicopper oxidases, including manganese oxidases and laccases . B . subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved . We demonstrate that CotA is a laccase . Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with DeltacotA spores . CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.

J Agric Food Chem, 2001 Aug, 49(8), 4083 - 9
Determination of antioxidant and antimicrobial activities of Rumex crispus L . extracts; Yildirim A et al.; The antioxidant activities, reducing powers, 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, amount of total phenolic compounds, and antimicrobial activities of ether, ethanol, and hot water extracts of the leaves and seeds of Rumex crispus L . were studied . The antioxidant activities of extracts increase with increasing amount of extracts (50-150 microg) . However, the water extracts of both the leaves and seeds have shown the highest antioxidant activities . Thus, addition of 75 microg of each of the above extracts to the linoleic acid emulsion caused the inhibition of peroxide formation by 96 and 94%, respectively . Although the antioxidant activity of the ethanol extract of seed was lower than the water extract, the difference between these was not statistically significant, P > 0.05 . Unlike the other extracts, 75 microg of the ether extract of seeds was unable to show statistically significant antioxidant activity, P > 0.05 (between this extract and control in that there is no extract in the test sample) . Among all of the extracts, the highest amount of total phenolic compound was found in the ethanol extract of seeds, whereas the lowest amount was found in the ether extract of seeds . Like phenolic compounds, the highest reducing power and the highest DPPH scavenging activity were found in the ethanol extract of seeds . However, the reducing activity of the ethanol extract of seeds was approximately 40% that of ascorbic acid, whereas in the presence of 400 microg of water and ethanol extracts of seeds scavenging activities were about 85 and 90%, respectively . There were statistically significant correlations between amount of phenolic compounds and reducing power and between amount of phenolic compounds and percent DPPH scavenging activities (r = 0.99, P < 0.01, and r = 0.864, P < 0.05, respectively) and also between reducing powers and percent DPPH scavenging activities (r = 0.892, P < 0.05) . The ether extracts of both the leaves and seeds and ethanol extract of leaves had shown antimicrobial activities on Staphylococcus aureus and Bacillus subtilis . However, none of the water extracts showed antimicrobial activity on the studied microorganisms.

Biochemistry, 2001 Aug 28, 40(34), 10392 - 401
The srhSR gene pair from Staphylococcus aureus: genomic and proteomic approaches to the identification and characterization of gene function; Throup JP et al.; Systematic analysis of the entire two-component signal transduction system (TCSTS) gene complement of Staphylococcus aureus revealed the presence of a putative TCSTS (designated SrhSR) which shares considerable homology with the ResDE His-Asp phospho-relay pair of Bacillus subtilis . Disruption of the srhSR gene pair resulted in a dramatic reduction in growth of the srhSR mutant, when cultured under anaerobic conditions, and a 3-log attenuation in growth when analyzed in the murine pyelonephritis model . To further understand the role of SrhSR, differential display two-dimensional gel electrophoresis was used to analyze the cell-free extracts derived from the srhSR mutant and the corresponding wild type . Proteins shown to be differentially regulated were identified by mass spectrometry in combination with protein database searching . An srhSR deletion led to changes in the expression of proteins involved in energy metabolism and other metabolic processes including arginine catabolism, xanthine catabolism, and cell morphology . The impaired growth of the mutant under anaerobic conditions and the dramatic changes in proteins involved in energy metabolism shed light on the mechanisms used by S . aureus to grow anaerobically and indicate that the staphylococcal SrhSR system plays an important role in the regulation of energy transduction in response to changes in oxygen availability . The combination of proteomics, bio-informatics, and microbial genetics employed here represents a powerful set of techniques which can be applied to the study of bacterial gene function.

J Synchrotron Radiat, 2001 Mar 1, 8(Pt 2), 946 - 8
XAFS determination of the bacterial cell wall functional groups responsible for complexation of Cd and U as a function of pH; Kelly SD et al.; Bacteria, which are ubiquitous in near-surface geologic systems, can affect the distribution and fate of metals in these systems through adsorption reactions between the metals and bacterial cell walls . Recently, Fein et al . (1997) developed a chemical equilibrium approach to quantify metal adsorption onto cell walls, treating the sorption as a surface complexation phenomenon . However, such models are based on circumstantial bulk adsorption evidence only, and the nature and mechanism of metal binding to cell walls for each metal system have not been determined spectroscopically . The results of XAFS measurements at the Cd K-edge and U L3-edge on Bacillus subtilis exposed to these elements show that, at low pH, U binds to phosphoryl groups while Cd binds to carboxyl functional groups.

Anal Chem, 2001 Aug 1, 73(15), 3784 - 9
Continuous spore disruption using radially focused, high-frequency ultrasound; Chandler DP et al.; We report on the development of a novel, continuous-flow, radially focused ultrasonic disruptor capable of lysing Bacillus spores in the absence of added chemical denaturants, enzymes, or microparticles . Greater than 99% disruption was achieved for Bacillus globigii spores and Escherichia coli and Bacillus subtilis vegetative cells with sample residence times of 62, 12, and 12 s, respectively . Microscopic and SEM images indicated that at equivalent power levels, the incidence of cell death or loss of viability typically exceeded the efficiency of (visible) cell lysis . However, semiquantitative PCR showed up to a 1,000-fold increase in intracellular DNA availability from ultrasonically disrupted spores, and liberated DNA was intact and available for subsequent detection.

Biotechnol Bioeng, 2001 Sep, 76(2), 144 - 56
Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis; Dauner M et al.; Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates . Using a mixture of 10% {U-(13)C} and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing . The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional {(13)C, (1)H} nuclear magnetic resonance (NMR) spectroscopy . To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B . subtilis central metabolism . Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids . Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate . Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected . The malic enzyme reaction, in contrast, was found to be inactive . A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results . Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data .

Biotechnol Bioeng, 2001 Sep, 76(2), 132 - 43
Stoichiometric growth model for riboflavin-producing Bacillus subtilis; Dauner M et al.; Rate equations for measured extracellular rates and macromolecular composition data were combined with a stoichiometric model to describe riboflavin production with an industrial Bacillus subtilis strain using errors in variables regression analysis . On the basis of this combined stoichiometric growth model, we explored the topological features of the B . subtilis metabolic reaction network that was assembled from a large amount of literature . More specifically, we simulated maximum theoretical yields of biomass and riboflavin, including the associated flux regimes . Based on the developed model, the importance of experimental data on building block requirements for maximum yield and flux calculations were investigated . These analyses clearly show that verification of macromolecular composition data is important for optimum flux calculations .

Mol Biol Evol, 2001 Sep, 18(9), 1789 - 99
Ongoing evolution of strand composition in bacterial genomes; Rocha EP et al.; We tried to identify the substitutions involved in the establishment of replication strand bias, which has been recognized as an important evolutionary factor in the evolution of bacterial genomes . First, we analyzed the composition asymmetry of 28 complete bacterial genomes and used it to test the possibility that asymmetric deamination of cytosine might be at the origin of the bias . The model showed significant correlation to the data but left unexplained a significant portion of the variance and indicated a systematic underestimation of GC skews in comparison with TA skews . Second, we analyzed the substitutions acting on the genes from five fully sequenced Chlamydia genomes that had not suffered strand switch since speciation . This analysis showed that substitutions were not at equilibrium in Chlamydia trachomatis or in C . muridarum and that strand bias is still an on-going process in these genes . Third, we identified substitutions involved in the adaptation of genes that had switched strands after speciation . These genes adapted quickly to the skewed composition of the new strand, mostly due to C-->T, A-->G, and C-->G asymmetric substitutions . This observation was reinforced by the analysis of genes that switched strands after divergence between Bacillus subtilis and B . halodurans . Finally, we propose a more extended model based on the analysis of the substitution asymmetries of CHLAMYDIA: This model fits well with the data provided by bacterial genomes presenting strong strand bias.

J Mol Biol, 2001 Jul 27, 310(5), 1027 - 37
Structural organisation of the head-to-tail interface of a bacterial virus; Lurz R et al.; In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure . The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail . The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1 . It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16 . The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity . The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail . The gp16 ring is exposed to the virion outside . During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation . gp16 is processed during or after tail attachment to the connector region . The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-naive gp6 exhibits 13-fold symmetry . We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.

J AOAC Int, 2001 Jul-Aug, 84(4), 1159 - 63
Culture age and drying time as variables of the AOAC Sporicidal Test; Miner NA et al.; In the United States, the AOAC Sporicidal Activity of Disinfectants Method 966.04 is the standard for identifying a liquid chemical germicide as a sterilant . Furthermore, the highest level of a disinfectant must also be a sterilant as defined by Method 966.04, when used in its sterilant mode for a longer exposure time . The AOAC Sporicidal Test is also used as a part of the standard test methods to define a sterilant for Australia and the European Union . Many laboratories have identified variables of this test that can affect the sterilization exposure time for sterilants, or even the ability to classify a chemical as a sterilant . Method 966.04 requires spore-labeled porcelain penicylinders (cylinders) and silk suture loops, collectively referred to as carriers, to be dried for 24 h, but allows these carriers to be used for at least 7 days, in effect allowing a drying time of 24 h to at least 7 days . We tested the resistance of cylinders that had been labeled with Bacillus subtilis spores cultured for 72, 96, and 120 h, and dried for 24, 48, and 72 h against a 60 min exposure to 2.0% alkaline glutaraldehyde, and 2, 5, 10, 15, and 20 min exposures to 2.5N HCl . All the culture incubation and drying times met the standard of resistance to 2.5N HCI for at least 2.0 min at 20 degrees C, and all carriers contained at least 10(5) colony-forming units (CFU) of B . subtilis per carrier . However, for 3 repeated tests, regardless of incubation time, an average of 96% of the carriers were sterilized by the 2.0% glutaraldehyde after drying for 24 h, and an average of 61 % were sterilized after drying for 48 or 72 h . We propose that the variable of drying time be eliminated from Method 966.04.

Microbiology, 2001 Aug, 147(Pt 8), 2275 - 84
Starvation survival in Listeria monocytogenes: characterization of the response and the role of known and novel components; Herbert KC et al.; The starvation survival response (SSR) of Listeria monocytogenes EGD is induced under glucose- or multiple-nutrient-, but not amino-acid limitation . 0.01-0.2% of the population remain viable even after 20 d and the survivors show a reduced cell size and increased cross-protection to several environmental stresses . The development of the SSR may therefore be important in L . monocytogenes survival in the food environment . The initiation, but not the maintenance, of the SSR involves both protein and cell wall biosynthesis . It is also likely that nutrients released from dead cells are recycled to allow survival of the remaining population . To define the molecular mechanisms involved in the initiation, maintenance and release from the SSR the role of known, and novel, components was examined . The well-characterized regulators SigB and PrfA are both required for the full SSR and effect stress resistance during growth and starvation . A transposon mutagenesis screen identified two novel loci with roles in the SSR and stress resistance . Characterization of the transposon insertion sites revealed a putative homologue of the gene yulB from Bacillus subtilis and a gene of unknown function . The potential individual and combined roles of the SSR components are discussed.

J Mol Biol, 2001 May 25, 309(1), 215 - 26
The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase fold enzyme; van Pouderoyen G et al.; The X-ray structure of the lipase LipA from Bacillus subtilis has been determined at 1.5 A resolution . It is the first structure of a member of homology family 1.4 of bacterial lipases . The lipase shows a compact minimal alpha/beta hydrolase fold with a six-stranded parallel beta-sheet flanked by five alpha-helices, two on one side of the sheet and three on the other side . The catalytic triad residues, Ser77, Asp133 and His156, and the residues forming the oxyanion hole (backbone amide groups of Ile12 and Met78) are in positions very similar to those of other lipases of known structure . However, no lid domain is present and the active-site nucleophile Ser77 is solvent-exposed . A model of substrate binding is proposed on the basis of a comparison with other lipases with a covalently bound tetrahedral intermediate mimic . It explains the preference of the enzyme for substrates with C8 fatty acid chains.

Arch Microbiol, 2001 Jun, 175(6), 441 - 9
Characterization of glucose-repression-resistant mutants of Bacillus subtilis: identification of the glcR gene; Stulke J et al.; In Bacillus subtilis, carbon catabolite repression (CCR) is mediated by the pleiotropic repressor CcpA and by ATP-dependent phosphorylation of the HPr protein of the phosphotransferase system (PTS) . In this study, we attempted to identify novel genes that are involved in the signal transduction pathway that ultimately results in CCR in the presence of repressing carbon sources such as glucose . Seven mutants resistant to glucose repression of the levanase operon were isolated and characterized . All mutations were trans-acting and pleiotropic as determined by analyzing CCR of beta-xylosidase and of the sacPA and bglPH operon . Moreover, all mutations specifically affected repression exerted by glucose but not by other sugars . The mutations were mapped to three different loci on the genetic map, ptsG, glcR, and pgi . These three genes encode proteins involved in glucose metabolism . A novel repressor gene, glcR (ywpI), defined by two mutations, was studied in more detail . The glcR mutants exhibit loss of glucose repression of catabolic operons, a deficiency in glucose transport, and absence of expression of the ptsG gene . The mutant GlcR proteins act as super-repressors of ptsG expression.

J Biol Chem, 2001 Oct 5, 276(40), 37289 - 98 Epub 2001 Aug 06.
4'-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis; Mootz HD et al.; 4'-Phosphopantetheine transferases (PPTases) transfer the 4'-phosphopantetheine moiety of coenzyme A onto a conserved serine residue of acyl carrier proteins (ACPs) of fatty acid and polyketide synthases as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases . This posttranslational modification converts ACPs and PCPs from their inactive apo into the active holo form . We have investigated the 4'-phosphopantetheinylation reaction in Bacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin . We identified and cloned ydcB encoding AcpS from B . subtilis, which complemented an Escherichia coli acps disruption mutant . B . subtilis AcpS and its substrate ACP were biochemically characterized . AcpS also modified the d-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that was not identified by the Bacillus genome project . On the other hand, Sfp was able to modify in vitro all acyl carrier proteins tested . We thereby extend the reported broad specificity of this enzyme to the homologous ACP . This in vitro cross-interaction between primary and secondary metabolism was confirmed under physiological in vivo conditions by the construction of a ydcB deletion in a B . subtilis sfp(+) strain . The genes coding for Sfp and its homolog Gsp from Bacillus brevis could also complement the E . coli acps disruption . These results call into question the essential role of AcpS in strains that contain a Sfp-like PPTase and consequently the suitability of AcpS as a microbial target in such strains.

J Bacteriol, 2001 Sep, 183(17), 5110 - 21
Regulation of the glv operon in Bacillus subtilis: YfiA (GlvR) is a positive regulator of the operon that is repressed through CcpA and cre; Yamamoto H et al.; Maltose metabolism and the regulation of the glv operon of Bacillus subtilis, comprising three genes, glvA (6-phospho-alpha-glucosidase), yfiA (now designated glvR), and glvC (EIICB transport protein), were investigated . Maltose dissimilation was dependent primarily upon the glv operon, and insertional inactivation of either glvA, glvR, or glvC markedly inhibited growth on the disaccharide . A second system (MalL) contributed to a minor extent to maltose metabolism . Northern blotting revealed two transcripts corresponding to a monocistronic mRNA of glvA and a polycistronic mRNA of glvA-glvR-glvC . Primer extension analysis showed that both transcripts started at the same base (G) located 26 bp upstream of the 5' end of glvA . When glvR was placed under control of the spac promoter, expression of the glv operon was dependent upon the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) . In regulatory studies, the promoter sequence of the glv operon was fused to lacZ and inserted into the amyE locus, and the resultant strain (AMGLV) was then transformed with a citrate-controlled glvR plasmid, pHYCM2VR . When cultured in Difco sporulation medium containing citrate, this transformant {AMGLV(pHYCM2VR)} expressed LacZ activity, but synthesis of LacZ was repressed by glucose . In an isogenic strain, {AMGLVCR(pHYCM2VR)}, except for a mutation in the sequence of a catabolite-responsive element (cre), LacZ activity was expressed in the presence of citrate and glucose . Insertion of a citrate-controlled glvR plasmid at the amyE locus of ccpA(+) and ccpA mutant organisms yielded strains AMCMVR and AMCMVRCC, respectively . In the presence of both glucose and citrate, AMCMVR failed to express the glv operon, whereas under the same conditions high-level expression of both mRNA transcripts was found in strain AMCMVRCC . Collectively, our findings suggest that GlvR (the product of the glvR gene) is a positive regulator of the glv operon and that glucose exerts its effect via catabolite repression requiring both CcpA and cre.

J Bacteriol, 2001 Sep, 183(17), 4994 - 5000
Identification and characterization of fhuD1 and fhuD2, two genes involved in iron-hydroxamate uptake in Staphylococcus aureus; Sebulsky MT et al.; Staphylococcus aureus can utilize several hydroxamate siderophores for growth under iron-restricted conditions . Previous findings have shown that S . aureus possesses a cytoplasmic membrane-associated traffic ATPase that is involved in the specific transport of iron(III)-hydroxamate complexes . In this study, we have identified two additional genes, termed fhuD1 and fhuD2, whose products are involved in this transport process in S . aureus . We have shown that fhuD2 codes for a posttranslationally modified lipoprotein that is anchored in the cytoplasmic membrane, while the deduced amino acid sequence predicts the same for fhuD1 . The predicted FhuD1 and FhuD2 proteins share 41.0% identity and 56.4% total similarity with each other, 45.9 and 49.1% total similarity with the FhuD homolog in Bacillus subtilis, and 29.3 and 24.6% total similarity with the periplasmic FhuD protein from Escherichia coli . Insertional inactivation and gene replacement of both genes showed that while FhuD2 is involved in the transport of iron(III) in complex with ferrichrome, ferrioxamine B, aerobactin, and coprogen, FhuD1 shows a more limited substrate range, capable of only iron(III)-ferrichrome and iron(III)-ferrioxamine B transport in S . aureus . Nucleotide sequences present upstream of both fhuD1 and fhuD2 predict the presence of consensus Fur binding sequences . In agreement, transcription of both genes was negatively regulated by exogenous iron levels through the activity of the S . aureus Fur protein.

J Bacteriol, 2001 Sep, 183(17), 4958 - 63
Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5 in Bacillus subtilis; Inaoka T et al.; To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system . Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome . Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain . We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant . These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels . Thus, the mutation which altered Lys-56 to Arg resulted in a ram phenotype in B . subtilis . The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.

Mol Microbiol, 2001 Jul, 41(2), 409 - 22
Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon; Ludwig H et al.; Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis . Although the biochemical activity of glycolytic enzymes has been studied in detail, no information about the expression of glycolytic genes has so far been available in this organism . Therefore, transcriptional analysis of all glycolytic genes was performed . The genes cggR, gapA, pgk, tpi, pgm and eno, encoding the enzymes required for the interconversion of triose phosphates, are transcribed as a hexacistronic operon as demonstrated by Northern analysis . This gapA operon is repressed by the regulator CggR . The presence of sugars and amino acids synergistically results in the induction of the gapA operon . The transcriptional start site upstream of cggR was mapped by primer extension . Transcripts originating upstream of cggR are processed near the 3' end of cggR . This endonucleolytic cleavage leads to differential stability of the resulting processing products: the monocistronic cggR message is very rapidly degraded, whereas the mRNA species encoding glycolytic enzymes exhibit much higher stability . An additional internal constitutive promoter was identified upstream of pgk . Thus, gapA is the most strongly regulated gene of this operon . The pfk pyk operon encoding phosphofructokinase and pyruvate kinase is weakly induced by glucose . In contrast, the genes pgi and fbaA, coding for phosphoglucoisomerase and fructose-1,6-bisphosphate aldolase, are constitutively expressed.

Pharmazie, 2001 Jul, 56(7), 561 - 6
Experimental data and theoretical considerations concerning the validity of the SAL concept to characterize non-thermal antimicrobial treatments; von Woedtke T et al.; For sterilization processes the pharmacopoeias demand a sterility assurance level (SAL) of 10(-6), i.e . a probability of not more than one viable microorganism among one million sterilized products . This SAL concept is based on the assumption that the inactivation of microorganisms by physical or chemical means generally follows first-order kinetics . In this paper it is demonstrated that this is not absolutely true for non-thermal antimicrobial processes . Using Bacillus subtilis spore test preparations the sporicidal efficacy of gamma and ultraviolet irradiation on the one hand as well as the treatment by glutaraldehyde and hydrogen peroxide containing solutions on the other hand was investigated . A range of mean spore contamination between 10(8) and 10(-2) spores per test item could be supported by experimental data . It was demonstrated that the antimicrobial treatment parameters which are sufficient to reduce a high spore burden were not valid for an adequate reduction of the remaining lower spore burden . It is concluded that any extrapolation of such experimental data to the SAL range as usual in the validation of sterilization process parameters may be not permitted . Possible theoretical explanations of the non-homogeneity of the spore inactivation by non-thermal methods as well as consequences for the safety evaluation of sterilization processes are discussed.

J Biol Chem, 2001 Oct 12, 276(41), 37922 - 8 Epub 2001 Aug 03.
Negative cooperativity of substrate binding but not enzyme activity in wild-type and mutant forms of CTP:glycerol-3-phosphate cytidylyltransferase; Sanker S et al.; CTP:glycerol-3-phosphate cytidylyltransferase (GCT) catalyzes the synthesis of CDP-glycerol for teichoic acid biosynthesis in certain Gram-positive bacteria . This enzyme is a model for a cytidylyltransferase family that includes the enzymes that synthesize CDP-choline and CDP-ethanolamine for phosphatidylcholine and phosphatidylethanolamine biosynthesis . We have used quenching of intrinsic tryptophan fluorescence to measure binding affinities of substrates to the GCT from Bacillus subtilis . Binding of either CTP or glycerol-3-phosphate to GCT was biphasic, with two binding constants of about 0.1-0.3 and 20-40 microm for each substrate . The stoichiometry of binding was 2 molecules of substrate/enzyme dimer, so the two binding constants represented distinctly different affinities of the enzyme for the first and second molecule of each substrate . The biphasic nature of binding was observed with the wild-type GCT as well as with several mutants with altered Km or kcat values . This negative cooperativity of binding was also seen when a catalytically defective mutant was saturated with two molecules of CTP and then titrated with glycerol-3-phosphate . Despite the pronounced negative cooperativity of substrate binding, negative cooperativity of enzyme activity was not observed . These data support a mechanism in which catalysis occurs only when the enzyme is fully loaded with 2 molecules of each substrate/enzyme dimer.

J Endod, 2001 Mar, 27(3), 172 - 4
Antibacterial properties of electron beam-sterilized Gutta-Percha cones; Attin T et al.; The purpose of the study was to determine the effect of electron beam sterilization on gutta-percha cones (GPCs) at different times after sterilization . An agar diffusion test was used with -one aerobic bacterium (Bacillus subtilis) and five oral anaerobic bacteria (Fusobacterium nucleatum, Peptostreptococcus micros, Porphyromonas gingivalis, Propionibacterium acnes, and Veillonella parvula) . With each microorganism 30 agar plates were prepared, evenly distributed among three groups (group 1: unsterilized GPCs; groups 2 and 3: GPCs sterilized by electron beam irradiation 5 months and 5 yr before, respectively) . One GPC of the selected group was placed in each plate . After incubation the area of inhibition was read on the agar plates . Inhibition of growth was significantly different for the tested microorganisms . However no significant difference was observed between the sterilized and unsterilized GPCs . Both the tested sterilized and unsterilized GPCs impair the growth of endodontic pathogens, with no influence of the time elapsed since sterilization.

Med Sci Sports Exerc, 2001 Aug, 33(8), 1385 - 6
Ultraviolet exposure in the Ironman triathlon; Moehrle M; PURPOSE: Skin cancer is increasing worldwide and exposure to ultraviolet (UV) radiation is thought to be the most important environmental risk factor . People practicing outdoor sports are exposed to considerable amounts of UV radiation from the sun . METHODS: Three triathletes participated in the Ironman Triathlon World Championships 1999 in Hawaii (3.9-km swim, 180.2-km bike, 42.4-km run) . They attached Bacillus subtilis spore film dosimeters (VioSpor) on the back between their shoulders . The dosimeter system measured cumulative biologically weighted erythemal UV exposure . UV exposure is given in minimal erythema doses (1 MED corresponds to 250 J x m(-2) at 298 nm) . RESULTS: The mean personal UV exposure was 8.3 MED (6.9--9.7 MED) after 8:43 to 9:44 h of competition corresponding to 0.8 to 1.3 MED x h(-1) (bike and run) . The athletes were sunburned despite the use of water-resistant sunscreen (SPF 25+) on sun exposed skin . CONCLUSION: The International Radiation Protection Agency has issued guidelines for professional UV exposure . Ironman triathletes considerably exceeded these limits of exposure similar to other outdoor sports . Professional and amateur athletes should be aware of hazards caused by UV radiation . Adequate protection by water-resistant sunscreens and clothing as well as training and competition schedules with low sun exposure seem to be a reasonable recommendation.

J Nat Prod, 2001 Jul, 64(7), 932 - 4
Antimicrobial cuparene-type sesquiterpenes, enokipodins C and D, from a mycelial culture of Flammulina velutipes; Ishikawa NK et al.; Two new cuparene-type sesquiterpenes, enokipodins C (1) and D (2), were isolated from culture medium of an edible mushroom, Flammulina velutipes, along with enokipodins A (3) and B (4) . The structures of 1 and 2 were determined using spectroscopic methods (HRMS, (1)H and (13)C, and 2D NMR) . The absolute configuration of enokipodin C was determined from the observed (1)H NMR chemical shifts and NOEs in NOESY experiments after conversion into the corresponding esters with the chiral reagent 2-(2'-methoxy-1'-naphthyl)-3,4-dichlorobenzoic acid . All the metabolites showed antimicrobial activity against a fungus, Cladosporium herbarum, and Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus.

Nat Struct Biol, 2001 Aug, 8(8), 674 - 8
Structure of the Bacillus subtilis D-aminopeptidase DppA reveals a novel self-compartmentalizing protease; Remaut H et al.; Bacillus subtilis DppA is a binuclear zinc-dependent, D-specific aminopeptidase . The X-ray structure of the enzyme has been determined at 2.4 A resolution by a three-wavelength MAD experiment . The structure reveals that DppA is a new example of a 'self-compartmentalizing protease', a family of proteolytic complexes . Proteasomes are the most extensively studied representatives of this family . The DppA enzyme is composed of identical 30 kDa subunits organized in a decamer with 52 point-group symmetry . A 20 A wide channel runs through the complex, giving access to a central chamber holding the active sites . The structure shows DppA to be a prototype of a new family of metalloaminopeptidases characterized by the SXDXEG key sequence.

J Struct Biol, 2001 Feb-Mar, 133(2-3), 233 - 45
A novel neural network technique for analysis and classification of EM single-particle images; Pascual-Montano A et al.; We propose a novel self-organizing neural network for the unsupervised classification of electron microscopy (EM) images of biological macromolecules . The radical novelty of the algorithm lies in its rigorous mathematical formulation that, starting from a large set of possibly very noisy input data, finds a set of "representative" data items, organized onto an ordered output map, such that the probability density of this set of representative items resembles at its possible best the probability density of the input data . In a way, it summarizes large amounts of information into a concise description that rigorously keeps the basic pattern of the input data distribution . In this application to the field of three-dimensional EM of single particles, two different data sets have been used; one comprised 2458 rotational power spectra of individual negative stain images of the G40P helicase of Bacillus subtilis bacteriophage SPP1, and the other contained 2822 cryoelectron images of SV40 large T-antigen . Our experimental results prove that this technique is indeed very successful, providing the user with the capability of exploring complex patterns in a succinct, informative, and objective manner . The above facts, together with the consideration that the integration of this new algorithm with commonly used software packages is immediate, prompt us to propose it as a valuable new tool in the analysis of large collections of noisy data .

J Biol Inorg Chem, 2001 Jun, 6(5-6), 523 - 33
Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis; Green AJ et al.; The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity . The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form . N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence . SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose) . The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance {g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)} . Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water . Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms . Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity . The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site . Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E . coli ferredoxin reductase . Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.

Biosci Biotechnol Biochem, 2001 Jun, 65(6), 1391 - 4
Characterization of aspartate kinase III of Bacillus subtilis; Kobashi N et al.; A search in the Bacillus subtilis genome sequence found that the gene designated yclM encode(s) a protein showing significant identity in amino acid sequence to aspartate kinases . When yclM was introduced into Escherichia coli cells deficient in all three aspartate kinase genes, production of a protein with molecular size 50 kDa, which was similar to the value deduced from the nucleotide sequence of the gene, was observed . Expectedly, the protein purified to homogeneity had aspartate kinase activity . The enzyme was significantly inhibited by simultaneous addition of both threonine and lysine, which is a typical feature of aspartate kinase III of B . subtilis . The enzyme was very unstable in 10 mM tris-HCl (pH 7.5) buffer, but was stabilized by addition of 500 mM ammonium sulfate . Although all the aspartate kinases so far investigated are oligomeric enzymes, this aspartate kinase was suggested to be a monomer.

Water Res, 2001 Aug, 35(12), 2950 - 60
Inactivation of Bacillus subtilis spores and formation of bromate during ozonation; Driedger A et al.; Inactivation of B . subtilis spores with ozone was investigated to assess the effect of pH and temperature, to compare the kinetics to those for the inactivation of C . parvum oocysts, to investigate bromate formation under 2-log inactivation conditions, and to assess the need for bromate control strategies . The rate of B . subtilis inactivation with ozone was independent of pH, decreased with temperature (activation energy of 42,100 Jmol(-1)), and was consistent with the CT concept . B . subtilis was found to be a good indicator for C . parvum at 20-30 degrees C, but at lower temperatures B . subtilis was inactivated more readily than C . parvum . Bromate formation increased as both pH and temperature increased . For water with an initial bromide concentration of 33 microgl(-1), achieving 2-logs of inactivation, without exceeding the 100 microg l(-1) bromate standard, was most difficult at 30 degrees C for B . subtilis and at midrange temperatures (10-20 degrees C) for C . partum . pH depression and ammonia addition were found to reduce bromate formation without affecting B . subtilis inactivation, and may be necessary for waters containing more than 50 microgl(-1) bromide.

Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 9038 - 43 Epub 2001 Jul 24.
The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase; Rebeil R et al.; The major DNA photoproduct of dormant, UV-irradiated Bacillus subtilis spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine {spore photoproduct (SP)} . During spore germination, SP is reversed to two intact thymines in situ by the DNA repair enzyme SP lyase, an S-adenosylmethionine (S-AdoMet)-dependent iron-sulfur ({Fe-S}) protein encoded by the splB gene . In the present work, cross-linking, SDS/PAGE, and size exclusion chromatography revealed that SplB protein dimerized when incubated with iron and sulfide under anaerobic reducing conditions . SplB isolated under aerobic conditions generated an EPR spectrum consistent with that of a partially degraded {3Fe-4S} center, and reduction of SplB with dithionite shifted the spectrum to that of a {4Fe-4S} center . Addition of S-AdoMet to SplB converted some of the {4Fe-4S} centers to an EPR-silent form consistent with electron donation to S-AdoMet . HPLC and electrospray ionization MS analyses showed that SP lyase cleaved S-AdoMet to generate 5'-deoxyadenosine . The results indicate that (i) SP lyase is a homodimer of SplB; (ii) dimer formation is coordinated by a {4Fe-4S} center; and (iii) the reduced {4Fe-4S} center is capable of donating electrons to S-AdoMet to generate a 5'-adenosyl radical that is then used for the in situ reversal of SP . Thus, SP lyase belongs to the "radical SAM" superfamily of enzymes that use {Fe-S} centers and S-AdoMet to generate adenosyl radicals to effect catalysis . SP lyase is unique in being the first and only DNA repair enzyme known to function via this novel enzymatic mechanism.

Structure (Camb), 2001 Jul 3, 9(7), 605 - 14
Structure of the Bacillus cell fate determinant SpoIIAA in phosphorylated and unphosphorylated forms; Seavers PR et al.; BACKGROUND: The asymmetric cell division during sporulation in Bacillus subtilis gives rise to two compartments: the mother cell and the forespore . Each follow different programs of gene expression coordinated by a succession of alternate RNA polymerase sigma factors . The activity of the first of these sigma factors, sigmaF, is restricted to the forespore although sigmaF is present in the predivisional cell and partitions into both compartments following the asymmetric septation . For sigmaF to become active, it must escape from a complex with its cognate anti-sigma factor, SpoIIAB . This relief from SpoIIAB inhibition requires the dephosphorylation of the anti-sigma factor antagonist, SpoIIAA . The phosphorylation state of SpoIIAA is thus a key determinant of sigmaF activity and cell fate . RESULTS: We have solved the crystal structures of SpoIIAA from Bacillus sphaericus in its phosphorylated and unphosphorylated forms . The overall structure consists of a central beta-pleated sheet, one face of which is buried by a pair of alpha helices, while the other is largely exposed to solvent . The site of phosphorylation, Ser57, is located at the N terminus of helix alpha2 . The phosphoserine is exceptionally well defined in the 1.2 A electron density maps, revealing that the structural changes accompanying phosphorylation are slight . CONCLUSIONS: Comparison of unphosphorylated and phosphorylated SpoIIAA shows that covalent modification has no significant effect on the global structure of the protein . The phosphoryl group has a passive role as a negatively charged flag rather than the active role it plays as a nucleus of structural reorganization in many eukaryotic signaling systems.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 285 - 90
Purification and characterisation of a serine peptidase from the marine psychrophile strain PA-43; Irwin JA et al.; An extracellular serine peptidase, purified from the culture supernatant of the sub-Arctic psychrophilic bacterium strain PA-43, is monomeric, with a relative molecular mass of 76000, and an unusually low pI of 3.8 . The peptidase is active towards N-succinyl AAPF p-nitroanilide and N-succinyl AAPL p-nitroanilide, indicating a chymotrypsin-like substrate specificity . It is inhibited by the serine peptidase inactivator phenylmethylsulfonyl fluoride, but not by EDTA or EGTA, suggesting that added metal ions are not necessary for activity . The enzyme is most active at pH 8.3 and at 55-60 degrees C, although it is unstable at 60 degrees C . It is nevertheless remarkably stable for an enzyme from a psychrophilic microorganism, remaining active after 1 week at 20 degrees C and after five freeze-thaw cycles . Comparison of the N-terminal 40 amino acid residues with other archived sequences revealed highest similarity to the alkaline serine protease (aprx) from Bacillus subtilis.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1138 - 40 Epub 2001 Jul 23.
Cloning, purification and characterization of the 6-phospho-3-hexulose isomerase YckF from Bacillus subtilis; Taylor EJ et al.; The enzyme 6-phospho-3-hexulose isomerase (YckF) from Bacillus subtilis has been prepared and crystallized in a form suitable for X-ray crystallographic analysis . Crystals were grown by the hanging-drop method at 291 K using polyethylene glycol 2000 monomethylether as precipitant . They diffract beyond 1.7 A using an in-house Cu Kalpha source and belong to either space group P6(5)22 or P6(1)22, with unit-cell parameters a = b = 72.4, c = 241.2 A, and have two molecules of YckF in the asymmetric unit.

J Bacteriol, 2001 Aug, 183(16), 4905 - 9
Control of a family of phosphatase regulatory genes (phr) by the alternate sigma factor sigma-H of Bacillus subtilis; McQuade RS et al.; A family of 11 phosphatases can help to modulate the activity of response regulator proteins in Bacillus subtilis . Downstream of seven of the rap (phosphatase) genes are phr genes, encoding secreted peptides that function as phosphatase regulators . By using fusions to lacZ and primer extension analysis, we found that six of the seven phr genes are controlled by the alternate sigma factor sigma-H . These results expand the potential of sigma-H to contribute to the output of several response regulators by controlling expression of inhibitors of phosphatases.

J Bacteriol, 2001 Aug, 183(16), 4894 - 9
Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination; Setlow B et al.; Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water . However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation . We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.

J Bacteriol, 2001 Aug, 183(16), 4886 - 93
Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate; Paidhungat M et al.; Dormant Bacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA) . Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown . As Ca(2+)-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination . We found that Ca(2+)-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core . Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex . Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca(2+)-DPA . Our findings thus define a mechanism for Ca(2+)-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.

J Bacteriol, 2001 Aug, 183(16), 4814 - 22
Developmental gene expression in Bacillus subtilis crsA47 mutants reveals glucose-activated control of the gene for the minor sigma factor sigma(H); Dixon LG et al.; The presence of excess glucose in growth media prevents normal sporulation of Bacillus subtilis . The crsA47 mutation, located in the gene for the vegetative phase sigma factor (sigma(A)) results in a glucose-resistant sporulation phenotype . As part of a study of the mechanisms whereby the mutation in sigma(A) overcomes glucose repression of sporulation, we examined the expression of genes involved in sporulation initiation in the crsA47 background . The crsA47 mutation had a significant impact on a variety of genes . Changes to stage II gene expression could be linked to alterations in the expression of the sinI and sinR genes . In addition, there was a dramatic increase in the expression of genes dependent on the minor sigma factor sigma(H) . This latter change was paralleled by the pattern of spo0H gene transcription in cells with the crsA47 mutation . In vitro analysis of RNA polymerase containing sigma(A47) indicated that it did not have unusually high affinity for the spo0H gene promoter . The in vivo pattern of spo0H expression is not predicted by the known regulatory constraints on spo0H and suggests novel regulation mechanisms that are revealed in the crsA47 background.

EMBO Rep, 2001 Aug, 2(8), 685 - 9 Epub 2001 Jul 19.
Specific polar localization of ribosomes in Bacillus subtilis depends on active transcription; Mascarenhas J et al.; The large subunit of ribosomes in Bacillus subtilis was tagged by generation of a fusion of ribosomal protein L1 to blue fluorescent protein (BFP) . The fusion was fully active and localized around the nucleoids, predominantly close to the cell poles, in growing cells . However, in stationary phase cells, and in growing cells treated with rifampicin, L1-BFP was distributed throughout the cells, in contrast to cells treated with chloramphenicol, in which ribosomes still localized around nucleoids . These data show that specific localization of ribosomes is not due to nucleoid exclusion, but is a dynamic process due to active synthesis of RNA . Dual labelling of ribosomes and cold shock proteins (CSPs) tagged with green fluorescent protein (GFP) revealed colocalization of both protein classes . CSPs are implicated in coupling of transcription with translation and may bridge the spatial separation of ribosomes and nucleoid-associated RNA polymerase.

J Food Prot, 2001 Jul, 64(7), 1000 - 11
Optimizing detection of heat-injured Listeria monocytogenes in pasteurized milk; Teo AY et al.; Optimal conditions for the detection of heat-injured cells of Listeria monocytogenes in modified Pennsylvania State University (mPSU) broth were determined using a response surface design generated by a computer program, EChip . Different combinations of incubation temperatures and lithium, magnesium, and D-serine concentrations were evaluated to determine the optimum conditions for the detection of heat-injured L . monocytogenes in filter-sterilized whole milk inoculated with selected problematic background microflora . A concentration of 212 mM lithium chloride completely inhibited the growth of Enterococcus faecium while permitting recovery and detection of L . monocytogenes . A concentration of 15.8 mM MgSO4 was found to be optimum for the recovery and detection of L . monocytogenes . A concentration of 140.2 mM D-serine was found to completely inhibit the germination of Bacillus subtilis var . globii spores but not recovery and detection of L . monocytogenes . Under optimum concentrations of LiCl, MgSO4, and D-serine and in the absence of background microflora, the effect of incubation temperature on percentage detection was described by a second-order polynomial model, and 28 degrees C was determined to be optimal . In the presence of background microflora, the effect of incubation temperature on percentage detection of heat-injured cells was described by a third-order polynomial model, and 30 degrees C was found to be optimal . Optimizing the levels of highly specific and selective agents, nutrients, and incubation temperature in one recovery enrichment system dramatically increased the Listeria/background microflora ratio . This resulting medium, optimized PSU (oPSU) broth, greatly improved the detection of heat-injured and nonheat-injured L . monocytogenes by both conventional and molecular methods (Oxoid's Listeria Rapid Test, Gen-Probe's Accuprobe Listeria monocytogenes Culture Identification Test, and Qualicon's BAX for screening Listeria monocytogenes).

Mol Microbiol, 2001 Jul, 41(1), 131 - 43
Exploring the minimal substrate requirements for trans-cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis; Hansen A et al.; We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes . We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5' flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site . Processing efficiencies decreased up to 5000-fold when the 5' flank was shortened from 3 to 1 nt . Reduction of the 5' flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex . These results indicate that the crucial contribution of nt -2 in the single-stranded 5' flank to productive interaction is a general feature of A- and B-type bacterial RNase P enzymes . We also showed that an Rp-phosphorothioate modification at nt -2 interferes with processing . Bacterial RNase P holoenzymes are also capable of cleaving single-stranded RNA oligonucleotides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH termini, with measured turnover rates of up to 0.7 min-1 . All cleavage sites were at least 2 nt away from the 5' and 3' ends of the oligonucleotides . Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.

Mol Microbiol, 2001 Jul, 41(1), 59 - 71
Alkaline shock induces the Bacillus subtilis sigma(W) regulon; Wiegert T et al.; When confronted with a stress factor, bacteria react with a specific stress response, a genetically encoded programme resulting in the transiently enhanced expression of a subset of genes . One of these stress factors is a sudden increase in the external pH . As a first step to understand the response of Bacillus subtilis cells towards an alkali shock at the transcriptional level, we attempted to identify alkali-inducible genes using the DNA macroarray technique . To define the appropriate challenging conditions, we used the ydjF gene, the orthologue of the Escherichia coli pspA, as a model gene for an alkali-inducible gene . Hybridization of 33P-labelled cDNA to a DNA macroarray revealed induction of more than 80 genes by a sudden increase in the external pH value from 6.3 to 8.9 . It was discovered that a large subset of these genes belong to the recently described sigmaW regulon, which was confirmed by the analysis of a sigW knockout . A comparison of B . subtilis wild type with the congenic sigW knockout also led to the discovery of new members of the sigmaW regulon . In addition, we found several genes clearly not belonging to that regulon . This analysis represents the first report of an extracellular stimulus inducing the sigmaW regulon.

J Biol Chem, 2001 Sep 14, 276(37), 34824 - 31 Epub 2001 Jul 11.
Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis; Eppelmann K et al.; Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products . However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters . In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes . In the B . subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method . Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B . subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes . This engineered and genetically amenable B . subtilis strain will facilitate the rational design of new bacitracin derivatives.

Infect Immun, 2001 Aug, 69(8), 5016 - 24
Identification of Listeria monocytogenes in vivo-induced genes by fluorescence-activated cell sorting; Wilson RL et al.; Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen capable of causing severe infections in immunocompromised or pregnant individuals, as well as numerous animal species . Genetic analysis of Listeria pathogenesis has identified several genes which are crucial for virulence . The transcription of most of these genes has been shown to be induced upon entry of Listeria into the host cell . To identify additional genes that are induced in vivo and may be required for L . monocytogenes pathogenesis, a fluorescence-activated cell-sorting technique was initiated . Random fragments of the L . monocytogenes chromosome were cloned into a plasmid carrying a promoterless green fluorescent protein (GFP) gene, and the plasmids were transformed into the L . monocytogenes actA mutant DP-L1942 . Fluorescence-activated cell sorting (FACS) was used to isolate L . monocytogenes clones that exhibited increased GFP expression within macrophage-like J774 cells but had relatively low levels of GFP expression when the bacteria were extracellular . Using this strategy, several genes were identified, including actA, that exhibited such an expression profile . In-frame deletions of two of these genes, one encoding the putative L . monocytogenes uracil DNA glycosylase (ung) and one encoding a protein with homology to the Bacillus subtilis YhdP hemolysin-like protein, were constructed and introduced into the chromosome of wild-type L . monocytogenes 10403s . The L . monocytogenes 10403s ung deletion mutant was not attenuated for virulence in mice, while the yhdP mutant exhibited a three- to sevenfold reduction in virulence.

EMBO J, 2001 Jul 16, 20(14), 3789 - 99
Crystal structure of an activated form of the PTS regulation domain from the LicT transcriptional antiterminator; van Tilbeurgh H et al.; The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism . It belongs to a newly characterized family of bacterial regulators whose activity is controlled by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . LicT contains an N-terminal RNA-binding domain (56 residues), and a PTS regulation domain (PRD, 221 residues) that is phosphorylated on conserved histidines in response to substrate availability . Replacement of both His207 and His269 with a negatively charged residue (aspartic acid) led to a highly active LicT variant that no longer responds to either induction or catabolite repression signals from the PTS . In contrast to wild type, the activated mutant form of the LicT regulatory domain crystallized easily and provided the first structure of a PRD, determined at 1.55 A resolution . The structure is a homodimer, each monomer containing two analogous alpha-helical domains . The phosphorylation sites are totally buried at the dimer interface and hence inaccessible to phosphorylating partners . The structure suggests important tertiary and quaternary rearrangements upon LicT activation, which could be communicated from the protein C-terminal end up to the RNA-binding domain.

Genes Dev, 2001 Jul 1, 15(13), 1662 - 73
Division site selection protein DivIVA of Bacillus subtilis has a second distinct function in chromosome segregation during sporulation; Thomaides HB et al.; DivIVA is a coiled-coil, tropomyosin-like protein of Gram-positive bacteria . Previous work showed that this protein is targeted to division sites and retained at the cell poles after division . In vegetative cells, DivIVA sequesters the MinCD division inhibitor to the cell poles, thereby helping to direct cell division to the correct midcell site . We now show that DivIVA has a second, quite separate role in sporulating cells of Bacillus subtilis . It again acts at the cell pole but in this case interacts with the chromosome segregation machinery to help position the oriC region of the chromosome at the cell pole, in preparation for polar division . We isolated mutations in divIVA that separate the protein's role in sporulation from its vegetative function in cell division . DivIVA therefore appears to be a bifunctional protein with distinct roles in division-site selection and chromosome segregation.

FEMS Microbiol Lett, 2001 Jul 10, 201(1), 29 - 35
Functional analysis of the Bacillus subtilis cysK and cysJI genes; van der Ploeg JR et al.; The function of the Bacillus subtilis cysK and cysJI (previously designated yvgQR) genes, expected to be involved in the assimilatory sulfate reduction pathway, was investigated . A B . subtilis mutant with a deletion in the cysJI genes was unable to use sulfate or sulfite as sulfur source, which confirmed that these genes encode sulfite reductase . A mutant with a transposon insertion in the cysK gene, whose deduced protein sequence showed similarity to cysteine synthases, grew poorly on sulfate and butanesulfonate . A strain in which cysK and yrhA, a cysK paralog, were inactivated was unable to grow with sulfate . Whereas expression of the cysJI genes was induced by sulfate, expression of cysK was repressed both by sulfate and by cysteine.

FEBS Lett, 2001 Jul 6, 500(3), 129 - 31
A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans; Salazar JC et al.; The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli . As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A . ferrooxidans . The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn) . Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A . ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism . These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A . ferrooxidans.

J Bacteriol, 2001 Aug, 183(15), 4648 - 51
Involvement of stringent factor RelA in expression of the alkaline protease gene aprE in Bacillus subtilis; Hata M et al.; Expression of Bacillus subtilis aprE, encoding an extracellular alkaline protease, is positively regulated by phosphorylated DegU, the regulator of a two-component regulatory system, DegS-DegU . We found that the expression of an aprE'-'lacZ fusion was greatly reduced in a disruption mutant with a mutation of relA, which encodes the stringent factor RelA . The level of DegU in the relA mutant was similar to that in the wild-type cell . A relA degU double mutation did not result in a further decrease of the aprE'-'lacZ level found in a degU single mutant . The expression of the aprE'-'lacZ fusion in the relA mutant was stimulated by multicopy degR or the degU32(Hy) and degS200(Hy) mutations that cause the stabilization of phosphorylated DegU . Furthermore, the expression of sacB'-'lacZ, which is also dependent on phosphorylated DegU, was stimulated by the relA mutation, and this stimulation was not seen in the relA degU double mutant . These results show that RelA (or its product guanosine-3',5'-bisdiphosphate {pp Gpp}) does not affect the phosphorylation of DegU and suggest that it participates in the expression of aprE and sacB through the regulation of DegU-dependent transcription.

Biosci Biotechnol Biochem, 2001 May, 65(5), 1218 - 22
Flagellin as a biomarker for Bacillus subtilis strains; application to the DB9011 strain and the study of interspecific diversity in amino-acid sequences; Asano Y et al.; Polymerase chain reaction (PCR) for the flagellin central domain coding region (FCD-PCR) was applied to the detection and discrimination of Bacillus subtilis DB9011, a strain with useful functions in agriculture . Cross-reactions were observed in 4 B . subtilis strains with similar flagellin genes (hag) . Alignment of partial amino-acid sequences of flagellin and the results of PCR for the 16S/23S rRNA spacer in 11 B . subtilis strains suggested the presence of a group including strains with antifungal activity (DB9011 and others).

Biosci Biotechnol Biochem, 2001 May, 65(5), 1112 - 8
Adenine deaminase activity of the yicP gene product of Escherichia coli; Matsui H et al.; During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine . This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E . coli chromosome, suggesting that, like Bacillus subtilis, E . coli has an adenine deaminase . As the yicP gene of E . coli shares about 35% identity with the B . subtilis adenine deaminase gene (ade), we cloned yicP from the E . coli genome and developed a strain that overexpressed its product . The enzyme was purified from a cell extract of E . coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase {EC 3.5.4.2} activity . It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa . The enzyme required manganese ions as a cofactor, and adenine was its only substrate . Its optimum pH was 6.5-7.0 and its optimum temperature was 60 degrees C . The apparent Km for adenine was 0.8 mM.

Insect Biochem Mol Biol, 2001 Jul 26, 31(9), 857 - 65
Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis; Johns R et al.; Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B . burgdorferi . A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge . Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma . The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry . N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K . A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion . This is the first report of a defensin from a tick . The peptide was stable at least up to 70 degrees C . Although the tick defensin alone was not immediately effective against B . burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B . burgdorferi within 1h.

Orig Life Evol Biosph, 2001 Jun, 31(3), 287 - 303
Survival of microorganisms under the extreme conditions of the Atacama Desert; Dose K et al.; Spores of Bacillus subtilis, conidia of Aspergillus niger, versicolor and ochraceus and cells of Deinococcus radiodurans have been exposed in the dark at two locations (at about 23 degrees S and 24 degrees S) in the Atacama Desert for up to 15 months . B . subtilis spores (survival approximately 15%) and A . niger conidia (survival approximately 30%) outlived the other species . The survival of the conidia and spores species was only slightly poorer than that of the corresponding laboratory controls . However, the Deinococcus radiodurans cells did not survive the desert exposure, because they are readily inactivated at relative humidities between 40 and 80% which typically occur during desert nights . Cellular monolayers of the dry spores and conidia have in addition been exposed to the full sun light for up to several hours . The solar fluences causing 63% loss in viability (F37-values) have been determined . These F37-values are compared with those determined at other global locations such as Punta Arenas (53 degrees S), Key Largo (25 degrees N) or Mainz (50 degrees N) during the same season . The solar UVB radiation kills even the most resistant microorganisms within a few hours due to DNA damages . The data are also discussed with respect to possible similarities between the climatic conditions of the recent Atacama Desert and the deserts of early Mars.

J Biochem (Tokyo), 2001 Jul, 130(1), 99 - 106
Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region; Takahashi M et al.; Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation . Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity . A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate . To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' {Wells et al . (1987) Proc . Natl . Acad . Sci . USA 84, 1219-1223} . The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency . A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu . Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant . These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.

Microbiology, 2001 Jul, 147(Pt 7), 1805 - 13
Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation; Grandvalet C et al.; The immune inhibitor A (InhA) metalloprotease from Bacillus thuringiensis specifically cleaves antibacterial proteins produced by the insect host, suggesting that it may contribute to the overall virulence of B . thuringiensis . The transcriptional regulation of the inhA gene in both B . thuringiensis and Bacillus subtilis was investigated . Using a transcriptional inhA'-lacZ fusion, it was shown that inhA expression is activated at the onset of sporulation . However, the transcriptional start site of inhA is similar to sigma(A)-dependent promoters, and deletion of the sporulation-specific sigma factors sigma(F) or sigma(E) had no effect on inhA expression in B . subtilis . The DNA region upstream from inhA contains two genes encoding polypeptides similar to the SinI and SinR regulators of B . subtilis . SinR is a DNA-binding protein regulating gene expression and SinI inhibits SinR activity . Overexpression of the sin genes affects the expression of the inhA'-lacZ transcriptional fusion in B . thuringiensis: early induction of inhA expression was observed when sinI was overexpressed, whereas inhA expression was reduced in a strain overexpressing sinR, suggesting that inhA transcription is repressed, directly or indirectly, by SinR . inhA transcription was greatly reduced in B . thuringiensis and B . subtilis spo0A mutants . Analysis of the inhA'-lacZ expression in abrB and abrB-spo0A mutants of B . subtilis indicates that the Spo0A-dependent regulation of inhA expression depends on AbrB, which is known to regulate expression of transition state and sporulation genes in B . subtilis.

Genome Biol . 2001;2(6):RESEARCH0019 . Epub 2001 Jun 01.
Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis; Sekowska A et al.; BACKGROUND: In global gene expression profiling experiments, variation in the expression of genes of interest can often be hidden by general noise . To determine how biologically significant variation can be distinguished under such conditions we have analyzed the differences in gene expression when Bacillus subtilis is grown either on methionine or on methylthioribose as sulfur source . RESULTS: An unexpected link between arginine metabolism and sulfur metabolism was discovered, enabling us to identify a high-affinity arginine transport system encoded by the yqiXYZ genes . In addition, we tentatively identified a methionine/methionine sulfoxide transport system which is encoded by the operon ytmIJKLMhisP and is presumably used in the degradation of methionine sulfoxide to methane sulfonate for sulfur recycling . Experimental parameters resulting in systematic biases in gene expression were also uncovered . In particular, we found that the late competence operons comE, comF and comG were associated with subtle variations in growth conditions . CONCLUSIONS: Using variance analysis it is possible to distinguish between systematic biases and relevant gene-expression variation in transcriptome experiments . Co-variation of metabolic gene expression pathways was thus uncovered linking nitrogen and sulfur metabolism in B . subtilis.

J Ind Microbiol Biotechnol, 2001 Mar, 26(3), 115 - 20
Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis; Sato T et al.; Efficient production of menaquinone (MK) by Bacillus subtilis was achieved . An edible strain of B . subtilis, isolated from the traditional Japanese food natto, was mutated to improve MK productivity . A menadione-resistant mutant producing 30% more MK than its parent strain was obtained . Soybean extract and glycerol were the best nitrogen and carbon sources, respectively, among the sources tested . Addition of yeast extract also increased MK productivity . The maximum concentration of MK reached about 35.0 mg/l after 4 days of culture in a jar fermenter . The pH of the medium decreased to 5.5 after the start of cultivation, then spontaneously increased to 7.7-8.0 . This pH change might be important in the production of MK because only small amounts of MK were obtained when pH was controlled at 5.7, 6.0, 7.0, 7.5 or 8.0.

J Bacteriol, 2001 Jul, 183(14), 4389 - 92
Multiple genes for the last step of proline biosynthesis in Bacillus subtilis; Belitsky BR et al.; The complete Bacillus subtilis genome contains four genes (proG, proH, proI, and comER) with the potential to encode Delta(1)-pyrroline-5-carboxylate reductase, a proline biosynthetic enzyme . Simultaneous defects in three of these genes (proG, proH, and proI) were required to confer proline auxotrophy, indicating that the products of these genes are mostly interchangeable with respect to the last step in proline biosynthesis.

J Bacteriol, 2001 Jul, 183(14), 4364 - 73
The PDZ domain of the SpoIVB serine peptidase facilitates multiple functions; Hoa NT et al.; During spore formation in Bacillus subtilis, the SpoIVB protein is a critical component of the sigma(K) regulatory checkpoint . SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms . These forms can signal proteolytic processing of the transcription factor sigma(K) in the outer mother cell chamber of the sporulating cell . This forms the basis of the sigma(K) checkpoint and ensures accurate sigma(K)-controlled gene expression . SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores . In addition to the serine peptidase domain, SpoIVB contains a PDZ domain . We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins . Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for trans proteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-sigma(K) processing, and (iv) signaling of the second function of SpoIVB.

J Bacteriol, 2001 Jul, 183(14), 4317 - 22
Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore; Hudson KD et al.; The GerAA, -AB, and -AC proteins of the Bacillus subtilis spore are required for the germination response to L-alanine as the sole germinant . They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spore's response; multiple homologues of the gerA genes are found in every spore former so far examined . The gerA operon is expressed in the forespore, and the level of expression of the operon appears to be low . The GerA proteins are predicted to be membrane associated . In an attempt to localize GerA proteins, spores of B . subtilis were broken and fractionated to give integument, membrane, and soluble fractions . Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores . The spore-specific penicillin-binding protein PBP5*, a marker for the outer forespore membrane, was absent from this fraction . Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores . Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore . The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.

J Bacteriol, 2001 Jul, 183(14), 4190 - 201
Control of the arabinose regulon in Bacillus subtilis by AraR in vivo: crucial roles of operators, cooperativity, and DNA looping; Mota LJ et al.; The proteins involved in the utilization of L-arabinose by Bacillus subtilis are encoded by the araABDLMNPQ-abfA metabolic operon and by the araE/araR divergent unit . Transcription from the ara operon, araE transport gene, and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR . The purified AraR protein binds cooperatively to two in-phase operators within the araABDLMNPQ-abfA (OR(A1) and OR(A2)) and araE (OR(E1) and OR(E2)) promoters and noncooperatively to a single operator in the araR (OR(R3)) promoter region . Here, we have investigated how AraR controls transcription from the ara regulon in vivo . A deletion analysis of the ara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes . Furthermore, OR(E1)-OR(E2) and OR(R3) are auxiliary operators for the autoregulation of araR and the repression of araE, respectively . Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators . This communication implicates the formation of a small loop by the intervening DNA . In an in vitro transcription system, AraR alone sufficed to abolish transcription from the araABDLMNPQ-abfA operon and araE promoters, strongly suggesting that it is the major protein involved in the repression mechanism of L-arabinose-inducible expression in vivo . The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.

J Bacteriol, 2001 Jul, 183(14), 4183 - 9
Identification of a DNA binding region in GerE from Bacillus subtilis; Crater DL et al.; Proteins that have a structure similar to those of LuxR and FixJ comprise a large subfamily of transcriptional activator proteins . Most members of the LuxR-FixJ family contain a similar amino-terminal receiver domain linked by a small region to a carboxy-terminal domain that contains an amino acid sequence similar to the helix-turn-helix (HTH) motif found in other DNA-binding proteins . GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family . Its 74-amino-acid sequence is similar over its entire length to the DNA binding region of this protein family, including the HTH motif . Therefore, GerE provides a simple model for studies of the role of this HTH domain in DNA binding . Toward this aim, we sought to identify the amino acids within this motif that are important for the specificity of binding to DNA . We examined the effects of single base pair substitutions in the high-affinity GerE binding site on the sigK promoter and found that nucleotides at positions +2, +3, and +4 relative to the transcription start site on the sigK promoter are important for a high-affinity interaction with GerE . We next examined the effects of single alanine substitutions at two positions in the HTH region of GerE on binding to wild-type or mutant target sites . We found that the substitution of an alanine for the threonine at position 42 of GerE produced a protein that binds with equal affinity to two sites that differ by 1 bp, whereas wild-type GerE binds with different affinities to these two sites . These results provide evidence that the amino acyl residues in or near the putative HTH region of GerE and potentially other members of the LuxR-FixJ family determine the specificity of DNA binding.

J Agric Food Chem, 2001 Jun, 49(6), 2982 - 6
m-Iodobenzoic acid complexes with selected metals: molecular structure and antimicrobial activity; Koczon P et al.; Complexes of lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, manganese, and zinc with m-iodobenzoic acid were studied . The FT-IR and FT-Raman spectra of the mentioned compounds in the solid state and water solutions were recorded and analyzed . Principal component analysis (PCA) was performed on the wavenumbers of selected bands (eight bands) occurring in the vibrational spectra . The numbers obtained as a result of this procedure characterize the electronic properties of the molecule of each complex . The antimicrobial activity of the studied compounds against selected bacteria (Escherichia coli and Bacillus subtilis) and yeast (Saccharomyces cerevisiae and Hansenula anomala) was estimated . The relationship between the chemical properties (as characterized by PCA of the IR spectra) and antimicrobial properties of the compounds was examined, and a good correlation between the two factors was found.

EMBO J, 2001 Jun 15, 20(12), 3238 - 50
Bimodal activation of SMC ATPase by intra- and inter-molecular interactions; Hirano M et al.; Structural maintenance of chromosomes (SMC) proteins play fundamental roles in higher-order chromosome dynamics from bacteria to humans . It has been proposed that the Bacillus subtilis SMC (BsSMC) homodimer is composed of two anti-parallel coiled-coil arms, each having an ATP-binding domain at its distal end . It remains totally unknown, however, how the two-armed structure supports ATP-dependent actions of BsSMC . By constructing a number of mutant derivatives including 'single-armed' BsSMC, we show here that the central hinge domain provides a structural flexibility that allows opening and closing of the two arms . This unique structure brings about bimodal regulation of the SMC ATPase cycle . Closing the arm can trigger ATP hydrolysis by allowing an end-end interaction within a dimer (intramolecular mode) . When bound to DNA, ATP promotes a dimer-dimer interaction, which in turn activates their DNA-dependent ATPase activity (intermolecular mode) . Our results reveal a novel mechanism of ATPase regulation and provide mechanistic insights into how eukaryotic SMC protein complexes could mediate diverse chromosomal functions, such as chromosome condensation and sister chromatid cohesion.

Adv Microb Physiol, 2001, 44, 93 - 140
Microbial molecular chaperones; Lund PA; Protein folding in the cell, long thought to be a spontaneous process, in fact often requires the assistance of molecular chaperones . This is thought to be largely because of the danger of incorrect folding and aggregation of proteins, which is a particular problem in the crowded environment of the cell . Molecular chaperones are involved in numerous processes in bacterial cells, including assisting the folding of newly synthesized proteins, both during and after translation; assisting in protein secretion, preventing aggregation of proteins on heat shock, and repairing proteins that have been damaged or misfolded by stresses such as a heat shock . Within the cell, a balance has to be found between refolding of proteins and their proteolytic degradation, and molecular chaperones play a key role in this . In this review, the evidence for the existence and role of the major cytoplasmic molecular chaperones will be discussed, mainly from the physiological point of view but also in relationship to their known structure, function and mechanism of action . The two major chaperone systems in bacterial cells (as typified by Escherichia coli) are the GroE and DnaK chaperones, and the contrasting roles and mechanisms of these chaperones will be presented . The GroE chaperone machine acts by providing a protected environment in which protein folding of individual protein molecules can proceed, whereas the DnaK chaperones act by binding and protecting exposed regions on unfolded or partially folded protein chains . DnaK chaperones interact with trigger factor in protein translation and with ClpB in reactivating proteins which have become aggregated after heat shock . The nature of the other cytoplasmic chaperones in the cell will also be reviewed, including those for which a clear function has not yet been determined, and those where an in vivo chaperone function has still to be proven, such as the small heat shock proteins IbpA and IbpB . The regulation of expression of the genes of the heat shock response will also be discussed, particularly in the light of the signals that are needed to induce the response . The major signals for induction of the heat shock response are elevated temperature and the presence of unfolded protein within the cell, but these are sensed and transduced differently by different bacteria . The best characterized example is the sigma 32 subunit of RNA polymerase from E . coli, which is both more efficiently translated and also transiently stabilized following heat shock . The DnaK chaperones modulate this effect . However, a more widely conserved system appears to be typified by the HrcA repressor in Bacillus subtilis, the activity of which is modulated by the GroE chaperone machine . Other examples of regulation of molecular chaperones will also be discussed . Finally, the likely future research directions for molecular chaperone biology in the post-genomic era will be briefly evaluated.

Adv Microb Physiol, 2001, 44, 35 - 91
General stress response of Bacillus subtilis and other bacteria; Hecker M et al.; One of the strongest and most noticeable responses of a Bacillus subtilis cell to a range of stress and starvation conditions is the dramatic induction of a large number of general stress proteins . The alternative sigma factor sigma B is responsible for the induction of the genes encoding these general stress proteins that occurs following heat, ethanol, salt or acid stress, or during energy depletion . sigma B was detected more than 20 years ago by Richard Losick and William Haldenwang as the first alternative sigma factor of bacteria, but interest in sigma B declined after it was realized that sigma B is not involved in sporulation . It later turned out that sigma B, whose activity itself is tightly controlled, is absolutely required for the induction of this regulon, not only in B . subtilis, but also in other Gram-positive bacteria . These findings may have been responsible for the recent revival of interest in sigma B . This chapter summarizes the current information on this sigma B response including the latest results on the signal transduction pathways, the structure of the regulon and its physiological role . More than 150 general stress proteins/genes belong to this sigma B regulon, which is believed to provide the non-growing cell with a non-specific, multiple and preventive stress resistance . sigma B-dependent stress proteins are involved in non-specific protection against oxidative stress and also protect cells against heat, acid, alkaline or osmotic stress . A cell in the transition from a growing to a non-growing state induced by energy depletion will be equipped with a comprehensive stress resistance machine to protect it against future stress . The protection against oxidative stress may be an essential part of this response . In addition, preloading of cells with sigma B-dependent stress proteins, induced by mild heat or salt stress, will protect cells against a severe, potentially lethal, future stress . Both the specific protection against an acute emerging stress, as well as the non-specific, prospective protection against future stress, are adaptive functions crucial for surviving stress and starvation in nature . We suggest that the sigma B response is one essential component of a survival strategy that ensures survival in a quiescent, vegetative state as an alternative to sporulation . The role of sigma B in related Gram-positive bacteria (including cyanobacteria) with special emphasis on pathogenic bacteria is discussed.

Chemotherapy, 2001 Jul-Aug, 47(4), 233 - 8
Investigation of oxacillin-hydrolyzing beta-lactamase in borderline methicillin-resistant clinical isolates of Staphylococcus aureus; Gal Z et al.; BACKGROUND: Mechanisms of borderline resistance of Staphylococcus aureus to penicillinase-resistant penicillins (PRPs) may include hyperproduction of classical penicillinase and/or production of beta-lactamase hydrolyzing also PRPs . METHODS: beta-Lactamase activity of whole cells and purified enzymes was estimated spectrophotometrically and in isolated cytoplasmic membranes by bioassay with Bacillus subtilis as test strain . RESULTS: Out of 53 clinical isolates of S . aureus, 18 showed oxacillin MIC values from 0.5 to 2 microg/ml, which were reduced by sulbactam and/or clavulanic acid in the case of four isolates producing large quantities of inducible, type A beta-lactamase . Cytoplasmic membranes isolated from these strains showed oxacillin-hydrolyzing activity . One of these strains was grown also in the presence of globomycin, an antibiotic known to interfere with the anchorage of membrane lipoproteins; this treatment eliminated the oxacillin-hydrolyzing activity . CONCLUSIONS: The resistance in these strains was due to a membrane-bound lipoprotein with oxacillin-hydrolyzing activity.

Mol Microbiol, 2001 May, 40(4), 963 - 75
Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence; Gominet M et al.; PlcR is a pleiotropic regulator of virulence factors in the insect pathogen Bacillus thuringiensis and in the opportunistic human pathogen Bacillus cereus . It activates the transcription of at least 15 genes encoding extracellular proteins, including phospholipases C, proteases and enterotoxins . Expression of the plcR gene is autoregulated and activated at the onset of stationary phase . Here, we used mini-Tn10 transposition to generate a library of B . thuringiensis mutants, with the goal of characterizing genes involved in the expression of the plcR gene . Three mutant strains were identified carrying distinct mini-Tn10 insertions . The mutations impaired plcR expression and caused a deficient haemolytic phenotype, similar to the phenotype of a B . thuringiensis strain in which the plcR gene had been disrupted . The insertion sites of the three mini-Tn10 transposons mapped in a five-gene operon encoding polypeptides homologous to the components of the oligopeptide permease (Opp) system of Bacillus subtilis, and with a similar structural organization . By analogy, the five B . thuringiensis genes were designated oppA, B, C, D and F . In vitro disruption of the B . thuringiensis oppB gene reproduced the effect of the mini-Tn10 insertions (i.e . the loss of haemolytic activity) and reduced the virulence of the strain against insects . These phenotypes are similar to those of a DeltaplcR mutant . Opp is required for the import of small peptides into the cell . Therefore, plcR expression might be activated at the onset of stationary phase by the uptake of a signalling peptide acting as a quorum-sensing effector . The opp mutations impaired the sporulation efficiency of B . thuringiensis when the cells were cultured in LB medium . Thus, Opp is on the pathway that ultimately regulates Spo0A phosphorylation, as is the case in B . subtilis . However, analysis of plcR expression in DeltaoppB, Deltaspo0A and DeltaoppB Deltaspo0A mutants indicates that Opp is required for plcR expression via a Spo0A-independent mechanism.

Mol Microbiol, 2001 May, 40(4), 795 - 803
Bacterial cell division: regulating Z-ring formation; Harry EJ; The earliest stage of cell division in bacteria is the formation of a Z ring, composed of a polymer of the FtsZ protein, at the division site . Z rings appear to be synthesized in a bi-directional manner from a nucleation site (NS) located on the inside of the cytoplasmic membrane . It is the utilization of a NS specifically at the site of septum formation that determines where and when division will occur . However, a Z ring can be made to form at positions other than at the division site . How does a cell regulate utilization of a NS at the correct location and at the right time? In rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, two factors involved in this regulation are the Min system and nucleoid occlusion . It is suggested that in B . subtilis, the main role of the Min proteins is to inhibit division at the nucleoid-free cell poles . In E . coli it is currently not clear whether the Min system can direct a Z ring to the division site at mid-cell or whether its main role is to ensure that division inhibition occurs away from mid-cell, a role analogous to that in B . subtilis . While the nucleoid negatively influences Z-ring formation in its vicinity in these rod-shaped organisms, the exact relationship between nucleoid occlusion and the ability to form a mid-cell Z ring is unresolved . Recent evidence suggests that in B . subtilis and Caulobacter crescentus, utilization of the NS at the division site is intimately linked to the progress of a round of chromosome replication and this may form the basis of achieving co-ordination between chromosome replication and cell division.

J Bacteriol, 2001 Jul, 183(13), 4094 - 8
DNA-binding activity of amino-terminal domains of the Bacillus subtilis AbrB protein; Xu K et al.; Two truncated variants of AbrB, comprising either its first 53 (AbrBN53) or first 55 (AbrBN55) amino acid residues, were constructed and purified . Noncovalently linked homodimers of the truncated variants exhibited very weak DNA-binding activity . Cross-linking AbrBN55 dimers into tetramers and higher-order multimers (via disulfide bonding between penultimate cysteine residues) resulted in proteins having DNA-binding affinity comparable to and DNA-binding specificity identical to those of intact, wild-type AbrB . These results indicate that the DNA recognition and specificity determinants of AbrB binding lie solely within its N-terminal amino acid sequence.

J Bacteriol, 2001 Jul, 183(13), 4052 - 60
Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis; Graumann PL et al.; Entry into sporulation in Bacillus subtilis is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell . Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell . Replication origin regions were visualized by the use of a cassette of tandem copies of lacO that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI . The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented . On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore . Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (smc) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division . A small proportion ( approximately 1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region . Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ . A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.

J Bacteriol, 2001 Jul, 183(13), 3982 - 90
Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores; Paidhungat M et al.; Dormant Bacillus subtilis spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the gerA family of operons, of which the gerB operon is a member . The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane . In this study we have used Western blot analysis to determine the precise location of GerBA, a gerB-encoded receptor protein, in various spore fractions . GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles . GerBA was also solubilized from the P100 fraction with detergent but not with high salt . These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane . Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane . Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore . We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of gerB operon expression and with the putative receptor function of the proteins encoded by the gerB operon.

J Bacteriol, 2001 Jul, 183(13), 3885 - 9
SsrA-mediated tagging in Bacillus subtilis; Wiegert T et al.; A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the ssrA gene . This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis . To study this ssrA-mediated mechanism in Bacillus subtilis, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the bgaB reporter gene coding for a heat-stable beta-galactosidase fused to an HrcA-controlled promoter . After the predicted proteolysis tag was fused to HrcA, the reporter beta-galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA . Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable . Replacement of the hrcA stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused by trans translational addition of the proteolytic tag . Inactivating the B . subtilis ssrA or smpB (yvaI) gene prevented the trans translational tagging reaction . Various protease-deficient strains of B . subtilis were tested for proteolysis of tagged HrcA . HrcA remained stable only in clpX or clpP knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in B . subtilis.

J Bacteriol, 2001 Jul, 183(13), 3833 - 41
Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis; Ogura Y et al.; In Escherichia coli, the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation . To examine the effects on replication initiation in B . subtilis, we constructed a strain in which a copy of the dnaA gene was integrated at the purA locus on the chromosome under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter . However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response . Transcription of the native dnaA-dnaN operon was greatly reduced at high DnaA levels, but it was increased in a dnaA-null mutant, indicating autoregulation of the operon by DnaA . When a copy of the dnaN gene was added downstream of the additional dnaA gene at purA, the cells grew at high DnaA levels, suggesting that depletion of DnaN (beta subunit of DNA polymerase III) within the cell by repression of the native dnaA-dnaN operon at high DnaA levels was the cause of the SOS induction . Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type . Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level . These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B . subtilis.

Anal Chem, 2001 May 15, 73(10), 2331 - 7
Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization; Ullom JN et al.; We demonstrate that molecular ions with mass-to-charge ratios (m/z) ranging from a few hundred to 19 050 can be desorbed from whole bacterial spores using infrared laser desorption and no chemical matrix . We have measured the mass of these ions using time-of-flight mass spectrometry and we observe that different ions are desorbed from spores of Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Bacillus niger . Our results raise the possibility of identifying microorganisms using mass spectrometry without conventional sample preparation techniques such as the addition of a matrix . We have measured the dependence of the ion yield from B . subtilis on desorption wavelength over the range 3.05-3.8 microm, and we observe the best results at 3.05 microm . We have also generated mass spectra from whole spores using 337-nm ultraviolet laser desorption, and we find that these spectra are inferior to spectra generated with infrared desorption . Since aerosol analysis is a natural application for matrix-free desorption, we have measured mass spectra from materials such as ragweed pollen and road dust that are likely to form a background to microbial aerosols . We find that these materials are readily differentiated from bacterial spores.

Microbiology, 2001 Jun, 147(Pt 6), 1631 - 40
Sulfur-limitation-regulated proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study; Coppee JY et al.; Little is known about the genes and enzymes involved in sulfur assimilation in Bacillus subtilis, or about the regulation of their expression or activity . To identify genes regulated by sulfur limitation, the authors used two- dimensional (2D) gel electrophoresis to compare the proteome of a wild-type strain grown with either sulfate or glutathione as sole sulfur source . A total of 15 proteins whose synthesis is modified under these two conditions were identified by matrix-assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry . In the presence of sulfate, an increased amount of proteins involved in the metabolism of C(1) units (SerA, GlyA, FolD) and in the biosynthesis of purines (PurQ, Xpt) and pyrimidines (Upp, PyrAA, PyrF) was observed . In the presence of glutathione, the synthesis of two uptake systems (DppE, SsuA), an oxygenase (SsuD), cysteine synthase (CysK) and two proteins of unknown function (YtmI, YurL) was increased . The changes in expression of the corresponding genes, in the presence of sulfate and glutathione, were monitored using slot-blot analyses and lacZ fusions . The ytmI gene is part of a locus of 12 genes which are co-regulated in response to sulfur availability . This putative operon is activated by a LysR-like regulator, YTLI: This is the first regulator involved in the control of expression in response to sulfur availability to be identified in B . subtilis.

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