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