J Bacteriol, 2004 Feb, 186(4), 1129 - 35 Assembly of multiple CotC forms into the Bacillus subtilis spore coat; Isticato R et al.; We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms . Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs . The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation . None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant . This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface. J Bacteriol, 2004 Feb, 186(4), 1120 - 8 Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis; Susanna KA et al.; The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK . ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination . In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter . In this study, we investigated the mechanism of this transcription activation . The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter . This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding . Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown . In vitro transcription with B . subtilis RNA polymerase reconstituted with wild-type alpha-subunits and with C-terminal deletion mutants of the alpha-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK . This indicates that ComK is not a type I activator . We also show that ComK is not required for open complex formation . A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase. J Bacteriol, 2004 Feb, 186(4), 1110 - 9 Interactions among CotB, CotG, and CotH during assembly of the Bacillus subtilis spore coat; Zilhao R et al.; Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides . One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface . The cotB gene was initially identified by reverse genetics as encoding an abundant coat component . cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66) . Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46) . Expression of cotB in sporulating cells of B . subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66 . These results suggest that soon after synthesis, CotB undergoes a posttranslational modification . Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci . We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants . Therefore, both cotH and cotG are required for the efficient conversion of CotB-46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat . We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG . Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG . We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected . Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact . We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB. J Bacteriol, 2004 Feb, 186(4), 1097 - 105 Differential expression of two paralogous genes of Bacillus subtilis encoding single-stranded DNA binding protein; Lindner C et al.; The Bacillus subtilis genome comprises two paralogous single-stranded DNA binding protein (SSB) genes, ssb and ywpH, which show distinct expression patterns . The main ssb gene is strongly expressed during exponential growth and is coregulated with genes encoding the ribosomal proteins S6 and S18 . The gene organization rpsF-ssb-rpsR as observed in B . subtilis is found in many gram-positive as well as some gram-negative bacteria, but not in Escherichia coli . The ssb gene is essential for cell viability, and like other SSBs its expression is elevated during SOS response . In contrast, the paralogous ywpH gene is transcribed from its own promoter at the onset of stationary phase in minimal medium only . Its expression is ComK dependent and its gene product is required for optimal natural transformation. J Bacteriol, 2004 Feb, 186(4), 1084 - 96 Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42; Koumoutsi A et al.; The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere . We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid level to the corresponding genes of Bacillus subtilis 168 . Six large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied 7.5% of the whole genome . Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B . subtilis 168 . Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D . The fengycin (fen) and the surfactin (srf) operons were organized and located as in B . subtilis 168 . A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D . The bmy island was found inserted close to the fen operon . The responsibility of the bmy, fen, and srf gene clusters for the production of the corresponding secondary metabolites was demonstrated by cassette mutagenesis, which led to the loss of the ability to produce these peptides . Although these single mutants still largely retained their ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner. J Bacteriol, 2004 Feb, 186(4), 1078 - 83 Alpha-helix E of Spo0A is required for sigmaA- but not for sigmaH-dependent promoter activation in Bacillus subtilis; Kumar A et al.; At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters . The first type includes the spoIIG and spoIIE promoters, which are used by sigma(A)-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor sigma(H) . Previous genetic analyses have identified specific amino acids in alpha-helix E of Spo0A that are important for activation of Spo0A-dependent, sigma(A)-dependent promoters . However, these amino acids are not required for activation of the sigma(H)-dependent spoIIA promoter . We now report the effects of additional single-amino-acid substitutions and the effects of deletions in alpha-helix E . The effects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the sigma(A)-dependent promoters . Based on the effects of a deletion mutation, we suggest that alpha-helix E in Spo0A is not directly involved in interaction with sigma(H)-RNA polymerase. J Bacteriol, 2004 Feb, 186(4), 1050 - 9 The ytkD (mutTA) gene of Bacillus subtilis encodes a functional antimutator 8-Oxo-(dGTP/GTP)ase and is under dual control of sigma A and sigma F RNA polymerases; Ramirez MI et al.; The regulation of expression of ytkD, a gene that encodes the first functional antimutator 8-oxo-dGTPase activity of B . subtilis, was studied here . A ytkD-lacZ fusion integrated into the ytkD locus of wild-type B . subtilis 168 revealed that this gene is expressed during both vegetative growth and early stages of sporulation . In agreement with this result, ytkD mRNAs were detected by both Northern blotting and reverse transcription-PCR during both developmental stages . These results suggested that ytkD is transcribed by the sequential action of RNA polymerases containing the sigma factors sigma(A) and sigma(F), respectively . In agreement with this suggestion, the spore-associated expression was almost completely abolished in a sigF genetic background but not in a B . subtilis strain lacking a functional sigG gene . Primer extension analysis mapped transcriptional start sites on mRNA samples isolated from vegetative and early sporulating cells of B . subtilis . Inspection of the sequences lying upstream of the transcription start sites revealed the existence of typical sigma(A)- and sigma(F)-type promoters . These results support the conclusion that ytkD expression is subjected to dual regulation and suggest that the antimutator activity of YtkD is required not only during vegetative growth but also during the early sporulation stages and/or germination of B . subtilis . While ytkD expression obeyed a dual pattern of temporal expression, specific stress induction of the transcription of this gene does not appear to occur, since neither oxidative damage (following either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment or sigma(B) general stress inducers (sodium chloride, ethanol, or heat) affected the levels of the gene product produced. J Bacteriol, 2004 Feb, 186(4), 989 - 1000 Novel roles of the master transcription factors Spo0A and sigmaB for survival and sporulation of Bacillus subtilis at low growth temperature; Mendez MB et al.; Spore development and stress resistance in Bacillus subtilis are governed by the master transcription factors Spo0A and sigma(B), respectively . Here we show that the coding genes for both regulatory proteins are dramatically induced, during logarithmic growth, after a temperature downshift from 37 to 20 degrees C . The loss of sigma(B) reduces the stationary-phase viability of cold-adapted cells 10- to 50-fold . Furthermore, we show that sigma(B) activity is required at a late stage of development for efficient sporulation at a low temperature . On the other hand, Spo0A loss dramatically reduces the stationary-phase viability of cold-adapted cells 10,000-fold . We show that the requirement of Spo0A for cellular survival during the cold is independent of the activity of the key transition state regulator AbrB and of the simple loss of sporulation ability . Furthermore, Spo0A, and not proficiency in sporulation, is required for the development of complete stress resistance of cold-adapted cells to heat shock (54 degrees C, 1 h), since a loss of Spo0A, but not a loss of the essential sporulation transcription factor sigma(F), reduced the cellular survival in response to heat by more than 1,000-fold . The overall results argue for new and important roles for Spo0A in the development of full stress resistance by nonsporulating cells and for sigma(B) in sporulation proficiency at a low temperature. FEMS Microbiol Lett, 2004 Jan 30, 230(2), 241 - 9 The yjoB gene of Bacillus subtilis encodes a protein that is a novel member of the AAA family; Kotschwar M et al.; The yjoB gene of Bacillus subtilis encodes a 48.8-kDa protein belonging to the AAA family . Members of this family contain a 200-250-amino acid residues AAA domain carrying a Walker A and B ATP-binding site assumed to be part of a molecular chaperone . The yjoB gene belongs to the sigmaW regulon, and members of this regulon have been reported to be transiently induced when cells enter the stationary growth phase . This assumption was confirmed here for yjoB by Western blot experiments and by analysis of a transcriptional fusion . Purified YjoB protein exhibited ATPase activity but was unable to prevent aggregation of denatured citrate synthase . An alignment of YjoB with a subgroup of AAA proteins present in Archaea suggests that YjoB might be involved in the modulation of the activity of one or more proteases. Biochim Biophys Acta, 2004 Jan 28, 1660(1-2), 16 - 23 Bacillus subtilis alpha-amylase: interactions of a partially folded conformer with small unilamellar vesicles; Colomer-Pallas A et al.; We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength . Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)) . This association prevented its subsequent folding . The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B . subtilis which displays a strong affinity for SUV (KA approximately 1.5x10(8) M(-1)) . Vesicles coated with PrsA lost their ability to bind the partially folded conformer . The approach in vitro, in which our aim was to mimic the last stage of alpha-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane. Biochemistry, 2004 Feb 10, 43(5), 1360 - 8 The side chain of aspartic acid 69 dictates the folding mechanism of Bacillus subtilis HPr; Schmittschmitt JP et al.; Many small, single-domain proteins show equilibrium and kinetic folding mechanisms that appear to be adequately described as two state . The two-state model makes several predictions that can be tested experimentally . First, the conformational stability determined at or extrapolated to a set of reference conditions should be independent of the measurement method (thermal or solvent denaturation or hydrogen exchange) . Second, model-independent measures of the cardinal thermodynamic parameters (T(m), DeltaH) as determined from direct calorimetric means should be identical to those determined from the two-state analysis of thermal unfolding data . Third, the ratio of the kinetic folding and unfolding rate constants should be equal to K(eq) determined from an equilibrium measurement under the same conditions . Here, we show that the wild-type HPr protein from Bacillus subtilis does not meet all of these criteria under our standard conditions . However, if we replace the side chain of Asp69, or add moderate concentrations of salt, we find excellent two-state behavior in both equilibrium and kinetic folding . Thus, for this protein and possibly others, very subtle changes in the primary structure or in the solution conditions can dramatically alter the relative stabilities of the native intermediate, and unfolded ensembles can cause an observable change in the nature of the folding mechanism. Food Addit Contam, 2004 Feb, 21(2), 145 - 53 Evaluation and establishing the performance of different screening tests for tetracycline residues in animal tissues; Okerman L et al.; Four methods intended for screening muscle tissue for residues belonging to the tetracycline group were compared using artificially contaminated as well as incurred samples . Two agar diffusion methods were studied: one with Bacillus subtilis as a test strain, the second with Bacillus cereus . Two variants of each method were compared: thin plates for analysis of intact or minced meat, and thick plates for analysis of meat fluid . The thin plate variants could not be evaluated with artificially contaminated samples because it was impossible to prepare homogeneously spiked, undiluted meat . The thick plates were suited for doxycycline and chlortetracycline, but they did not detect oxytetracycline or tetracycline in spiked meat fluid . The results of these tests done on incurred meat were very good for doxycycline and satisfying or just failing for oxytetracycline, while the best detection capability was obtained when intact frozen meat was examined on thin plates seeded with B . cereus . Two commercially available screening tests were also evaluated . The Premi(R) test, an inhibitor test with Bacillus stearothermophilus as a test strain and an indicator for growth, was not suited for detection of tetracyclines up to the maximum residue limit . Tetrasensor(R), a receptor test specific for tetracyclines, proved a quick and simple test able to detect meat samples artificially contaminated with tetracycline, oxytetracycline, doxycycline or chlortetracycline, as well as meat incurred with oxytetracycline or doxycycline. Bioinformatics, 2004 Mar 22, 20(5), 709 - 17 Epub 2004 Jan 29. Using functional and organizational information to improve genome-wide computational prediction of transcription units on pathway-genome databases; Romero PR et al.; MOTIVATION: The prediction of transcription units (TUs, which are similar to operons) is an important problem that has been tackled using many different approaches . The availability of complete microbial genomes has made genome-wide TU predictions possible . Pathway-genome databases (PGDBs) add metabolic and other organizational (i.e . protein complexes) information to the annotated genome, and are able to capture TU organization information . These characteristics of PGDBs make them a suitable framework for the development and implementation of TU predictors . RESULTS: We implemented a TU predictor that uses only intergenic distance and functional classification of genes to predict TU boundaries, and applied it to EcoCyc, our PGDB of Escherichia coli . To this original predictor, we added information on metabolic pathways, protein complexes and transporters, all readily available in EcoCyc, in order to generate an enhanced predictor . The enhanced predictor correctly predicted 80% of the known E.coli TUs (69% of the known operons), a moderate improvement over the original predictor's performance (75% of TUs and 65% of operons correctly predicted), demonstrating that the extra information available in the PGDB does indeed improve prediction performance . Performance of this E.coli-based predictor on a genome other than that of E.coli was tested on BsubCyc, our computationally generated PGDB for Bacillus subtilis, for which a set of 100 known operons is available . Prediction accuracy decreased substantially (46% of the known operons correctly predicted) . This was due in part to missing information in BsubCyc, which prevented full use of the predictor's features . The augmented predictor has been implemented as part of our Pathway Tools software suite, and can be used to populate a PGDB with predicted TUs . AVAILABILITY: The TU predictor is included in version 7.0 of the Pathway Tools software suite . Pathway Tools 7.0 is available free of charge to academic institutions and for a fee to commercial enterprises . It runs on Sun Solaris 8, Linux and Windows . TUs predicted on the Caulobacter crescentus and Mycobacterium tuberculosis (H37Rv) genomes are available in our CauloCyc and MtbrvCyc databases, available at the BioCyc web site . To obtain version 7.0 of Pathway Tools, follow the directions in our web site, http://biocyc.org/download.shtml. Farmaco, 2004 Jan, 59(1), 13 - 20 Biodegradation of DNA and nucleotides to nucleosides and free bases; Kruszewska H et al.; Thirty-two different microorganisms were examined in order to check their ability to degrade an exogenous DNA . Bacteria from species: Stenotrophomonas maltophilia, Brevundimonas diminuta, Bacillus subtilis, Mycobacterium butyricum and fungus Fusarium moniliforme were capable to degrade DNA to nucleic bases or their derivatives . Degradation of DNA by S . maltophilia resulted in formation of free bases, such as hypoxanthine, thymine, uracil and xanthine . The optimum concentration of DNA seemed to be 3 mg ml(-1) . The mode of degradation of DNA nucleotides depended on the type of nucleotide and its concentration, but nucleic bases or their derivatives were always formed at the end of the reaction process. J Org Chem, 2004 Feb 6, 69(3), 601 - 12 Design, synthesis, and evaluation of 9-D-ribitylamino-1,3,7,9-tetrahydro-2,6,8-purinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents as inhibitors of tiboflavin synthase and lumazine synthase; Cushman M et al.; Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport processes . To obtain structural probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a series of ribitylpurinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents were synthesized . Since the purinetrione ring system and the ribityl hydroxyl groups can be alkylated, the synthesis required the generation of these two moieties in protected form before the desired alkylation reaction could be carried out . These substances were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site . All of the compounds were found to be effective inhibitors of both Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase . Molecular modeling of the binding of 3-(1,3,7,9-tetrahydro-9-D-ribityl-2,6,8-trioxopurin-7-yl)propane 1-phosphate provided a structural explanation for how these compounds are able to effectively inhibit both enzymes . Interestingly, the enzyme kinetics of these new compounds in comparison with the parent purinetrione demonstrated unexpectedly that the phosphate and phosphonate substituents contributed negatively to the binding . A possible explanation for these effects on lumazine synthase would be that the inorganic phosphate in the assay buffer competes with the substituted purinetriones for binding to the enzyme . This would be consistent with the observed increase in K(m) of the 3,4-dihydroxybutanone-4-phosphate substrate from 5.2 microM in Tris buffer or from 6.7 microM in MOPS buffer to 50 microM in phosphate buffer when tested on Bacillus subtilis lumazine synthase . However, when tested in Tris buffer vs Mycobacterium tuberculosis lumazine synthase, three of the phosphate inhibitors displayed inhibition constants in the 4-5 nM range, indicating that they are much more potent than the parent purinetrione . Under these conditions, the phosphate moieties of the inhibitors do contribute positively to their binding . The alpha,alpha-difluorophosphonate analogue, which is expected to have enhanced metabolic stability relative to the phosphates, was also found to be an inhibitor of Mycobacterium tuberculosis lumazine synthase with a K(i) of 60 nM. J Biol Chem, 2004 May 21, 279(21), 21787 - 92 Epub 2004 Jan 27. Bacillus subtilis CheC and FliY are members of a novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction cascade; Szurmant H et al.; Rapid restoration of prestimulus levels of the chemotactic response regulator, CheY-P, is important for preparing bacteria and archaea to respond sensitively to new stimuli . In an extension of previous work (Szurmant, H., Bunn, M . W., Cannistraro, V . J., and Ordal, G . W . (2003) J . Biol . Chem . 278, 48611-48616), we describe a new family of CheY-P phosphatases, the CYX family, that is widespread among the bacteria and archaea . These proteins provide another pathway, in addition to the ones involving CheZ of the gamma- and beta-proteobacteria (e.g . Escherichia coli) or the alternative CheY that serves as a "phosphate sink" among the alpha-proteobacteria (e.g . Sinorhizobium meliloti), for dephosphorylating CheY-P . In particular, we identify CheC, known previously to be involved in adaptation to stimuli in Bacillus subtilis, as a CheY-P phosphatase . Using an in vitro assay used previously to demonstrate that the switch protein FliY is a CheY-P phosphatase, we have shown that increasing amounts of CheC accelerate the hydrolysis of CheY-P . In vivo, a double mutant lacking cheC and the region of fliY that encodes the CheY-P binding domain is almost completely smooth swimming, implying that these cells contain very high levels of CheY-P . CheC appears to be primarily involved in restoring normal CheY-P levels following the addition of attractant, whereas FliY seems to act on CheY-P constitutively . The activity of CheC is relatively low compared to that of FliY, but we have shown that the chemotaxis protein CheD enhances the activity of CheC 5-fold . We suggest a model for how FliY, CheC, and CheD work together to regulate CheY-P levels in the bacterium. Proteins, 2004 Feb 15, 54(3), 424 - 32 Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68; Pons T et al.; Multiple-sequence alignment of glycoside hydrolase (GH) families 32, 43, 62, and 68 revealed three conserved blocks, each containing an acidic residue at an equivalent position in all the enzymes . A detailed analysis of the site-directed mutations so far performed on invertases (GH32), arabinanases (GH43), and bacterial fructosyltransferases (GH68) indicated a direct implication of the conserved residues Asp/Glu (block I), Asp (block II), and Glu (block III) in substrate binding and hydrolysis . These residues are close in space in the 5-bladed beta-propeller fold determined for Cellvibrio japonicus alpha-L-arabinanase Arb43A {Nurizzo et al., Nat Struct Biol 2002;9:665-668} and Bacillus subtilis endo-1,5-alpha-L-arabinanase . A sequence-structure compatibility search using 3D-PSSM, mGenTHREADER, INBGU, and SAM-T02 programs predicted indistinctly the 5-bladed beta-propeller fold of Arb43A and the 6-bladed beta-propeller fold of sialidase/neuraminidase (GH33, GH34, and GH83) as the most reliable topologies for GH families 32, 62, and 68 . We conclude that the identified acidic residues are located at the active site of a beta-propeller architecture in GH32, GH43, GH62, and GH68, operating with a canonical reaction mechanism of either inversion (GH43 and likely GH62) or retention (GH32 and GH68) of the anomeric configuration . Also, we propose that the beta-propeller architecture accommodates distinct binding sites for the acceptor saccharide in glycosyl transfer reaction . Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 329 - 30 Epub 2004 Jan 23. Crystallization of YloQ, a GTPase of unknown function essential for Bacillus subtilis viability; Cladiere L et al.; YloQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome . A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism . Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 A spacing. Biosci Biotechnol Biochem, 2004 Jan, 68(1), 132 - 7 Bacillus subtilis ypgA gene is fni, a nonessential gene encoding type 2 isopentenyl diphosphate isomerase; Takagi M et al.; We previously identified the fni gene of Streptomyces sp . strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity . An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome . However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product . A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene . The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product . The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail . The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH . The enzyme also catalyzed the reverse reaction in the presence of both the cofactors . Disruption of the ypgA3 gene was not lethal to B . subtilis . These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase. J Biol Chem, 2004 Apr 9, 279(15), 14860 - 70 Epub 2004 Jan 26. A threshold mechanism governing activation of the developmental regulatory protein sigma F in Bacillus subtilis; Carniol K et al.; The RNA polymerase sigma factor sigma(F) is a developmental regulatory protein that is activated in a cell-specific manner following the formation of the polar septum during the process of spore formation in the bacterium Bacillus subtilis . Activation of sigma(F) depends on the membrane-bound phosphatase SpoIIE, which localizes to the septum, and on the formation of the polar septum itself . SpoIIE is responsible for dephosphorylating and thereby activating the phosphoprotein SpoIIAA, which, in turn, triggers the release of sigma(F) from the anti-sigma(F) factor SpoIIAB . Paradoxically, however, the presence of unphosphorylated SpoIIAA is insufficient to cause sigma(F) activation as SpoIIAA reaches substantial levels in mutants blocked in polar septation . We now describe mutants of SpoIIE, SpoIIAA, and SpoIIAB that break the dependence of sigma(F) activation on polar division . Analysis of these mutants indicates that unphosphorylated SpoIIAA must reach a threshold concentration in order to trigger the release of sigma(F) from SpoIIAB . Evidence is presented that this threshold is created by the action of SpoIIAB, which can form an alternative, long lived complex with SpoIIAA . We propose that formation of the SpoIIAA-SpoIIAB complex serves as a sink that traps SpoIIAA in an inactive state and that only when unphosphorylated SpoIIAA is in excess to the sink does activation of sigma(F) take place. Antimicrob Agents Chemother, 2004 Feb, 48(2), 575 - 88 Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces ambofaciens linear chromosome; Pang X et al.; Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats . Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters . Sequence analysis identified 26 open reading frames, including genes encoding the beta-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS . These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics . The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase . Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats) . Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis . Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional . Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes . The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway . High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants . Two additional genes encoding KS and CLF were present in the cluster . However, disruption of the second KS gene had no effect on either pigment or antibiotic production. Antimicrob Agents Chemother, 2004 Feb, 48(2), 484 - 90 The ybxI gene of Bacillus subtilis 168 encodes a class D beta-lactamase of low activity; Colombo ML et al.; The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues . The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D beta-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors . To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned the ybxI gene in the pET-26b expression vector . Transformation of Escherichia coli BL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein . The recombinant protein was purified to 95% homogeneity . YbxI interacts with several beta-lactam antibiotics and can hydrolyze some of them . YbxI is not inactivated by clavulanic acid . The YbxI function and its enzymatic activity in B . subtilis remain unknown . The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by beta-lactam antibiotics . YbxI does not hydrolyze some of the standard substrates of D-alanyl-D-alanine peptidases, the targets of penicillin . YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a beta-lactamase. J Biotechnol, 2004 Feb 19, 108(1), 41 - 9 Substrate specificity of native and mutated cytochrome P450 (CYP102A3) from Bacillus subtilis; Lentz O et al.; Within the Bacillus subtilis genome sequencing project, two monooxygenases (CYP102A2 and CYP102A3) were discovered which revealed a similarity of 76% to the well-known cytochrome P450 BM-3 (CYP102A1) of Bacillus megaterium . All enzymes are natural fusion proteins consisting of a heme domain and a reductase domain . We here report the cloning, expression and characterization of B . subtilis enzyme CYP102A3 . The substrate specificity of this enzyme is similar to that of B . megaterium CYP102A1, which hydroxylates medium-chain fatty acids in subterminal positions . A double mutant was prepared that hydroxylates a number of other substrates, which do not bear any resemblance to the natural substrate of this enzyme family. FEBS Lett, 2004 Jan 16, 557(1-3), 133 - 5 In vivo formation of glutamyl-tRNA(Gln) in Escherichia coli by heterologous glutamyl-tRNA synthetases; Nunez H et al.; Two types of glutamyl-tRNA synthetase exist: the discriminating enzyme (D-GluRS) forms only Glu-tRNA(Glu), while the non-discriminating one (ND-GluRS) also synthesizes Glu-tRNA(Gln), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli) . Testing the capacity to complement a thermosensitive E . coli gltX mutant and to suppress an E . coli trpA49 missense mutant we examined the properties of heterologous gltX genes . We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu-tRNA(Glu), A . ferrooxidans and Helicobacter pylori GluRS2 form Glu-tRNA(Gln) in E . coli in vivo. Bioorg Med Chem Lett, 2004 Feb 9, 14(3), 757 - 60 Synthesis and antibiotic activity of the tricyclic furo{3,2-c} isochromen-2-trione unit of the pyranonaphthoquinones; Bianchi DA et al.; The elaboration and biological activity of 15, containing the proposed pharmacophore for the antibiotic activity of the pyranonaphthoquinones, are reported . The synthetic strategy involved acid-catalyzed lactonization of mandelate 17 for isochroman ring formation, in combination with a Wittig-oxa-Michael functionalization of isochroman-3-ol derivative 20, a lactonization involving configurational inversion of a benzylic alcohol and a final AgO oxidation . Compound 15 showed activity against Staphylococcus aureus and Bacillus subtilis with MIC of 64 and 32 microg/mL, respectively. Chem Commun (Camb), 2004 Jan 7, (1), 86 - 7 Epub 2003 Nov 17. Carbon-carbon bond cleavage by cytochrome p450(BioI)(CYP107H1); Cryle MJ et al.; Cytochrome p450(BioI)(CYP107H1) is believed to supply pimelic acid equivalents for biotin biosynthesis in Bacillus subtilis: we report here that the mechanistic pathway adopted by this multifunctional p450 for the in-chain cleavage of fatty acids is via consecutive formation of alcohol and threo-diol intermediates, with the likely absolute configuration of the intermediates also reported. Analyst, 2003 Dec, 128(12), 1462 - 6 Epub 2003 Oct 30. Determination of DNA by Rayleigh light scattering enhancement of molecular "light switches"; Chen F et al.; Base on the enhancement of Rayleigh light scattering signals of molecular "light switches" by DNA under acidic condition, a sensitive and convenient method for DNA determination was proposed . The experiments indicated that, under optimum conditions, good linear relationships were obtained between the Rayleigh light scattering intensity and the concentration of nucleic acids . The detect limits of calf thymus DNA (ctDNA) were 13.0 ng ml(-1), 4.2 ng ml(-1), 51.5 ng ml(-1) and 3.0 ng ml(-1) with four "light switches", respectively . Plasmid DNA extracted from Bacillus subtilis were determined by the proposed method with satisfactory results, and the recovery rates of calf thymus DNA were in the range of 94.6-110.7%. FEMS Microbiol Lett, 2004 Jan 15, 230(1), 41 - 6 Genetic evidence for the temperature-sensing ability of the membrane domain of the Bacillus subtilis histidine kinase DesK; Hunger K et al.; A decrease in environmental temperature leads to the synthesis of Delta5-unsaturated fatty acids in Bacillus subtilis by the fatty acid desaturase Des . Des is regulated by the two-component system DesKR . To understand the mechanism of cold signal perception and transduction by the membrane domain and the cytosolic domain of DesK, we expressed the cytosolic domain of DesK in trans under the control of a xylose-inducible promoter without the membrane domain . We performed growth experiments and a Northern blot analysis . Our results show that the kinase function of the cytosolic domain of DesK is temperature-independent, leading to a constitutive expression of the des gene . These findings support the conclusion that the membrane domain of DesK is the temperature-sensing element of the two-component system. Mol Microbiol, 2004 Feb, 51(3), 827 - 35 Initiation of intracellular offspring in Epulopiscium; Angert ER et al.; Epulopiscium spp . are the largest heterotrophic bacteria yet described . A distinguishing feature of the Epulopiscium group is their viviparous production of multiple, internal offspring as a means of cellular reproduction . Based on their phylogenetic position, among low G + C Gram-positive endospore-forming bacteria, and the remarkable morphological similarity between developing endospores and Epulopiscium offspring, we hypothesized that intracellular offspring production in Epulopiscium evolved from endospore formation . These observations also raise the possibility that a cell with the capacity to form multiple intracellular offspring was the ancestor of all contemporary endospore-forming bacteria . In an effort to characterize mechanisms common to both processes, we describe the earliest stages of offspring formation in Epulopiscium . First, in anticipation of polar division, some of the mother cell DNA coalesces at the cell poles . FtsZ then localizes in a bipolar pattern and the cell divides . A portion of the pole-associated DNA is trapped within the small cells formed by division at both poles . As development progresses, more pole-associated DNA is apparently packaged into the offspring primordia . These results illustrate three mechanisms, the reorganization of cellular DNA, asymmetric division and DNA packaging, that are common to both endospore formation in Bacillus subtilis and the production of active, intracellular offspring in Epulopiscium . Unlike most endospore formers, Epulopiscium partitions only a small proportion of mother cell DNA into the developing offspring. Mol Microbiol, 2004 Feb, 51(3), 749 - 64 Several distinct localization patterns for penicillin-binding proteins in Bacillus subtilis; Scheffers DJ et al.; Bacterial cell shape is determined by a rigid external cell wall . In most non-coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl . To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin-binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis . The localization of these fusions was studied in a wild-type background as well as in strains deficient in FtsZ, MreB or Mbl . PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum . All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum . This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall . The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth . Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP-PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl. Mol Microbiol, 2004 Feb, 51(3), 721 - 8 Receptor conformational changes enhance methylesterase activity during chemotaxis by Bacillus subtilis; Bunn MW et al.; Addition and removal of the attractant asparagine causes methanol formation as a consequence of methylation and demethylation of conserved glutamate residues in the Bacillus subtilis chemotaxis receptor McpB C-terminal domain . We found that methanol was released on both addition and removal of asparagine even when the response regulator domain of CheB was removed (to produce CheB(141-357)) . Thus, in undergoing the transition from unbound receptor to ligand-bound adapted receptor, the receptor must pass through a state of heightened susceptibility to demethylation by CheB that is independent of phosphorylation . The same result occurred when the aspartate phosphorylation site of CheB, Asp54, had been mutated to an asparagine residue, provided the enzyme was sufficiently induced . However, no methanol release was observed for an active site point mutant, cheB(S173C), in response to addition or removal of asparagine even when induced . Finally, methanol release was observed only for attractant addition in a mutant background lacking the coupling proteins, CheW and CheV, provided CheB(141-357) was present . Thus, on attractant addition, methanol must arise from a transient conformation of the receptor C-terminal domain that is an intrinsic property of the receptor; on attractant removal, however, methanol must arise from a different transient conformation, one dependent on the presence of coupling proteins. Biochemistry, 2004 Jan 27, 43(3), 748 - 58 Reactions of 4-oxalocrotonate tautomerase and YwhB with 3-halopropiolates: analysis and implications; Wang SC et al.; 4-Oxalocrotonate tautomerase (4-OT) and YwhB, a 4-OT homologue found in Bacillus subtilis, exhibit a low level hydratase activity that converts trans-3-haloacrylates to acetaldehyde, presumably through a malonate semialdehyde intermediate . The mechanism for the initial transformation of the 3-haloacrylate to malonate semialdehyde involves Pro-1 as well as an arginine, two residues that play critical roles in the 4-OT-catalyzed isomerization reaction and the YwhB-catalyzed tautomerization reaction . These residues are also critical for the trans-3-chloroacrylic acid dehalogenase (CaaD)-catalyzed conversion of trans-3-haloacrylates to malonate semialdehyde . Recently, 3-bromo- and 3-chloropropiolate, the acetylene analogues of 3-haloacrylates, were characterized as potent irreversible inhibitors of CaaD due to the covalent modification of the catalytic proline . In view of these observations, an investigation of the behavior of 4-OT and YwhB with the 3-halopropiolates was undertaken . The results show that these compounds are potent irreversible inhibitors of 4-OT and YwhB with Pro-1 being the sole site of covalent modification by 3-bromopropiolate . The inactivation process could involve the enzyme-catalyzed addition of water to the 3-halopropiolate yielding an acyl halide, which would inactivate the enzyme or be initiated by the nucleophilic attack of Pro-1 at the C-3 position of the 3-halopropiolate in a Michael type reaction . The presence of the halogen along with Arg-11 could facilitate both reactions with the latter causing the polarization of the alpha,beta-unsaturated acids . The 3-halopropiolates are the first identified inhibitors of YwhB and confirm the importance of Pro-1 in its mechanism . In addition, the results set the stage for the use of these compounds as mechanistic probes of the primary as well as low level activities of 4-OT and YwhB. Electrophoresis, 2004 Jan, 25(1), 141 - 55 Profiling and comprehensive expression analysis of ABC transporter solute-binding proteins of Bacillus subtilis membrane based on a proteomic approach; Bunai K et al.; We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach . We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner . The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile . Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins . We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY . Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome . We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis . We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7 . The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium). J Bacteriol, 2004 Feb, 186(3), 818 - 28 Regulation of the tryptophan biosynthetic genes in Bacillus halodurans: common elements but different strategies than those used by Bacillus subtilis; Szigeti R et al.; In Bacillus subtilis, an RNA binding protein called TRAP regulates both transcription and translation of the tryptophan biosynthetic genes . Bacillus halodurans is an alkaliphilic Bacillus species that grows at high pHs . Previous studies of this bacterium have focused on mechanisms of adaptation for growth in alkaline environments . We have characterized the regulation of the tryptophan biosynthetic genes in B . halodurans and compared it to that in B . subtilis . B . halodurans encodes a TRAP protein with 71% sequence identity to the B . subtilis protein . Expression of anthranilate synthetase, the first enzyme in the pathway to tryptophan, is regulated significantly less in B . halodurans than in B . subtilis . Examination of the control of the B . halodurans trpEDCFBA operon both in vivo and in vitro shows that only transcription is regulated, whereas in B . subtilis both transcription of the operon and translation of trpE are controlled . The attenuation mechanism that controls transcription in B . halodurans is similar to that in B . subtilis, but there are some differences in the predicted RNA secondary structures in the B . halodurans trp leader region, including the presence of a potential anti-antiterminator structure . Translation of trpG, which is within the folate operon in both bacilli, is regulated similarly in the two species. J Inorg Biochem, 2004 Feb, 98(2), 322 - 32 Synthesis, X-ray crystal structure, antimicrobial activity and photodynamic effects of some thiabendazole complexes; Mothilal KK et al.; An interesting series of metal complexes of thiabendazole (tbz) is synthesized and characterized by elemental analyses and spectroscopic studies . The crystal structure of the hydrogen bonded one dimensional Co(II) complex, namely {Co(tbz)(2)(NO(3))(H(2)O)}(NO(3)) is solved by single crystal X-ray diffraction . The complex crystallizes in monoclinic space group P2(1)/a with unit cell parameters, a=14.366(2), b=11.459(4), c=15.942(3) A, beta=113.78(3) degrees and z=4 . The unit cell packing reveals an extensive hydrogen bonding involving a water molecule, nitrate ligands and the protonated nitrogen atoms of the tbz ligands, resulting in a one dimensional hydrogen bonding pattern . The antimicrobial activity of the complexes against selected bacteria (Escherichia coli and Bacillus subtilis) and yeast (Aspergillus flavues) is estimated . The relationship between the enzymatic production of ROS and antimicrobial activity of the complexes is examined, and a good correlation between two factors is found . Photodynamic quantum yields of singlet oxygen production (RNO bleaching assay) and rate of superoxide generation (SOD inhibitable ferricytochrome c reduction assay and EPR spin trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide as spin trap) by the metal complexes have been studied. J Appl Microbiol, 2004, 96(2), 289 - 301 Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents; Young SB et al.; AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to the general biological decontamination agents, Decon and Oxone . METHODS AND RESULTS: Spores of B . subtilis treated with Decon or Oxone did not accumulate DNA damage and were not mutagenized . Spore killing by these agents was increased if spores were decoated . Spores prepared at higher temperatures were more resistant to these agents, consistent with a major role for spore coats in this resistance . Neither Decon nor Oxone released the spore core's depot of dipicolinic acid (DPA), but Decon- and Oxone-treated spores more readily released DPA upon a subsequent normally sublethal heat treatment . Decon- and Oxone-killed spores initiated germination with dodecylamine more rapidly than untreated spores, but could not complete germination triggered by nutrients or Ca(2+)-DPA and did not degrade their peptidoglycan cortex . However, lysozyme treatment did not recover these spores . CONCLUSIONS: Decon and Oxone do not kill B . subtilis spores by DNA damage, and a major factor in spore resistance to these agents is the spore coat . Spore killing by both agents renders spores defective in germination, possibly because of damage to the inner membrane of spore . SIGNIFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of the killing of bacterial spores by Decon and Oxone. Mikrobiol Z, 2003 Sep-Oct, 65(5), 13 - 9 Antiscleroma effectiveness of certain Bacillus subtilis strains studied in vitro and in vivo; Lysetska MV et al.; Antagonistic activity of approximately 200 Bacillus subtilis cultures vs . Klebsiella rhinoscleromatis clinical strains in experiments on solid and liquid nutrient media, and in experiments on laboratory animals has been studied . 16 B . subtilis strains characterized by a vivid antiscleroma activity in vivo have been selected . Maximum preventive and therapeutical influence of the B . subtilis strain 1119 upon the flow of experimental acute septic and lung scleroma infection has been determined . While co-cultivating the test cultures under study (B . subtilis 1119 and K . rhinoscleromatis 230), a significant titer decline and complete klebsiella's growth suppression on the 8th-12th days provided the certain concentrations of the antagonistic microbes have been shown. Appl Environ Microbiol, 2004 Jan, 70(1), 475 - 82 Species differentiation of a diverse suite of Bacillus spores by mass spectrometry-based protein profiling; Dickinson DN et al.; In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of Bacillus spores . MALDI-TOFMS protein profiles of 11 different strains of Bacillus spores, encompassing nine different species, were evaluated . Bacillus species selected for MALDI-TOFMS analysis represented the spore-forming bacterial diversity of typical class 100K clean room spacecraft assembly facilities . A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time . A library of MALDI-TOFMS spectra was created from these nine Bacillus species, the most diverse protein profiling study of the genus reported to date . Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study . The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations . The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis . Proteomic studies of Bacillus subtilis 168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum . Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified . The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the Bacillus genus. Anal Chem, 2003 Oct 15, 75(20), 5480 - 7 Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry; Steele PT et al.; Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time . Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var . niger were obtained in a bipolar aerosol time-of-flight mass spectrometer using a pulsed 266-nm laser for molecular desorption and ionization . Spectra from single spores collected at an average fluence of approximately 0.1 J/cm2 frequently contain prominent peaks attributed to arginine, dipicolinic acid, and glutamic acid, but the shot-to-shot (spore-to-spore) variability in the data may make it difficult to consistently distinguish closely related Bacillus species with an automated routine . Fortunately, a study of the laser power dependence of the mass spectra reveals clear trends and a finite number of "spectral types" that span most of the variability . This, we will show, indicates that a significant fraction of the variability must be attributed to fluence variations in the profile of the laser beam. Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 534 - 9 Epub 2004 Jan 02. Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme; Xie Z et al.; The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET) . Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-I(eq)-to-N, and focused on the I(eq)-to-N transition . The present study focuses on the U-to-I(eq) transition . Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5' end and Cy3 at the 3' end show that modifications required for labeling do not interfere with folding and help to define the Mg(2+) concentration range for the U-to-I(eq) transition . Histogram analysis of the Mg(2+)-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates . The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg(2+) concentrations on the time scale of seconds . The trajectories at intermediate Mg(2+) concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range . This heterogeneity, together with the observation of "nonsudden jump" FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many "local" conformational switches . A free energy contour is constructed to illustrate the complex folding surface. Microbiology, 2004 Jan, 150(Pt 1), 205 - 15 Chlamydia trachomatis sigma28 recognizes the fliC promoter of Escherichia coli and responds to heat shock in chlamydiae; Shen L et al.; The rpsD gene of Chlamydia trachomatis encodes the alternative sigma factor sigma28, which bears strong homology to many bacterial sigma factors, including Escherichia coli sigma8 and Bacillus subtilis sigmaB and sigmaD . Recently, a sigma28 promoter was identified upstream of the late-cycle-expressed gene hctB, which encodes the Chlamydia-histone-like protein 2 (Yu & Tan, 2003) . In this study it is shown that the product of chlamydial rpsD is an E . coli sigma28 homologue . It was found that recombinant chlamydial sigma8, in combination with E . coli core RNA polymerase, initiates transcription in vitro from the E . coli sigma28-dependent promoter of fliC . It was also demonstrated that the recombinant chlamydial sigma28 does not recognize major sigma factor sigma70-consensus-like sequences in vitro . In C . trachomatis-infected cells, two rpsD transcripts were detected with 5' ends located 18 (transcript I) and 54 bp (transcript II) upstream of the translational initiation codon at 16 and 30 h post-infection . When the temperature of cultures infected with C . trachomatis was shifted from 35 to 42 degrees C, the rpsD transcript I increased dramatically . The levels of chlamydial sigma28, relative to EF-Tu, were greater throughout the exponential growth phase of the reticulate body, but lower late in the developmental cycle . These data support the hypothesis that sigma28 plays a role in the regulatory network that allows chlamydiae to survive changes in its environment, enabling it to complete its unique developmental cycle. Microbiology, 2004 Jan, 150(Pt 1), 163 - 70 Functional relationship between SpoVIF and GerE in gene regulation during sporulation of Bacillus subtilis; Kuwana R et al.; The sporulation-specific SpoVIF (YjcC) protein of Bacillus subtilis is essential for the development of heat-resistant spores . The GerE protein, the smallest member of the LuxR-FixJ family, contains a helix-turn-helix (HTH) motif and is involved in the expression of various sporulation-specific genes . In this study, the gene expression and protein composition of sporulating spoVIF-negative cells were analysed . CgeA, CotG and CotS, which are GerE-dependent coat proteins, were not expressed in the spoVIF-negative cells . Northern blotting showed that SpoVIF regulated the transcription of cgeA, cotG and cotS in a manner similar to that of GerE . In spoVIF-negative cells, gerE mRNA was transcribed normally, but immunoblot analysis using anti-GerE antiserum showed that the quantity of GerE protein was considerably less than that in wild-type controls . Using GFP (green fluorescent protein) fusion proteins, the localization of SpoVIF and GerE was observed by fluorescence microscopy . SpoVIF-GFP was detectable in the mother cell compartment, as was GerE-GFP . These results suggest that SpoVIF directly or indirectly controls the function of the GerE protein, and that SpoVIF is required for gene regulation during the latter stages of sporulation. J Bacteriol, 2004 Jan, 186(2), 278 - 86 The trp RNA-binding attenuation protein of Bacillus subtilis regulates translation of the tryptophan transport gene trpP (yhaG) by blocking ribosome binding; Yakhnin H et al.; Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA {trpG}) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein . TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms . TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter . Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs . We found that regulation of trpP and pabA is unaltered in a rho mutant strain . Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region . While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA . RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon . Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit . Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding . Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts. Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 452 - 7 Epub 2003 Dec 30. Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA; Ayora S et al.; Bacillus subtilis RecU protein is involved in homologous recombination, DNA repair, and chromosome segregation . Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) ( approximately 10- and approximately 40-fold lower efficiency, respectively) . RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the epsilon epistatic group (namely ruvA, recD, and recU) . In the presence of Mg(2+), RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate . These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly. Int J Food Microbiol, 2004 Jan 15, 90(2), 197 - 205 Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkia biglobosa) to produce Soumbala; Ouoba LI et al.; Bacillus spp . are the predominant microorganisms in fermented African locust bean called Soumbala in Burkina Faso . Ten strains selected as potential starter cultures were characterised by PCR amplification of the16S-23S rDNA intergenic transcribed spacer (ITS-PCR), restriction fragment length polymorphism of the ITS-PCR (ITS-PCR RFLP), pulsed field gel electrophoresis (PFGE) and sequencing of the 968-1401 region of the 16S rDNA . In previous studies, the isolates were identified by phenotyping as Bacillus subtilis and Bacillus pumilus . The phenotyping was repeated as a reference in the present study.The ITS-PCR and ITS-PCR RLFP allowed a typing at species level . The PFGE was more discriminative and allowed a typing at strain level . Full agreement with the phenotyping was observed in all cases . The sequencing of the 16S rDNA allowed the identification at species level with an identity from 97% to 100% comparing the sequences to those from the GenBank databases . The desired cultures of B . subtilis and B . pumilus from African locust bean fermentation were distinguished by ITS-PCR and ITS-PCR RLFP from Bacillus cereus and Bacillus sphaericus which sometimes occur in the beginning of the fermentation. J Biochem (Tokyo), 2003 Nov, 134(5), 691 - 7 Inhibition of Bacillus subtilis aprE expression by lincomycin at the posttranscriptional level through inhibition of ppGpp synthesis; Arai A et al.; Expression of the Bacillus subtilis alkaline protease gene aprE is controlled by many positive and negative regulators at the transcriptional level . During the course of screening for organic compounds that affect the expression of a translational aprE'-'lacZ fusion, we found that lincomycin (Lm), erythromycin and chloramphenicol exhibited an inhibitory effect in concentrations that hardly affected cell growth . The antibiotics are known to inhibit protein synthesis by binding to ribosomes . We chose one of them, Lm, for further study . We have previously shown that aprE expression requires guanosine 3',5'-bisdiphosphate (ppGpp) synthesized on the ribosome by the stringent factor RelA . An examination of Lm-treated cells showed that the levels of ppGpp were greatly reduced in these cells, and the inhibitory effect of the antibiotic was not seen in relA-disruption mutants . Transcriptional levels of aprE, however, were not influenced by Lm treatment as shown by using a transcriptional aprE-lacZ fusion as well as quantitative RT-PCR . Furthermore, disruption of relA did not affect the expression of transcriptional aprE-lacZ . From these results, we conclude that aprE expression is controlled by the stringent control at the posttranscriptional level, and that Lm inhibits this process by inhibiting ppGpp synthesis on the ribosome. J Biochem (Tokyo), 2003 Nov, 134(5), 655 - 60 Purification and characterization of YfkN, a trifunctional nucleotide phosphoesterase secreted by Bacillus subtilis; Chambert R et al.; YfkN isolated from the culture supernatant of Bacillus subtilis in the exponential phase of growth is a protein of 143.5 kDa that derives from a putative large precursor of 159.6 kDa processed at both the N- and C-terminal ends . Pulse-chase experiments indicated that the release occurs slowly with a half-time longer than 30 min, suggesting that the event is coupled with wall turnover . YfkN exhibits 2',3' cyclic nucleotide phosphodiesterase, 2' (or 3') nucleotidase and 5' nucleotidase activities . In vitro the protein is reduced by subtilisin digestion to a shorter polypeptide (68 kDa), displaying phosphodiesterase activity but devoid of any 5'nucleotidase activity . This proteolytic processing led us to localize the potential active sites of the various nucleotidase activities . When bacteria were grown in low phosphate medium, the exocellular production of the enzyme was enhanced, suggesting that it plays a role in phosphate metabolism . Comparison with nucleotidase databases suggests that yfkN resulted from gene fusion. Int J Biochem Cell Biol, 2004 Mar, 36(3), 535 - 44 GS-Finder: a program to find bacterial gene start sites with a self-training method; Ou HY et al.; In this paper, a self-training method is proposed to recognize translation start sites in bacterial genomes without a prior knowledge of rRNA in the genomes concerned . Many features with biological meanings are incorporated, including mononucleotide distribution patterns near the start codon, the start codon itself, the coding potential and the distance from the most-left start codon to the start codon . The proposed method correctly predicts 92% of the translation start sites of 195 experimentally confirmed Escherichia coli CDSs, 96% of 58 reliable Bacillus subtilis CDSs and 82% of 140 reliable Synechocystis CDSs . Moreover, the self-training method presented might also be used to relocate the translation start sites of putative CDSs of genomes, which are predicted by gene-finding programs . After post-processing by the method presented, the improvement of gene start prediction of some gene-finding programs is remarkable, e.g., the accuracy of gene start prediction of Glimmer 2.02 increases from 63 to 91% for 832 E . coli reliable CDSs . An open source computer program to implement the method, GS-Finder, is freely available for academic purposes from http://tubic.tju.edu.cn/GS-Finder/. J Mol Biol, 2004 Jan 16, 335(3), 707 - 22 Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity; Li PT et al.; TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration . When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression . Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan . In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V) . We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits . The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength . Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA . Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently. Arch Microbiol, 2004 Feb, 181(2), 137 - 43 Epub 2003 Dec 19. Escherichia coli RNase E and RNase G cleave a Bacillus subtilis transcript at the same site in a structure-dependent manner; Hambraeus G et al.; The decay of Bacillus subtilis aprE leader- lacZ mRNA was examined in Escherichia coli wild-type and in mutants deficient in RNase E, RNase G, or both . Two versions of the mRNA were studied: the wild-type mRNA, which has a stem-loop at the 5' end, and a mutant mRNA, with a single-stranded 5' end . The half-life of both transcripts was determined by RNase E, the half-life of the mutant transcript being one-third of that of the wild-type transcript . RNase G cleaved both transcripts at a site within an AU-rich sequence in the stem-loop region, but cleavage was much more efficient when the stem-loop was destabilized . RNase E cleaved at the same site, but less efficiently and only in the mutant transcript. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 175 - 7 Epub 2003 Dec 18. Crystallization of the oligopeptide-binding protein AppA from Bacillus subtilis; Wright L et al.; AppA is the membrane-anchored extracellular receptor component of an ABC transporter responsible for the uptake of oligopeptides into Bacillus subtilis . AppA has been overexpressed as a cleavable maltose-binding protein fusion in Escherichia coli . Following removal of the fusion portion, AppA has been crystallized from morpholinoethanesulfonic acid-buffered solutions at pH 6.5 containing polyethylene glycol and zinc acetate . A complete X-ray diffraction data set extending to 2.3 A spacing has been collected. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 166 - 8 Epub 2003 Dec 18. Expression, purification, crystallization and preliminary crystallographic analysis of a putative GTP-binding protein, YsxC, from Bacillus subtilis; Das SK et al.; Bacillus subtilis YsxC has been putatively identified as a member of the GTP-binding protein family . Gene-knockout/deletion analysis has suggested that this protein is essential for survival of the microorganism and hence may represent a target for the development of a novel anti-infective agent . The B . subtilis ysxC gene was cloned and the protein was overexpressed in Escherichia coli and subsequently purified . Using hanging-drop vapour-diffusion crystallization techniques, two different crystal forms of YsxC were obtained in the presence and absence of GDP and which have one and two copies of YsxC in the asymmetric unit, respectively . Both crystal forms diffract to beyond 2.0 A resolution and are suitable for structure determination. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 160 - 2 Epub 2003 Dec 18. Crystallization and preliminary X-ray crystallographic investigations on several thermostable forms of a Bacillus subtilis lipase; Rajakumara E et al.; Bacillus subtilis lipase loses activity above pH 10.5 and below pH 6.0 . However, at low pH, i.e . below pH 5.0, the lipase acquires remarkable thermostability . Activity was unaltered for 2 h at 323 K at pH 4.0-5.0, although at pH values above 7.0 the activity was lost rapidly within minutes . Circular-dichroism studies indicate significant changes in the tertiary structure of the lipase, whereas the secondary-structural content remained unaltered . To elucidate the structural basis of the enhanced thermostability, three different forms have been crystallized at low pH along with three crystal forms of two thermostable mutants obtained using a directed-evolution approach. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 116 - 9 Epub 2003 Dec 18. Crystallization and preliminary X-ray analysis of 5'-methylthioribose kinase from Bacillus subtilis and Arabidopsis thaliana; Ku SY et al.; Recombinant Bacillus subtilis 5'-methylthioribose (MTR) kinase has been expressed, purified and subsequently crystallized using the hanging-drop vapor-diffusion technique . With PEG 2000MME as the precipitant, two different crystal forms have been grown in the absence and presence of the detergent CHAPS . These crystals belong to space groups P2(1)2(1)2(1) (unit-cell parameters a = 193.7, b = 83.2, c = 51.6 A) and P2(1)2(1)2 (unit-cell parameters a = 213.8, b = 83.2, c = 51.5 A), respectively . The crystals grown in the presence of CHAPS diffract to 2.2 A resolution at Station X8C, National Synchrotron Light Source (NSLS) . For both crystal forms, the presence of two monomers per asymmetric unit is predicted (Matthews coefficient V(M) = 2.29 and 2.52 A(3) Da(-1), respectively) . Recombinant C-terminally histidine-tagged Arabidopsis thaliana MTR kinase has also been expressed, purified and refolded into its active form . Rod-shaped crystals of this protein were grown from PEG 8000 using the hanging-drop vapor-diffusion technique . These crystals exhibit the symmetry of space group C2 (unit-cell parameters a = 162.3, b = 83.3, c = 91.0 A, beta = 117.8 degrees ) and diffract to 1.9 A resolution at Station X8C, NSLS . Two monomers are estimated to be present in the asymmetric unit (V(M) = 2.82 A(3) Da(-1)). Protein Expr Purif, 2004 Jan, 33(1), 57 - 65 Expression, purification, and characterization of BsSco, an accessory protein involved in the assembly of cytochrome c oxidase in Bacillus subtilis; Andrews D et al.; The studies described here were performed to characterize further the plasma membrane associated protein BsSco, which is the product of the gene ypmQ, in Bacillus subtilis . BsSco is a member of the Sco family of proteins found in the inner mitochondrial membrane of yeast and humans and implicated as an accessory protein in the assembly of the Cu(A) site of cytochrome c oxidase . We have cloned the gene expressing BsSco, placed a six-histidine tag on its C-terminus, and over-expressed this protein in B . subtilis . Recombinant BsSco with the his-tag has been purified from Triton X-100-solubilized plasma membranes by nickel metal affinity chromatography . Mass spectral analysis of the purified protein is consistent with processing of BsSco by signal peptidase II removing an N-terminal putative transmembrane sequence to leave an acyl-glyceryl moiety at cysteine residue 19 . Antibodies, raised against purified, recombinant BsSco, were used to characterize the timing of the level of native BsSco in batch cultures of wild-type B . subtilis . There is a marked lag in the level of native BsSco, but it does appear prior to cytochrome c oxidase, which is expressed in late stage growth . This work supports a role for BsSco in the assembly of the Cu(A) site of cytochrome c oxidase and its functional relationship to the Sco proteins found in eukaryotic cells. Curr Biol, 2003 Dec 16, 13(24), 2196 - 200 Assembly of the SpoIIIE DNA translocase depends on chromosome trapping in Bacillus subtilis; Ben-Yehuda S et al.; Sporulation in Bacillus subtilis is an attractive system in which to study the translocation of a chromosome across a membrane . Sporulating cells contain two sister chromosomes that are condensed in an elongated axial filament with the origins of replication anchored at opposite poles of the sporangium . The subsequent formation of a septum near one pole divides the sporangium unequally into a forespore (the smaller compartment) and a mother cell . The septum forms around the filament, trapping the origin-proximal region of one chromosome in the forespore . As a consequence, the trapped chromosome transverses the septum with the remainder being left in the mother cell . Next, SpoIIIE assembles at the middle of the septum to create a translocase that pumps the origin-distal, two-thirds of the chromosome into the forespore . Here, we address the question of how the DNA translocase assembles and how it localizes to the septal midpoint . We present evidence that DNA transversing the septum is an anchor that nucleates the formation of the DNA translocase . We propose that DNA anchoring is responsible for the assembly of other SpoIIIE-like DNA translocases, such as those that remove trapped chromosomes from the division septum of cells undergoing binary fission. Mikrobiologiia, 2003 Sep-Oct, 72(5), 581 - 93 {Conjugation in bacilli}; Prozorov AA; The review considers experimental data on the conjugal transfer of plasmids in the Bacillus cereus and Bacillus subtilis groups (the transfer of large self-transmissible plasmids and the mobilization of small plasmids) . Conjugation in bacilli is compared with conjugation in E . coli dependent on the F factor . Conjugation of bacilli in their natural habitats is also discussed. J Bacteriol, 2004 Jan, 186(1), 258 - 61 A mother cell-specific class B penicillin-binding protein, PBP4b, in Bacillus subtilis; Wei Y et al.; The Bacillus subtilis genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan . The pbpI (yrrR) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing sigma(E) . Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure. J Bacteriol, 2004 Jan, 186(1), 200 - 6 Surfaces of Spo0A and RNA polymerase sigma factor A that interact at the spoIIG promoter in Bacillus subtilis; Kumar A et al.; In Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, sigma(A) (e.g., spoIIG), and those used by RNA polymerase containing the secondary sigma factor, sigma(H) (e.g., spoIIA) . Several single amino acid substitutions in region 4 of sigma(A) define positions in sigma(A) that are specifically required for Spo0A-dependent promoter activation . Similarly, several single amino acid substitutions in Spo0A define positions in Spo0A that are required for sigma(A)-dependent promoter activation but not for other functions of Spo0A . It is unknown whether these amino acids in Spo0A interact directly with those in region 4 of sigma(A) or whether they interact with another subunit of RNA polymerase to effect promoter activation . Here we report the identification of a new amino acid in region 4 of sigma(A), arginine at position 355 (R355), that is involved in Spo0A-dependent promoter activation . To further investigate the role of R355, we used the coordinates of Spo0A and sigma region 4, each in complex with DNA, to build a model for the interaction of sigma(A) and Spo0A at the spoIIG promoter . We tested the model by examining the effects of amino acid substitutions in the putative interacting surfaces of these molecules . As predicted by the model, we found genetic evidence for interaction of R355 of sigma(A) with glutamine at position 221 of Spo0A . These results appear to define the surfaces of Spo0A and sigma(A) that directly interact during activation of the spoIIG promoter. J Bacteriol, 2004 Jan, 186(1), 179 - 91 Fine-tuning in regulation of Clp protein content in Bacillus subtilis; Gerth U et al.; Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions . Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell . According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP . Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions . ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation . In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level . Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B . subtilis. J Bacteriol, 2004 Jan, 186(1), 80 - 9 Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan; Gilmore ME et al.; Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex . A peptidoglycan structural modification found uniquely in spores is the formation of muramic delta-lactam . Production of muramic delta-lactam in Bacillus subtilis requires removal of a peptide side chain from the N-acetylmuramic acid residue by a cwlD-encoded muramoyl-L-Alanine amidase . Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives . Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK . We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation . Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation . The product of pdaA (yfjS), which encodes a putative deacetylase, has recently been shown to also be required for muramic delta-lactam formation . Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic delta-lactam formation . Expression of PdaA alone in E . coli had no effect on E . coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic delta-lactam . CwlD and PdaA are necessary and sufficient for muramic delta-lactam production, and no other B . subtilis gene product is required . PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate. J Bacteriol, 2004 Jan, 186(1), 15 - 21 Diversifying selection at the Bacillus quorum-sensing locus and determinants of modification specificity during synthesis of the ComX pheromone; Ansaldi M et al.; The competence quorum-sensing system of Bacillus subtilis consists of two-component regulatory proteins, ComP (histidine kinase) and the response regulator, ComA, an extracellular pheromone (ComX), and a protein that is needed for the proteolytic cleavage and modification of pre-ComX (ComQ) . ComQ and pre-ComX are both necessary and sufficient for the production of active pheromone, which is released as an isoprenylated peptide . Laboratory strain 168 and a number of natural isolates of bacilli differ in the primary sequences of their pheromones as well as in the masses of their isoprenyl adducts . We have shown that ComX, ComQ, and the membrane-localized sensor domain of ComP are highly polymorphic in natural isolates of bacilli all closely related to the laboratory strain of B . subtilis . In this study, we used two statistical tests (the ratio of synonymous and nonsynonymous substitution rates and the Tajima D test) to demonstrate that these polymorphic sequences evolved by diversifying selection rather than by neutral drift . We show that the choice of isoprenyl derivative is determined by the C-terminal (mature) sequence of pre-ComX rather than by the ComQ protein . The implications of these findings for the evolution of the quorum-sensing system and for the protein-protein interactions involved in determining specificity are discussed. Curr Opin Struct Biol, 2003 Dec, 13(6), 739 - 47 Structural biology of enzymes of the thiamin biosynthesis pathway; Settembre E et al.; Thiamin pyrophosphate is an essential cofactor of carbohydrate and branched-chain amino acid metabolism . Although its mechanistic role is well studied, the biosynthesis of thiamin has only recently been understood . Thiamin biosynthesis in Escherichia coli and Bacillus subtilis show some similarities, but diverge at key steps of thiazole formation . The biosynthesis of thiamin in eukaryotes is at a very early stage of understanding . Structural and mechanistic studies on thiamin biosynthetic enzymes have played a key role in increasing our understanding of thiamin pyrophosphate biosynthesis and have revealed unexpected evolutionary ties. J Mol Biol, 2004 Jan 9, 335(2), 655 - 63 A mechanism for polar protein localization in bacteria; Howard M; We investigate a mechanism for the polar localization of proteins in bacteria . We focus on the MinCD/DivIVA system regulating division site placement in the rod-shaped bacterium Bacillus subtilis . Our model relies on a combination of geometric effects and reaction-diffusion dynamics to direct proteins to both cell poles, where division is then blocked . We discuss similarities and differences with related division models in Escherichia coli and also develop extensions of the model to asymmetric polar protein localization . We propose that our mechanism for polar localization may be employed more widely in bacteria, especially in outgrowing spores, which do not possess any pre-existing polar division apparatus from prior division events. Appl Microbiol Biotechnol, 2004 May, 64(4), 551 - 5 Epub 2003 Dec 11. An easy method for screening and isolating rod mutants of Bacillus subtilis; Cheung SH et al.; A convenient and rapid method for screening and identifying rod mutants of Bacillus subtilis is described . At the restrictive temperature (45 degrees C), all rod mutants of B . subtilis screened lost their ability to sporulate . The morphology and colour of mutant colonies grown on sporulation agar plates differed from those of rod+ cells, which were able to sporulate even at elevated temperature . These characteristics provide an alternative approach for the identification of rod mutants in B . subtilis culture by streaking the cells onto a minimal glucose agar plate and incubating at the restrictive temperature . After 30 h of incubation at this temperature, rod mutants are easily identified . This method will facilitate the screening and isolation of rod mutants of B . subtilis . J Med Chem, 2003 Dec 18, 46(26), 5803 - 11 Generation of bis-cationic heterocyclic inhibitors of Bacillus subtilis HPr kinase/phosphatase from a ditopic dynamic combinatorial library; Bunyapaiboonsri T et al.; Ditopic dynamic combinatorial libraries were generated and screened toward inhibition of the bifunctional enzyme HPr kinase/phosphatase from Bacillus subtilis . The libraries were composed of all possible combinations resulting from the dynamic interconversion of 16 hydrazides and five monoaldehyde or dialdehyde building blocks, resulting in libraries containing up to 440 different constituents . Of all possible acyl hydrazones formed, active compounds containing two terminal cationic heterocyclic recognition groups separated by a spacer of appropriate structure could be rapidly identified using a dynamic deconvolution procedure . Thus, parallel testing of sublibraries where one specific component was excluded basically revealed all the essential components . A potent ditopic inhibitor, based on 2-aminobenzimidazole, was identified from the process. Syst Appl Microbiol, 2003 Nov, 26(4), 495 - 501 Effect of food processing on the fate of DNA with regard to degradation and transformation capability in Bacillus subtilis; Kharazmi M et al.; Soymilk, tofu, corn masa, and cooked potato were produced from transgenic raw materials and the effect of processing on the degradation of DNA was studied . Major degrading factors were for soymilk and tofu the mechanical treatment of soaked soybeans and for corn masa and cooked potatoes the thermal treatment . In the processed foods no DNA fragments > 1.1 kb were detected . We included in our studies the effect of the size of donor DNA and length of the homologous sequence on the marker rescue transformation of B . subtilis LTH 5466, which was monitored by restoration of deleted nptII . When DNA fragments (168, 414, 658, and 792 bp) of nptII and linearized plasmid DNA (pGEM-T-1, 3168 bp and pGEM-T-2, 3792 bp) containing the 168 bp or 792 bp fragments, respectively, were used as donor DNA, it was observed that the efficiency of marker rescue decreased with decreasing length of homologous sequence . The use of a larger plasmid (pMR2, 5786 bp) containing the 792 bp fragment revealed higher efficiency of marker rescue compared to pGEM-T-2 . The nptII fragments resulted in lower efficiencies compared to plasmid DNA containing the same fragment . For the 792 bp fragment and the linearized plasmid pMR2 a first-order dependency of the frequency of marker rescue transformation on the DNA concentration was observed . Based on the acquired data, the hypothetical frequency of transformation of transgenic DNA to B . subtilis in cooked potatoes was calculated to be equal to 8.5 x 10(-19) and 1.2 x 10(-27) for homologous and illegitimate recombination, respectively . These data permit to roughly estimate the time after which a person (10(8) years) or the world population (15 days) is exposed to one transformant generated by homologous recombination event, when the daily consumption per person is 130 g of cooked potatoes. Appl Microbiol Biotechnol, 2004 Apr, 64(3), 382 - 6 Epub 2003 Dec 09. Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability; Yakhnin H et al.; We describe a method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability . This method utilizes a chloramphenicol-resistant plasmid containing a temperature-sensitive (ts) replication origin . In this method, the target gene is first cloned into the ts vector and the recombinant plasmid is used to transform wild-type B . subtilis . The second step involves transformation of the resulting strain with a linear DNA fragment containing a second antibiotic resistance marker (tet) that disrupts the gene of interest . Selection for tetracycline resistance forces a double crossover between the chromosomal and fragment-borne copies of the gene, thereby replacing the wild-type gene in the chromosome with the disrupted allele . Cells survive even if the gene is essential by virtue of the complementing plasmid . Transformants are then grown at the non-permissive temperature for plasmid replication under various growth conditions . Isolation of chloramphenicol-sensitive colonies indicates that the gene is dispensable, whereas the inability to isolate chloramphenicol-sensitive colonies indicates that the gene is essential . The general utility of this method is demonstrated by allowing disruption of mtrA and trpE under conditions that render each gene non-essential, but not under growth conditions in which each gene is essential. Microbiology, 2003 Dec, 149(Pt 12), 3413 - 21 Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon; Gaballa A et al.; Copper ions induce expression of the Bacillus subtilis copZA operon encoding a metallochaperone, CopZ, and a CPx-type ATPase efflux protein, CopA . The copZA promoter region contains an inverted repeat sequence similar to that recognized by the mercury-sensing MerR protein . To investigate the possible involvement of MerR homologues in copZA regulation, null mutations were engineered affecting each of four putative MerR-type regulators: yyaN, yraB, yfmP and yhdQ . Two of these genes affected copper regulation . Mutation of yhdQ (hereafter renamed cueR) dramatically reduced copper induction of copZA, and purified CueR bound with high affinity to the copZA promoter region . These results suggest that CueR is a direct regulator of copZA transcription that mediates copper induction . Surprisingly, a yfmP mutation also reduced copper induction of copZA . Sequence analysis suggested that yfmP was cotranscribed with yfmO, encoding a putative multidrug efflux protein . The yfmPO operon is autoregulated: a yfmP mutation derepressed the yfmP promoter and purified YfmP bound the yfmP promoter region, but not the copZA promoter region . Since the yfmP mutant strain was predicted to express elevated levels of the YfmO efflux pump, it was hypothesized that copper efflux might be responsible for the reduced copZA induction . Consistent with this model, in a yfmP yfmO double mutant copper induction of copZA was normal . The results demonstrate the direct regulation of the B . subtilis copper efflux system by CueR, and indirect regulation by a putative multidrug efflux system. Appl Spectrosc, 2003 Aug, 57(8), 893 - 9 Identification of bacterial spores using statistical analysis of Fourier transform infrared photoacoustic spectroscopy data; Thompson SE et al.; Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) has been applied for the first time to the identification and speciation of bacterial spores . A total of forty specimens representing five strains of Bacillus spores (Bacillus subtilis ATCC 49760, Bacillus atrophaeus ATCC 49337, Bacillus subtilis 6051, Bacillus thuringiensis subsp . kurstaki, and Bacillus globigii Dugway) were analyzed . Spores were deposited, with minimal preparation, into the photoacoustic sample cup and their spectra recorded . Principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations were used on this spectral library to develop algorithms for step-wise classification at three levels: (1) bacterial/nonbacterial, (2) membership within the spore library, and (3) bacterial strain . Internal cross-validation studies on library spectra yielded classification success rates of 87% or better at each of these three levels . Analysis of fifteen blind samples, which included five samples of spores already in the spectral library, two samples of closely related Bacillus globigii 01 spores not in the library, and eight samples of nonbacterial materials, yielded 100% accuracy in distinguishing among bacterial/nonbacterial samples, membership in the library, and bacterial strains within the library. J Mol Biol, 2004 Jan 2, 335(1), 357 - 69 Analysis of thermal adaptation in the HSL enzyme family; Mandrich L et al.; The recently solved three-dimensional (3D) structures of two thermostable members of the carboxylesterase/lipase HSL family, namely the Alicyclobacillus (formerly Bacillus) acidocaldarius and Archaeoglobus fulgidus carboxylesterases (EST2 and AFEST, respectively) were compared with that of the mesophilic homologous counterpart Brefeldine A esterase from Bacillus subtilis . Since the 3D homology models of other members of the HSL family were also available, we performed a structural alignment with all these sequences . The resulting alignment was used to assess the amino acid "traffic rule" in the HSL family . Quite surprisingly, the data were in very good agreement with those recently reported from two independent groups and based on the comparison of a huge number of homologous sequences from the genus Bacillus, Methanococcus and Deinococcus/Thermus . Taken as a whole, the data point to the statistical meaning of defined amino acid conversions going from psychrophilic to hyperthermophilic sequences . We identified and mapped several such changes onto the EST2 structure and observed that such mutations were localized mostly in loops regions or alpha-helices and were mostly excluded from the active site . A site-directed mutagenesis of two of the identified residues confirmed they were involved in thermal stability. Appl Opt, 2003 Nov 20, 42(33), 6696 - 703 Passive standoff detection of Bacillus subtilis aerosol by Fourier-transform infrared radiometry; Theriault JM et al.; An analysis is presented on the passive standoff detection and identification of Bacillus subtilis (BG) clouds with the Compact ATmospheric Sounding Interferometer (CATSI) sensor . This research is based on recent spectral measurements obtained during the Technology Readiness Evaluation trial held July 2002 at Dugway Proving Ground, Utah . Results obtained from three trial BG cloud episodes are used to explain and demonstrate the detection capability of the CATSI sensor . The BG clouds were measured at a distance of 3 km from the sensor in a near-horizontal path scenario . It was found that the low thermal contrast of approximately 0.2 K between the BG cloud and the background yielded weak but observable spectral signatures . The processing of the spectral signatures with the GASeous Emission Monitoring (GASEM) algorithm has provided a rough estimate of BG cloud column densities . The results of a series of simulations with the FASCOD3 transmission model have shown that the detection sensitivity for BG can be greatly improved for both slant path uplooking and downlooking scenarios. Biotechnol Prog, 2003 Nov-Dec, 19(6), 1689 - 96 Macro-level and genetic-level responses of Bacillus subtilis to shear stress; Sahoo S et al.; Responses of bacterial (Bacillus subtilis) cells under different shear levels, from both the macro and genetic viewpoints, have been presented . The responses were studied using a novel, couette flow bioreactor (CFB), in which the entire cultivation can be performed under defined shear conditions . Oxygen supply, the normal limiting factor for entire cultivations under defined shear conditions, has been achieved by passing air through a poly(tetrafluoroethylene) (PTFE) membrane fixed on the inner cylinder of the CFB . More importantly, analyses of the oxygen transfer capabilities as well as the shear rates show that in this CFB, the effects of defined shear can be studied without interference from the effects of oxygen supply . Further, the shake flask can be used as a proper control for studying the shear effects, mainly because the shear rate in the shake flask under normal shaker operating conditions of 190 rpm has been estimated to be a negligible 0.028 s(-1) compared to a value of 445 s(-1) at the lowest rpm employed in the CFB . At the macro level the cell size decreased by almost 50% at 1482 s(-1) compared to that at 0.028 s(-1), the growth rate increased by 245%, and the maximum cell concentration increased by 190% when the shear rate was increased from 0.028 to 1482 s(-1) . The specific intracellular catalase level increased by 335% and protease by 87% at 1482 s(-1) as compared to the control cultures at a shear rate 0.028 s(-1) . In addition, the specific intracellular reactive oxygen species level (siROS) at the highest shear rate was 9.3-fold compared to the control conditions . At the genetic level we have established the involvement of the transcription factor, sigma(B), in the bacterial responses to shear stress, which was unknown in the literature thus far; the sigma(B) expression correlated inversely with the siROS . Further, through experiments with ROS quenchers, we showed that ROS regulated sigma(B) expression under shear. Anal Biochem, 2004 Jan 1, 324(1), 84 - 91 Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips; Gabig-Ciminska M et al.; Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories . A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here . Different phages of Escherichia coli and Bacillus subtilis were analyzed . Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips . The assay resulted in specific signals from all four tested phages without significant background . Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min . A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample . These analyses confirmed the specificity of the assay. Mol Genet Genomics, 2004 Feb, 271(1), 40 - 9 Epub 2003 Dec 02. LexA-binding sequences in Gram-positive and cyanobacteria are closely related; Mazon G et al.; The lexA gene of the cyanobacterium Anabaena sp . strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe . After over-expression in E . coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene . Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum . Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein . These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related. Arch Microbiol, 2004 Jan, 181(1), 60 - 7 Epub 2003 Dec 02. Identification of aryl-phospho-beta-D-glucosidases in Bacillus subtilis; Setlow B et al.; Four aryl-phospho-beta- d-glucosidases were identified in Bacillus subtilis by using 4-methylumbelliferyl-phospho-beta- d-glucopyranoside as a substrate . Two of these enzymes are the products of the bglA and bglH genes, previously suggested to encode aryl-phospho-beta- d-glucosidases, while the other enzymes are encoded by the yckE and ydhP genes . Together, these four genes account for >99.9% of the glucosidase activity in B . subtilis on aryl-phospho-beta- d-glucosides . yckE was expressed at a low and constant level during growth, sporulation, and spore germination, and was not induced by aryl-beta- d-glucosides . ydhP was also not induced by aryl-beta- d-glucosides . However, while ydhP was expressed at only a very low level in exponential-phase cells and germinating spores, this gene was expressed at a higher levels upon entry into the stationary phase of growth . Strains lacking yckE or ydhP exhibited no defects in growth, sporulation, or spore germination or in growth on aryl-beta- d-glucosides . However, a strain lacking bglA, bglH and yckE grew poorly if at all on aryl-beta- d-glucosides as the sole carbon source. Mol Microbiol, 2003 Dec, 50(5), 1683 - 701 The Spo0A regulon of Bacillus subtilis; Molle V et al.; The master regulator for entry into sporulation in Bacillus subtilis is the DNA-binding protein Spo0A, which has been found to influence, directly or indirectly, the expression of over 500 genes during the early stages of development . To search on a genome-wide basis for genes under the direct control of Spo0A, we used chromatin immunoprecipitation in combination with gene microarray analysis to identify regions of the chromosome at which an activated form of Spo0A binds in vivo . This information in combination with transcriptional profiling using gene microarrays, gel electrophoretic mobility shift assays, using the DNA-binding domain of Spo0A, and bioinformatics enabled us to assign 103 genes to the Spo0A regulon in addition to 18 previously known members . Thus, in total, 121 genes, which are organized as 30 single-gene units and 24 operons, are likely to be under the direct control of Spo0A . Forty of these genes are under the positive control of Spo0A, and 81 are under its negative control . Among newly identified members of the regulon with transcription that was stimulated by Spo0A are genes for metabolic enzymes and genes for efflux pumps . Among members with transcription that was in-hibited by Spo0A are genes encoding components of the DNA replication machinery and genes that govern flagellum biosynthesis and chemotaxis . Also in-cluded in the regulon are many (25) genes with products that are direct or indirect regulators of gene transcription . Spo0A is a master regulator for sporulation, but many of its effects on the global pattern of gene transcription are likely to be mediated indirectly by regulatory genes under its control. Mol Microbiol, 2003 Dec, 50(5), 1591 - 604 Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon; Mascher T et al.; In response to sublethal concentrations of antibiotics, bacteria often induce an adaptive response that can contribute to antibiotic resistance . We report the response of Bacillus subtilis to bacitracin, an inhibitor of cell wall biosynthesis found in its natural environment . Analysis of the global transcriptional profile of bacitracin-treated cells reveals a response orchestrated by two alternative sigma factors (sigmaB and sigmaM) and three two-component systems (YvqEC, YvcPQ and BceRS) . All three two-component systems are located next to target genes that are strongly induced by bacitracin, and the corresponding histidine kinases share an unusual topology: they lack about 100 amino acids in their extracellular sensing domain, which is almost entirely buried in the cytoplasmic membrane . Sequence analysis indicates that this novel N-terminal sensing domain is a characteristic feature of a subfamily of histidine kinases, found almost entirely in Gram-positive bacteria and frequently linked to ABC transporters . A systematic mutational analysis of bacitracin-induced genes led to the identification of a new bacitracin-resistance determinant, bceAB, encoding a putative ABC transporter . The bcrC bacitracin resistance gene, which is under the dual control of sigmaX and sigmaM, was also induced by bacitracin . By comparing the bacitracin and the vancomycin stimulons, we can differentiate between loci induced specifically by bacitracin and those that are induced by multiple cell wall-active antibiotics. Biosci Biotechnol Biochem, 2003 Nov, 67(11), 2477 - 9 Inhibition of sortase, a bacterial surface protein anchoring transpeptidase, by beta-sitosterol-3-O-glucopyranoside from Fritillaria verticillata; Kim SH et al.; A glucosylsterol, beta-sitosterol-3-O-glucopyranoside, has been isolated as an active principle with sortase inhibitory effect from the bulbs of Fritillaria verticillata by bioassay-guided chromatographic fractionation . The isolate was a potent inhibitor of sortase, with an IC(50) value of 18.3 microg/ml and had antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Micrococcus leuteus with MIC values of 50, 200, and 400 microg/ml, respectively, indicating that this compound is a possible candidate for the development of a bacterial sortase inhibitor . In addition, sitosterol was found to be inactive upon sortase and bacterial cell growth . These results suggest that the inhibitory potency of beta-sitosterol-3-O-glucopyranoside is sensitively dependent upon the glucopyranoside side chain moiety. Biochim Biophys Acta, 2003 Dec 5, 1624(1-3), 109 - 14 Nucleotide and deduced amino acid sequences of a high-molecular-mass subtilisin from an alkaliphilic Bacillus isolate; Ogawa A et al.; A high-molecular-mass subtilisin was found in culture broth of the alkaliphilic Bacillus sp . strain KSM-KP43 . The gene encoding the enzyme (FT protease) was determined using a mixed primer designed from the N-terminal amino acid (aa) sequence of the purified enzyme . The determi