Bacillus subtilis

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 determined nucleotide sequence of the gene consisted of a 2427-bp open reading frame (ORF) that encoded a putative prepro-peptide (152 aa) and a mature enzyme (656 aa; 68,506 Da) . The deduced aa of the mature enzyme revealed a moderate homology to a subtilisin-type proteinase from Bacillus halodurans and a minor extracellular protease, Vpr, from Bacillus subtilis with 64% and 57% identity, respectively . The molecular mass of the purified recombinant FT protease was approximately 72 kDa as judged by both SDS-polyacrylamide gel electrophoresis (PAGE) and gel filtration . FT protease showed maximal activity toward glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide at pH 10.5 and at 45 degrees C . The enzyme was rapidly inactivated by incubation over 45 degrees C for 15 min at both pH 7 and 10 . Calcium ions were slightly protective for thermoinactivation of the enzyme.

Metab Eng, 2003 Oct, 5(4), 246 - 54
Genetic engineering of Escherichia coli for production of tetrahydrobiopterin; Yamamoto K et al.; Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals . In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR) . BH4 is a medicine used to treat atypical hyperphenylalaninemia . It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures . To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E . coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes . These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4 . In order to increase BH4 productivity we made further improvements . First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced . Second, to augment the activity of GCHI, the folE gene from E . coli was replaced by the mtrA gene from Bacillus subtilis . These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth . The results suggest the possibility of commercial BH4 production by our method.

Expert Opin Biol Ther, 2003 Dec, 3(8), 1263 - 70
Bacterial spores as heat stable vaccine vehicles; Duc le H et al.; Recently, the first use of bacterial spores as vaccine vehicles was reported, showing that mice orally immunised with Bacillus subtilis spores expressing a tetanus antigen could be protected against lethal challenge with tetanus toxin . Unlike many second generation vaccine systems currently under development, none offer the heat stability of bacterial spores or the flexibility for genetic manipulation . The current use of Bacillus spores as probiotics for both humans and animals may facilitate their eventual licensing as oral vaccines . This review reports the progress that has been made in the establishment of bacterial spores as vaccine vehicles and outlines the potential advantages of the spore vaccine approach.

J Agric Food Chem, 2003 Dec 3, 51(25), 7338 - 45
Impact of xylanases with different substrate selectivity on gluten-starch separation of wheat flour; Frederix SA et al.; The influence on wheat flour gluten-starch separation of a xylanase from Aspergillus aculeatus (XAA) with hydrolysis selectivity toward water extractable arabinoxylan (WE-AX) and that is not inhibited by wheat flour xylanase inhibitors was compared to that of a xylanase from Bacillus subtilis (XBS) with hydrolysis selectivity toward water unextractable arabinoxylan (WU-AX) and that is inhibited by such inhibitors . XAA improved gluten agglomeration through degradation of WE-AX and concomitant reduction in viscosity, which in the laboratory scale batter procedure with a set of vibrating sieves (400, 250, and 125 microm), increased protein recoveries on the 400 microm sieve . In contrast, XBS had a negative effect as it decreased gluten protein recovery on this sieve, probably as a result of the viscosity increase that accompanied WU-AX solubilization . Hence, it was active even if most likely a considerable part of its activity was prevented by xylanase inhibitors . A combination of XAA and XBS at a low dosage yielded a distribution of gluten proteins on the different sieves comparable to that of the control . At a high combined dosage, the gluten agglomeration was better than that with XAA alone, indicating that both WE-AX and WU-AX have a negative impact on gluten agglomeration . Finally, experiments with endoxylanase addition at different moments during the separation process suggest that the status of the arabinoxylan population during dough mixing is far less critical for its impact on gluten agglomeration than that during the batter phase.

Chem Commun (Camb), 2003 Nov 7, (21), 2718 - 9
Production of vancomycin aglycone conjugated to a peptide carrier domain derived from a biosynthetic non-ribosomal peptide synthetase; Vitali F et al.; A method for attaching the vancomycin aglycone to a peptide carrier domain (PCD) is reported which involves reacting the apo-PCD produced in Escherichia coli with vancomycin aglycone-coenzyme A thioester, catalyzed by the phosphopantetheinyl transferase Sfp from Bacillus subtilis.

Icarus, 2003 Oct, 165(2), 253 - 76
Survival of endospores of Bacillus subtilis on spacecraft surfaces under simulated martian environments: implications for the forward contamination of Mars; Schuerger AC et al.; Experiments were conducted in a Mars simulation chamber (MSC) to characterize the survival of endospores of Bacillus subtilis under high UV irradiation and simulated martian conditions . The MSC was used to create Mars surface environments in which pressure (8.5 mb), temperature (-80, -40, -10, or +23 degrees C), gas composition (Earth-normal N2/O2 mix, pure N2, pure CO2, or a Mars gas mix), and UV-VIS-NIR fluence rates (200-1200 nm) were maintained within tight limits . The Mars gas mix was composed of CO2 (95.3%), N2 (2.7%), Ar (1.7%), O2 (0.2%), and water vapor (0.03%) . Experiments were conducted to measure the effects of pressure, gas composition, and temperature alone or in combination with Mars-normal UV-VIS-NIR light environments . Endospores of B . subtilis, were deposited on aluminum coupons as monolayers in which the average density applied to coupons was 2.47 x 10(6) bacteria per sample . Populations of B . subtilis placed on aluminum coupons and subjected to an Earth-normal temperature (23 degrees C), pressure (1013 mb), and gas mix (normal N2/O2 ratio) but illuminated with a Mars-normal UV-VIS-NIR spectrum were reduced by over 99.9% after 30 sec exposure to Mars-normal UV fluence rates . However, it required at least 15 min of Mars-normal UV exposure to reduce bacterial populations on aluminum coupons to non-recoverable levels . These results were duplicated when bacteria were exposed to Mars-normal environments of temperature (-10 degrees C), pressure (8.5 mb), gas composition (pure CO2), and UV fluence rates . In other experiments, results indicated that the gas composition of the atmosphere and the temperature of the bacterial monolayers at the time of Mars UV exposure had no effects on the survival of bacterial endospores . But Mars-normal pressures (8.5 mb) were found to reduce survival by approximately 20-35% compared to Earth-normal pressures (1013 mb) . The primary implications of these results are (a) that greater than 99.9% of bacterial populations on sun-exposed surfaces of spacecraft are likely to be inactivated within a few tens of seconds to a few minutes on the surface of Mars, and (b) that within a single Mars day under clear-sky conditions bacterial populations on sun-exposed surfaces of spacecraft will be sterilized . Furthermore, these results suggest that the high UV fluence rates on the martian surface can be an important resource in minimizing the forward contamination of Mars . c2003 Elsevier Inc . All rights reserved.

J Mol Biol, 2003 Dec 5, 334(4), 609 - 24
Mechanism of repression by Bacillus subtilis CcpC, a LysR family regulator; Kim SI et al.; Bacillus subtilis CcpC is a LysR family transcriptional regulatory protein that negatively regulates genes encoding enzymes of the tricarboxylic acid branch of the Krebs cycle . In the present work, the promoter region of the aconitase (citB) gene was used to investigate the mechanism of repression by CcpC . The binding of CcpC to the citB promoter region was shown to depend on DNA elements located near positions -66 and -27 . Binding to these elements induced a bend in the DNA at position -41 . Introduction of mutations in the -27 region and the presence of citrate, the inducer, had similar effects . In either case, citB expression was derepressed in vivo, the affinity of CcpC binding was reduced in vitro, the angle of the bend was relaxed, and RNA polymerase gained greater access to the -35 region of the promoter.

Biochemistry, 2003 Dec 2, 42(47), 13969 - 76
TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution; Safferling M et al.; The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane . In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution . Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species . Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane . Rather, oligomeric forms established in vivo persisted after solubilization . Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications . Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution . In addition, TetL dimers were found in a number of other detergents and over a wide pH range . It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.

Lett Appl Microbiol, 2003, 37(5), 399 - 404
Validation of a polynomial regression model: the thermal inactivation of Bacillus subtilis spores in milk; Jagannath A et al.; AIMS: The predicted survival of Bacillus subtilis 168 spores from a polynomial regression equation was validated in milk . METHODS AND RESULTS: Bias factor suggested as an index of model performance was used to validate the polynomial model predictions in ultrahigh temperature (UHT) treated and sterilized whole and skim milk . Model predictions were fail safe, predicting higher D-values (decimal reduction times) in buffer than actually noted in milk . CONCLUSIONS: The D-values for spores were lower in milk as compared with those predicted in potassium phosphate buffer contrary to the popular expectation of better spore survival in complex food systems . The Bias factor, a quantitative measure of the model performance, indicated that on average the model predictions exceed the observations by 40% in the case of whole milk and by 60% in the case of skim milk . SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is an attempt to ascertain the extent of reliability that one can safely place in polynomial model predictions, without compromising on the safety or palatability of foods where it is eventually intended to be applied . The work has also highlighted the differences in the thermal inactivation pattern of spores in buffer and in milk with a possible influence of the various constituents of milk . The work will assist the dairy industry to better design thermal processes to ensure longer shelf life of dairy foods.

Fitoterapia, 2003 Dec, 74(7-8), 682 - 5
Antioxidant and antimicrobial activities of Onosma argentatum and Rubia peregrina; Ozgen U et al.; The n-hexane-dichloromethane (1:1) extract of the roots of Onosma argentatum and the methanol extract (partitioned between water and chloroform, ethyl acetate and n-butanol, respectively), of the underground parts (roots and rhizomes) of Rubia peregrina were tested in vitro for their antioxidant and antimicrobial activities . The highest antioxidant activity (98%) was observed at 0.1% concentration for the roots of O . argentatum . It was 96% at 0.25% concentration on the ethyl acetate fraction of R . peregrina . O . argentatum extract was effective on Staphylococcus aureus, Bacillus subtilis and Escherichia coli . The ethyl acetate and chloroform fractions of R . peregrina were effective on S . aureus and E . coli, respectively . These two species did not have any antifungal activity.

Curr Microbiol, 2003 Oct, 47(4), 327 - 36
ScoC mediates catabolite repression of sporulation in Bacillus subtilis; Shafikhani SH et al.; Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation . Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation . Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and sigmaH in controlling the catabolite repression of sporulation gene expression . In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation . In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci . Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression . Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system . We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucose.

Curr Microbiol, 2003 Oct, 47(4), 300 - 8
Catabolite-induced repression of sporulation in Bacillus subtilis; Shafikhani SH et al.; In response to nutrient limitations, Bacillus subtilis cells undergo a series of morphological and genetic changes that culminate in the formation of endospores . Conversely, excess catabolites inhibit sporulation . It has been demonstrated previously that excess catabolites caused a decrease in culture medium pH in a process that required functional AbrB . Culture medium acidification was also shown to inhibit sigmaH-dependent sporulation gene expression . The studies reported here investigate the effects of AbrB-mediated pH sensing on B . subtilis developmental competence . We have found that neither addition of a pH stabilizer, MOPS (pH 7.5), nor null mutations in abrB blocked catabolite repression of sporulation . Moreover, catabolite-induced culture medium acidification was observed in cultures of catabolite-resistant sporulation mutants, crsA47, rvtA11, and hpr-16, despite their efficient sporulation . These results suggest that AbrB-mediated pH sensing is not the only mechanism regulating catabolite repression of sporulation . The AbrB pathway may function to channel cells toward genetic competence, as opposed to other postexponential differentiation pathways.

Curr Microbiol, 2003 Oct, 47(4), 272 - 7
Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis; Yao S et al.; Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea . A 3642-bp genomic region of B . subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B . subtilis 168 was cloned, sequenced, and characterized . Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B . subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B . subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system . The genetic regions between the srfD gene and lpa gene from B . subtilis B3 and B . subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B . subtilis 168 and B . subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins . There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.

J Biol Chem, 2004 Feb 27, 279(9), 7850 - 5 Epub 2003 Nov 19.
Characterization and reconstitution of a 4Fe-4S adenylyl sulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis; Berndt C et al.; CysH1 from Bacillus subtilis encodes a 3'-phospho/adenosine-phosphosulfate-sulfonucleotide reductase (SNR) of 27 kDa . Recombinant B . subtilis SNR is a homodimer, which is bispecific and reduces adenylylsulfate (APS) and 3'-phosphoadenylylsulfate (PAPS) alike with thioredoxin 1 or with glutaredoxin 1 as reductants . The enzyme has a higher affinity for PAPS (K(m)PAPS 6.4 microm Trx-saturating, 10.7 microm Grx-saturating) than for APS (K(m) APS 28.7 microm Trx-saturating, 105 microm Grx-saturating) at a V(max) ranging from 280 to 780 nmol sulfite mg(-1) min(-1) . The catalytic efficiency with PAPS as substrate is higher by a factor of 10 (K(cat)/K(m) 2.7 x 10(4)-3.6 x 10(4) liter mol(-1) s(-1) . B . subtilis SNR contains one 4Fe-4S cluster per polypeptide chain . SNR activity and color were lost rapidly upon exposure to air or upon dilution . Mossbauer and absorption spectroscopy revealed that the enzyme contained a 4Fe-4S cluster when isolated, but degradation of the 4Fe-4S cluster produced an inactive intermediate with spectral properties of a 2Fe-2S cluster . Activity and spectral properties of the 4Fe-4S cluster were restored by preincubation of SNR with the iron-sulfur cluster-assembling proteins IscA1 and IscS . Reconstitution of the 4Fe-4S cluster of SNR did not affect the reductive capacity for PAPS or APS . The interconversion of the clusters is thought to serve as oxygen-sensitive switch that suppresses SO(3) formation under aerobiosis.

RNA, 2003 Dec, 9(12), 1502 - 15
Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus; Schaak JE et al.; Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms . We recently determined that the B . subtilis trp leader readthrough transcript can adopt a Mg(2+)-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE . In the present study, sequence comparisons to trp leaders from three other Bacillus sp . were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved . To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus . Structure mapping experiments confirmed the predicted secondary structure . Native gel experiments identified a faster mobility species in the presence of Mg(2+), suggesting that a Mg(2+)-dependent tertiary structure forms . Mg(2+)-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions . Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop . Thermal denaturation experiments revealed a Mg(2+)- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats . The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined . These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14351 - 6 Epub 2003 Nov 17.
Nonorthologous replacement of lysyl-tRNA synthetase prevents addition of lysine analogues to the genetic code; Jester BC et al.; Insertion of lysine during protein synthesis depends on the enzyme lysyl-tRNA synthetase (LysRS), which exists in two unrelated forms, LysRS1 and LysRS2 . LysRS1 has been found in most archaea and some bacteria, and LysRS2 has been found in eukarya, most bacteria, and a few archaea, but the two proteins are almost never found together in a single organism . Comparison of structures of LysRS1 and LysRS2 complexed with lysine suggested significant differences in their potential to bind lysine analogues with backbone replacements . One such naturally occurring compound, the metabolic intermediate S-(2-aminoethyl)-L-cysteine, is a bactericidal agent incorporated during protein synthesis via LysRS2 . In vitro tests showed that S-(2-aminoethyl)-L-cysteine is a poor substrate for LysRS1, and that it inhibits LysRS1 200-fold less effectively than it inhibits LysRS2 . In vivo inhibition by S-(2-aminoethyl)-L-cysteine was investigated by replacing the endogenous LysRS2 of Bacillus subtilis with LysRS1 from the Lyme disease pathogen Borrelia burgdorferi . B . subtilis strains producing LysRS1 alone were relatively insensitive to growth inhibition by S-(2-aminoethyl)-L-cysteine, whereas a WT strain or merodiploid strains producing both LysRS1 and LysRS2 showed significant growth inhibition under the same conditions . These growth effects arising from differences in amino acid recognition could contribute to the distribution of LysRS1 and LysRS2 in different organisms . More broadly, these data demonstrate how diversity of the aminoacyl-tRNA synthetases prevents infiltration of the genetic code by noncanonical amino acids, thereby providing a natural reservoir of potential antibiotic resistance.

J Mol Biol, 2003 Nov 28, 334(3), 403 - 19
Nucleotide dependent monomer/dimer equilibrium of OpuAA, the nucleotide-binding protein of the osmotically regulated ABC transporter OpuA from Bacillus subtilis; Horn C et al.; The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions . Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro . OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC) . Dynamic monomer/dimer equilibrium was observed for OpuAA, and the K(D) value was determined to be 6 microM . Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA . In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(2)=2.3 min(-1)), whereas dimeric OpuAA showed little ATPase activity under this condition . The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle . The monomer was the preferred species under post-hydrolysis conditions (e.g . ADP/Mg(2+)), whereas the dimer dominated the nucleotide-free and ATP-bound states . The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP . One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the dimeric state of OpuAA . Binding of TNP-ADP/Mg(2+) to dimeric OpuAA induced a conformational change that led to the decay of the dimer . On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.

FEBS Lett, 2003 Nov 20, 554(3), 231 - 6
Sub-classification of response regulators using the surface characteristics of their receiver domains; Kojetin DJ et al.; The omnipresent bacterial switch known as a two-component system is comprised of a response regulator and a sensor kinase with which it interacts . Sensor kinases have been classified and further sub-classified into groups based on their sequence similarity, loop lengths and domain organization . Response regulators have been classified predominantly by the identity and function of their output domains . Here, comparative based homology modeling of the receiver domains of the OmpR sub-family of response regulators in Bacillus subtilis and Escherichia coli suggests further sub-classification is possible . A color-coded scale is used to show trends in surface hydrophobicity . For the OmpR receiver domains modeled these trends allow further sub-classification . The specific surface regions used for this sub-classification procedure correlate with clusters of residues that are important for interaction with cognate four helix bundle HisKA/Hpt domains.

Biochemistry, 2003 Nov 25, 42(46), 13422 - 8
Solution structure of apo CopZ from Bacillus subtilis: further analysis of the changes associated with the presence of copper; Banci L et al.; The solution structure of apo CopZ from Bacillus subtilis has been determined with the aim of investigating the changes in the hydrophobic interactions around the M-X-C-X-X-C copper(I) binding motif upon metal binding . The methionine of this motif (Met 11 in CopZ) points toward the solvent in apo CopZ, whereas its sulfur atom is close to the metal ion in the metal-loaded protein, though probably not at binding distance . This change is associated with the weakening of the interaction between Leu 37 and Cys 16, present in the apo form, and the formation of an interaction between Met 11 and Tyr 65 . Loops 1, 3, and 5 are affected by metal binding . Comparison with the structure of other homologous proteins confirms that often metal binding affects a hydrophobic patch around the metal site, possibly for optimizing and tuning the hydrophobic interactions with the partners . It is also shown that copper(I) exchanges among apo CopZ molecules in slow exchange on the NMR time scale, whereas it is known that such exchange between partner molecules (i.e., metallochaperones and metal pumps) is fast.

J Bacteriol, 2003 Dec, 185(23), 6981 - 4
Crystal structure of Bacillus subtilis alpha-amylase in complex with acarbose; Kagawa M et al.; The crystal structure of Bacillus subtilis alpha-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation . After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process.

APMIS, 2003 Oct, 111(10), 926 - 30
Association of the phi nucleotide with codon bias, amino acid usage and expressivity: differences between Bacillus subtilis and Escherichia coli; Fuglsang A; By measuring the non-randomness in Shine-Dalgarno regions it was recently shown that the compositional non-randomness peaks approximately 10 nucleotides upstream of the start codons . This position, termed the phi position, was furthermore shown to be associated with certain characteristics of the gene/protein and start codon usage . This raises the question whether codon usage in general is associated with the phi position . In this study, the connection between the phi nucleotide and general codon usage, both gene-wide and at the level of individual amino acids, was studied in Eschericia coli and Bacillus subtilis . E . coli but not B . subtilis shows a strong general association between the phi position and codon usage bias . In both species, the genes with higher expressivity show stronger conservation in the Shine-Dalgarno region compared to the genes with lower expressivity.

J Biol Chem, 2004 Jan 30, 279(5), 3885 - 92 Epub 2003 Nov 11.
RNA polymerase mutation activates the production of a dormant antibiotic 3,3'-neotrehalosadiamine via an autoinduction mechanism in Bacillus subtilis; Inaoka T et al.; Bacillus and Streptomyces species possess the ability to produce a variety of commercially important metabolites and extracellular enzymes . We previously demonstrated that antibiotic production in Streptomyces coeli-color A3(2) and Streptomyces lividans can be enhanced by RNA polymerase (RNAP) mutations selected for the rifampicin-resistant (Rif(r)) phenotype . Here, we have shown that the introduction of a certain Rif(r) rpoB mutation into a B . subtilis strain resulted in cells that overproduce an aminosugar antibiotic 3,3'-neotrehalosadiamine (NTD), the production of which is dormant in the wild-type strain . Mutational and recombinant gene expression analyses have revealed a polycistronic gene ntdABC (formally yhjLKJ) and a monocistronic gene ntdR (formally yhjM) as the NTD biosynthesis operon and a positive regulator for ntdABC, respectively . Analysis of transcriptional fusions to a lacZ reporter revealed that NTD acts as an autoinducer for its own biosynthesis genes via NtdR protein . Our results also showed that the Rif(r) rpoB mutation causes an increase in the activity of sigma(A)-dependent promoters including ntdABC promoter . Therefore, we propose that unlike the wild-type RNAP, the mutant RNAP efficiently recognized the sigma(A)-dependent promoters, resulting in the dramatic activation of the NTD biosynthesis pathway by an autoinduction mechanism.

FEMS Microbiol Lett, 2003 Nov 7, 228(1), 93 - 7
YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis; Bernard R et al.; The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system . When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin . Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator . The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium . When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one . We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.

Arch Pharm Res, 2003 Oct, 26(10), 816 - 20
Antimycobacterial and antioxidant flavones from Limnophila geoffrayi; Suksamrarn A et al.; The chloroform extract of the aerial part of Limnophila geoffrayi showed antimycobacterial and antioxidant activities . Bioassay-guided fractionation has led to the isolation of the flavones nevadensin (5,7-dihydroxy-6,8,4'-trimethoxyflavone, 1) and isothymusin (6,7-dimethoxy-5,8,4'-trihydroxyflavone, 2) . Both compounds 1 and 2 exhibited inhibition activity against Mycobacterium tuberculosis, with equal MIC value of 200 microg/mL . Only compound 2 exhibited antioxidant activity against the radical scavenging ability of DPPH, with the IC50 value of 7.7 microg/mL . The crude hexane, chloroform and methanol extracts as well as the pure compounds 1 and 2 did not exhibit mutagenic activity in the Bacillus subtilis recassay.

J Biochem (Tokyo), 2003 Oct, 134(4), 513 - 9
Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector; Itaya M et al.; Direct cloning of a long continuous genome segment in a Bacillus subtilis genome vector was demonstrated for the first time . Two small DNA fragments had to be installed in the vector prior to cloning . The DNA between these two fragments was cloned via homologous recombination . The efficiency of cloning was estimated using the 3,573-kb genome of a cyanobacterium, Synechocystis sp . PCC 6803 . Recombinants were selected using the internal selection system of the Bacillus genome vector or with the antibiotic resistance marker in the cyanobacterial genome . Designated genomic segments as large as 77-kb were cloned by means of a single procedure . Cloning efficiency is affected by the molecular weight of the donor DNA and the size of the DNA to be cloned . The method is suitable for direct target cloning of large-sized DNA.

Structure (Camb), 2003 Nov, 11(11), 1431 - 43
Solution structure of Sco1: a thioredoxin-like protein Involved in cytochrome c oxidase assembly; Balatri E et al.; Sco1, a protein required for the proper assembly of cytochrome c oxidase, has a soluble domain anchored to the cytoplasmic membrane through a single transmembrane segment . The solution structure of the soluble part of apoSco1 from Bacillus subtilis has been solved by NMR and the internal mobility characterized . Its fold places Sco1 in a distinct subgroup of the functionally unrelated thioredoxin proteins . In vitro Sco1 binds copper(I) through a CXXXCP motif and possibly His 135 and copper(II) in two different species, thus suggesting that copper(II) is adventitious more than physiological . The Sco1 structure represents the first structure of this class of proteins, present in a variety of eukaryotic and bacterial organisms, and elucidates a link between copper trafficking proteins and thioredoxins . The availability of the structure has allowed us to model the homologs Sco1 and Sco2 from S . cerevisiae and to discuss the physiological role of the Sco family.

Structure (Camb), 2003 Nov, 11(11), 1313 - 4
Let's Sco1, Oxidase! Let's Sco!
Winge DR.
The solution structure of Sco1 from Bacillus subtilis is the first structure of a protein important in the assembly of cytochrome c oxidase (CcO) . The assembly of CcO requires the insertion of multiple cofactors . Sco1 is a conserved protein implicated in formation of the binuclear Cu(A) center.

Nucleic Acids Res, 2003 Nov 15, 31(22), 6570 - 7
Gene essentiality determines chromosome organisation in bacteria; Rocha EP et al.; In Escherichia coli and Bacillus subtilis, essentiality, not expressivity, drives the distribution of genes between the two replicating strands . Although essential genes tend to be coded in the leading replicating strand, the underlying selective constraints and the evolutionary extent of these findings have still not been subject to comparative studies . Here, we extend our previous analysis to the genomes of low G + C firmicutes and gamma-proteobacteria, and in a second step to all sequenced bacterial genomes . The inference of essentiality by homology allows us to show that essential genes are much more frequent in the leading strand than other genes, even when compared with non- essential highly expressed genes . Smaller biases were found in the genomes of obligatory intracellular bacteria, for which the assignment of essentiality by homology from fast growing free-living bacteria is most problematic . Cross-comparisons used to assess potential errors in the assignment of essentiality by homology revealed that, in most cases, variations in the assignment criteria have little influence on the overall results . Essential genes tend to be more conserved in the leading strand than average genes, which is consistent with selection for this positioning and may impose a strong constraint on chromosomal rearrangements . These results indicate that essentiality plays a fundamental role in the distribution of genes in most bacterial genomes.

Appl Environ Microbiol, 2003 Nov, 69(11), 6841 - 7
Peptide ligands that bind selectively to spores of Bacillus subtilis and closely related species; Knurr J et al.; As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis . All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7 . We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding . We observed equal 7-mer peptide binding to spores of B . subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii . These three species comprise one branch on the Bacillus phylogenetic tree . We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B . subtilis . The sequence Asn-His-Phe-Leu-Pro was used to identify B . subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation . One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat . SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding . Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.

Microbiology, 2003 Nov, 149(Pt 11), 3289 - 97
Ammonium utilization in Bacillus subtilis: transport and regulatory functions of NrgA and NrgB; Detsch C et al.; Bacillus subtilis uses glutamine as the best source of nitrogen . In the absence of glutamine, alternative nitrogen sources such as ammonium can be used . Ammonium utilization involves the uptake of the gas or the ammonium ion, the synthesis of glutamine by the glutamine synthetase and the recycling of the glutamate by the glutamate synthase . In this work, ammonium transport in B . subtilis was studied . At high ammonium concentrations, a large fraction of the ammonium is present as ammonia, which may enter the cell via diffusion . In contrast, the ammonium transporter NrgA is required for ammonium utilization at low concentrations or at low pH values when the equilibrium between uncharged ammonia and the ammonium ion is shifted towards ammonium . Moreover, a functional NrgA is essential for the transport of the ammonium analogue methylammonium . NrgA is encoded in the nrgAB operon . The product of the second gene, NrgB, is a member of the PII family of regulatory proteins . In contrast to PII proteins from other organisms, there is no indication for a covalent modification of NrgB in response to the nitrogen supply of the cell . It is demonstrated here that NrgB is localized at the membrane, most likely in association with the ammonium transporter NrgA . The presence of a functional NrgB is required for full-level expression of the nrgAB operon in response to nitrogen limitation, suggesting that NrgB might relay the information on ammonium availability to downstream regulatory factors and thus fine-tune their activity.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13603 - 8 Epub 2003 Nov 03.
Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis; Nakano S et al.; The Spx protein of Bacillus subtilis represses activator-stimulated transcription by interacting with the C-terminal domain of RNA polymerase (RNAP) alpha subunit . Its concentration increases in cells lacking the ATP-dependent protease, ClpXP, resulting in severe effects on growth and developmental processes . Microarray analysis was undertaken to identify genes that are induced or repressed when Spx interacts with RNAP . The induced genes included those encoding products known to function in maintaining thiol homeostasis . Two genes, thioredoxin (trxA) and thioredoxin reductase (trxB), are transcriptionally induced under conditions of thiol-specific oxidative (disulfide) stress by a mechanism involving Spx-RNAP interaction . Disulfide stress also results in an increase in Spx-dependent transcriptional repression . The increase in Spx activity in cells encountering disulfide stress is due in part to a posttranscriptional mechanism of spx control resulting in an increase in Spx concentration . An spx null mutant and a strain bearing an allele of rpoA that prevents Spx-RNAP interaction show hypersensitivity to disulfide stress . From these results, it is proposed that Spx is an activator that mobilizes the operations necessary to reverse the effects of oxidative damage, but it also serves as a negative regulator that causes the postponement of developmental programs and energy-consuming growth-related functions while the cell copes with the period of stress.

Genes Dev, 2003 Nov 1, 17(21), 2688 - 97
An mRNA structure in bacteria that controls gene expression by binding lysine; Sudarsan N et al.; Riboswitches are metabolite-responsive genetic control elements that reside in the untranslated regions (UTRs) of certain messenger RNAs . Herein, we report that the 5'-UTR of the lysC gene of Bacillus subtilis carries a conserved RNA element that serves as a lysine-responsive riboswitch . The ligand-binding domain of the riboswitch binds to L-lysine with an apparent dissociation constant (KD) of approximately 1 micro M, and exhibits a high level of molecular discrimination against closely related analogs, including D-lysine and ornithine . Furthermore, we provide evidence that this widespread class of riboswitches serves as a target for the antimetabolite S-(2-aminoethyl)-L-cysteine (AEC) . These findings add support to the hypotheses that direct sensing of metabolites by messenger RNAs is a fundamental form of genetic control and that riboswitches represent a new class of antimicrobial drug targets.

Gene, 2003 Nov 13, 319, 65 - 9
The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP-N-acetylglucosamine 4-epimerisation; Soldo B et al.; Mutations in the Bacillus subtilis gene that affect the activity of the uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 4-epimerase (EC 5.1.3.7) were shown to map to galE, the structural gene of the UDP-glucose (Glc) 4-epimerase (EC 5.1.3.2) . This genetic evidence that the same enzyme can catalyse the epimerisation of hexoses as well as of their N-acetylated forms is confirmed by in vitro assays with purified enzyme . It appears that in B . subtilis, Gne (GneA, GalE) is involved in two distinct and essential functions, i.e., cell detoxification under certain growth conditions and the biosynthesis of anionic cell wall polymers . We discuss the evidence that such enzymes capable of utilizing both UDP-hexoses and UDP-N-acetylhexosamines are present in other organisms.

Mol Biol Evol, 2004 Jan, 21(1), 108 - 16 Epub 2003 Oct 31.
An analysis of determinants of amino acids substitution rates in bacterial proteins; Rocha EP et al.; The variation of amino acid substitution rates in proteins depends on several variables . Among these, the protein's expression level, functional category, essentiality, or metabolic costs of its amino acid residues may play an important role . However, the relative importance of each variable has not yet been evaluated in comparative analyses . To this aim, we made regression analyses combining data available on these variables and on evolutionary rates, in two well-documented model bacteria, Escherichia coli and Bacillus subtilis . In both bacteria, the level of expression of the protein in the cell was by far the most important driving force constraining the amino acids substitution rate . Subsequent inclusion in the analysis of the other variables added little further information . Furthermore, when the rates of synonymous substitutions were included in the analysis of the E . coli data, only the variable expression levels remained statistically significant . The rate of nonsynonymous substitution was shown to correlate with expression levels independently of the rate of synonymous substitution . These results suggest an important direct influence of expression levels, or at least codon usage bias for translation optimization, on the rates of nonsynonymous substitutions in bacteria . They also indicate that when a control for this variable is included, essentiality plays no significant role in the rate of protein evolution in bacteria, as is the case in eukaryotes.

J Bacteriol, 2003 Nov, 185(22), 6728 - 31
In vivo effect of mutations at the PRPP binding site of the Bacillus subtilis purine repressor; Rappu P et al.; The Bacillus subtilis PurR mediates adenine repression and guanosine induction of purA . PRPP inhibits binding of PurR to DNA in vitro . Mutations in the PRPP binding motif of PurR caused strong repression regardless of purine exclusions or additions, establishing the role of PRPP as regulator of PurR.

J Bacteriol, 2003 Nov, 185(22), 6666 - 77
Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases; Yamamoto H et al.; LytF, LytE, and LytC are vegetative cell wall hydrolases in Bacillus subtilis . Immunofluorescence microscopy showed that an epitope-tagged LytF fusion protein (LytF-3xFLAG) in the wild-type background strain was localized at cell separation sites and one of the cell poles of rod-shaped cells during vegetative growth . However, in a mutant lacking both the cell surface protease WprA and the extracellular protease Epr, the fusion protein was observed at both cell poles in addition to cell separation sites . This suggests that LytF is potentially localized at cell separation sites and both cell poles during vegetative growth and that WprA and Epr are involved in LytF degradation . The localization pattern of LytE-3xFLAG was very similar to that of LytF-3xFLAG during vegetative growth . However, especially in the early vegetative growth phase, there was a remarkable difference between the shape of cells expressing LytE-3xFLAG and the shape of cells expressing LytF-3xFLAG . In the case of LytF-3xFLAG, it seemed that the signals in normal rod-shaped cells were stronger than those in long-chain cells . In contrast, the reverse was found in the case of LytE-3xFLAG . This difference may reflect the dependence on different sigma factors for gene expression . The results support and extend the previous finding that LytF and LytE are cell-separating enzymes . On the other hand, we observed that cells producing LytC-3xFLAG are uniformly coated with the fusion protein after the middle of the exponential growth phase, which supports the suggestion that LytC is a major autolysin that is not associated with cell separation.

J Biol Chem, 2004 Jan 16, 279(3), 1757 - 67 Epub 2003 Oct 30.
Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis; Le Chatelier E et al.; In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases . These are designated pol C and DnaE in Bacillus subtilis . We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis . The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5'-DNA end . DnaE is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication . Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases . These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 460 - 4
A novel extracellular protein of Streptomyces peucetius binds to daunorubicin but does not inhibit the bioactivity of the antibiotic; Prasad R et al.; Extracellular proteins of Streptomyces peucetius that bind to a red pigment were identified during the course of isolation of mutants defective in daunorubicin production . Two pigment-protein complexes were partially purified and this complex inhibited the growth of Bacillus subtilis . Routine solvent extraction could not separate the pigment from purified pigment-protein complex . Treatment with 2% SDS at 100 degrees C followed by solvent extraction also failed to separate the protein from the pigment . However, the pigment could be separated from the purified pigment-protein complex by heating it in 0.1M HCl at 100 degrees C followed by solvent extraction . The pigment extracted from the complex was analyzed by HPLC and found to be daunorubicin.

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 267 - 71
3-alkoxy-5-isoxazolidinones mimic beta-lactams; Cao X et al.; 3-Alkoxy-5-isoxazolidinones mimic the action of beta-lactams . They bind to the penicillin-binding proteins . They inhibit Class A, Class B, and Class D beta-lactams . They inhibit the growth of Bacillus subtilis . We give a novel synthesis for these compounds.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2245 - 53
Expression profiling of translation-associated genes in sporulating Bacillus subtilis and consequence of sporulation by gene inactivation; Ohashi Y et al.; A DNA microarray technique was used to demonstrate global changes in the transcription pattern of translation-associated genes that encode fifty-four ribosomal proteins including a putative ribosomal gene, and eleven translation factors in sporulating B . subtilis . We found that the mRNA levels of nine genes involved in the translation system, which include the genes for three ribosomal proteins (rpmA, rpmGB, and ctc) and two translation factors (efp, and frr), were maintained at a high level at the onset of sporulation . The ypfD gene, which encodes the ribosomal protein S1 homologue, was also found to be expressed significantly during the early sporulation stage . In order to demonstrate the significance of these genes for sporulation, mutants were constructed using the pMutinT3 disruption vector . We detected an impaired sporulation in the mutants of rpmA (gene for the ribosomal protein L27), efp (elongation factor P), frr (ribosome recycling factor), and ypfD . The effect was especially pronounced in the efp mutant, sporulation of which was entirely abolished without affecting growth . The reduced expression of rpmGB (ribosomal protein L33) resulted in an impaired sporulation only at a high temperature (47 degrees C) . Only the rplI mutant, which encodes the ribosomal protein L9, could not be obtained, implying that its function is essential for viability . Thus, we successfully demonstrated the significance of several translation-associated genes in sporulation by using the results of the gene expression profiling.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2154 - 9
Purification and identification of antimicrobial sesquiterpene lactones from yacon (Smallanthus sonchifolius) leaves; Lin F et al.; The extraction of yacon {Smallanthus sonchifolius (Poepp . and Endl.) H . Robinson; Asteraceae} leaves and chromatographic separation yielded two new antibacterial melampolide-type sesquiterpene lactones, 8beta-tigloyloxymelampolid-14-oic acid methyl ester and 8beta-methacryloyloxymelampolid-14-oic acid methyl ester, as well as the four known melampolides, sonchifolin, uvedalin, enhydrin and fluctuanin . The newly identified compound, 8beta-methacryloyloxymelampolid-14-oic acid methyl ester, exhibited potent antimicrobial activity against Bacillus subtilis and Pyricularia oryzae, while 8beta-tigloyloxymelampolid-14-oic acid methyl ester showed lower activity . Fluctuanin exhibited the strongest antibacterial activity against B . subtilis among these six sesquiterpene lactones.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2106 - 14
Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids; Oku H et al.; It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay . This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli . To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation . The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody . The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B . subtilis . Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS . However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer . Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity . This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.

J Biol Chem, 2004 Jan 23, 279(4), 2704 - 11 Epub 2003 Oct 28.
Three-dimensional structure of YaaE from Bacillus subtilis, a glutaminase implicated in pyridoxal-5'-phosphate biosynthesis; Bauer JA et al.; The structure of YaaE from Bacillus subtilis was determined at 2.5-A resolution . YaaE is a member of the triad glutamine aminotransferase family and functions in a recently identified alternate pathway for the biosynthesis of vitamin B(6) . Proposed active residues include conserved Cys-79, His-170, and Glu-172 . YaaE shows similarity to HisH, a glutaminase involved in histidine biosynthesis . YaaD associates with YaaE . A homology model of this protein was constructed . YaaD is predicted to be a (beta/alpha)(8) barrel on the basis of sequence comparisons . The predicted active site includes highly conserved residues 211-216 and 233-235 . Finally, a homology model of a putative YaaD-YaaE complex was prepared using the structure of HisH-F as a model . This model predicts that the ammonia molecule generated by YaaE is channeled through the center of the YaaD barrel to the putative YaaD active site.

Biotechnol Lett, 2003 Oct, 25(19), 1609 - 12
Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores; Hirose J et al.; A strain of B . subtilis produced 1.8 times more laccase on sporulation medium than on non-sporulation medium . Spores oxidized mono- and di-methoxyphenols (0.1 mM) at 50 degrees C . The half-life of laccase bound to spores was about 2 d and the substrate was repeatedly removed by spores recovered from the reaction mixture.

J Proteome Res, 2003 Sep-Oct, 2(5), 488 - 94
Quantitative metabolome analysis using capillary electrophoresis mass spectrometry; Soga T et al.; A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed . Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values . This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B . subtilis sporulation.

Toxicology, 2003 Nov 5, 192(2-3), 237 - 48
DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E; Abid-Essefi S et al.; Zearalenone (ZEN) is a non-steroidal oestrogenic mycotoxin produced by several Fusarium species growing on cereals . ZEN and its metabolites bind to human oestrogen receptors and hence display oestrogenic and anabolic properties . Several lines of investigation suggest that ZEN may be genotoxic in vivo . ZEN damages DNA in Bacillus subtilis recombination tests, and it induces sister chromatid exchange and chromosomal aberration in CHO cells . ZEN also induces DNA-adduct formation in mouse tissues and SOS repair process in lysogenic bacteria . In the present study, ZEN genotoxicity has been confirmed in three cell-lines, Vero, Caco-2 and DOK at concentrations of 10, 20 and 40 microM . Under these conditions, ZEN induces concentration-dependent DNA fragmentation resulting in DNA laddering patterns on agarose gel electrophoresis . This observation is consistent with apoptosis, which was confirmed by observations of formation of apoptotic bodies . Moreover, ZEN induces cell cycle arrest in the three cell-lines characterised by an increase of the number of cells in the G2/M phase of the cell cycle . Vitamin E (25 microM) added simultaneously with ZEN partially reduces DNA fragmentation and apoptotic body formation after 24h incubation . Vitamin E may act by maintaining prolonged cell cycle arrest during which time DNA repair takes place.

Biochemistry, 2003 Nov 4, 42(43), 12634 - 42
DNA-binding and oligomerization studies of the manganese(II) metalloregulatory protein MntR from Bacillus subtilis; Lieser SA et al.; The metalloregulatory protein MntR from Bacillus subtilis acts as a transcriptional regulator of manganese homeostasis . MntR is a member of a subfamily of DtxR-related proteins that perform analogous regulatory functions in a variety of pathogenic organisms . Metal ions activate MntR to bind DNA and repress the transcription of the mntH gene, which encodes for a proton-coupled metal ion transporter . Size-exclusion chromatography and sedimentation equilibrium ultracentrifugation studies show that apo MntR is predominantly a homodimer in solution . Using fluorescence anisotropy measurements, the DNA binding properties of MntR have been examined . In the strict absence of divalent transition metal ions MntR has a low affinity for the mntH control sequence (K(d) > 8.0 microM) . However, binding of MntR is stimulated by the presence of Mn(2+) and Cd(2+) to generate high affinity binding with K(d) values of 16.0 and 7.3 nM, respectively . MntR is also shown to bind the mntH control sequence in the presence of other divalent transition metals, including Ni(2+), Cu(2+), and Zn(2+), but with much lower affinity (K(d) approximately 1.3-2.3 microM) . The data here demonstrate that differences in metal-activated DNA binding plays a role in the mechanism of manganese(II)-selective transcription factors and that the oligomerization of MntR is metal-independent, which distinguishes this protein from iron(II)-responsive homologues in the DtxR protein family.

Nat Prod Res, 2003 Dec, 17(6), 389 - 95
Microbial transformation of cortisol and prolyl endopeptidase inhibitory activity of its transformed products; Choudhary MI et al.; Incubation of cortisol (1) with Gibberella fujikuruoi for 12 days yielded an oxidatively cleaved product, 11beta-hydroxyandrost-4-en-3,17-dione (2), while incubation with Bacillus subtilis and Rhizopus stolonifer yielded the reduced product, 11beta, 17alpha,20,21-tetrahydroxy-(20S)-pregn-4-en-3-one (3) . Other reduced products, 11beta, 17alpha, 21-trihydroxy-5alpha-pregnan-3, 20-dione (4) and 3beta, 11beta, 17alpha, 21-tetrahydroxy-5alpha-pregnan-20-one (5) were obtained by incubation of compound 1 with Bacillus cerus . The inhibitory activity of compounds 1-5 against prolyl endopeptidase enzyme (PEP) was also assayed . Compounds 2 (IC50 162.8 microM) and 4 (IC50 157 microM) have shown significant inhibitory activity against PEP.

Nucleic Acids Res . 2003 Nov 1;31(21):e133.
One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid; Tsuge K et al.; A universal method to reconstitute sets of genes was developed . Owing to the intrinsic nature of the plasmid establishment mechanism in Bacillus subtilis, the assembly of five antibiotic resistance genes with a defined order and orientation was achieved . These five fragments and the plasmid have three-base protruding sequences at both ends . The protruding sequences are designed so that each fragment is ligated once in a row according to the pairing . Ligation by T4 DNA ligase in the presence of 150 mM NaCl and 10% polyethylene glycol at 37 degrees C yielded high molecular tandem repeat linear form DNA . This multimeric form of DNA was preferentially used for plasmid establishment in B.subtilis . The method, referred to as Ordered Gene Assembly in B.subtilis (OGAB), allows for the design of multiple fragments with very high efficiency and great fidelity.

Genome Inform Ser Workshop Genome Inform, 2002, 13, 71 - 81
Origin of most primitive mRNAs and genetic codes via interactions between primitive tRNA ribozymes; Ohnishi K et al.; The origin and early evolution of genetic codon system and early mRNAs were analyzed from a viewpoint of primordial gene theory and the poly-tRNA theory . A hypothetical 25-amino acid (aa)-primordial peptide was deduced from internal aa-sequence homology of adenylate kinases . Theoretical models were made which can reasonably explain how primitive tRNA(s) could have had converted to be earliest mRNAs via interactions between presumptive anticodons and (poly-)tRNA ribozyme . Transfer-RNA gene clusters in the trrnD- and rrnB-operons of Bacillus subtilis seemed to be relics of early peptide-synthesizing RNA machine . Detailed analyses revealed that the poly-tRNA regions in these operons are true relics of RNA-machine for making a 16-aa trrnD-peptide and a 21-aa rrnB-peptide, whose aa sequences are in the order of aa specificities of tRNAs in the tRNA gene clusters of the trrnD-operon and rrnB-operon, respectively . The primordial gene-encoded peptide deduced from adenylate kinases were found to be a genuine homologue of the rrnB-peptide . Various protein superfamilies were found to have evolved from either of these two types of primitive peptides . Earliest mRNAs were concluded to have evolved from tRNAGly (trrnD-mRNA) or tRNAHis (rrnB-mRNA), where trrnD- and rrnB-mRNAs are hypothetical primitive mRNAs complementary to the tandem arrangement of 16 or 21 anticodons of tRNAs in the trrnD-operon and rrnB-operon, respectively . The poly-tRNA model is considered to be an excellent theory, because it can reasonably explain origins of both genetic codes and earliest mRNAs, and because the hypothesis can be statistically evaluated by base-identity levels in proper alignments . The genetic codon system is a typical mature semeiotic system within a cell, and the genesis of the genetic codon system was discussed from an aspect of de Saussure's semeiology . Arbitrary correspondence between (anti)codon and aa would be most plausibly a result of semeiotic culture system of intracellular tRNA-riboorganismic society.

Annu Rev Cell Dev Biol, 2003, 19, 565 - 87
Proteolysis in bacterial regulatory circuits; Gottesman S; Proteolysis by cytoplasmic, energy-dependent proteases plays a critical role in many regulatory circuits, keeping basal levels of regulatory proteins low and rapidly removing proteins when they are no longer needed . In bacteria, four families of energy-dependent proteases carry out degradation . In all of them, substrates are first recognized and bound by ATPase domains and then unfolded and translocated to a sequestered proteolytic chamber . Substrate selection depends not on ubiquitin but on intrinsic recognition signals within the proteins and, in some cases, on adaptor or effector proteins that participate in delivering the substrate to the protease . For some, the activity of these adaptors can be regulated, which results in regulated proteolysis . Recognition motifs for proteolysis are frequently found at the N and C termini of substrates . Proteolytic switches appear to be critical for cell cycle development in Caulobacter crescentus, for proper sporulation in Bacillus subtilis, and for the transition in and out of stationary phase in Escherichia coli . In eukaryotes, the same proteases are found in organelles, where they also play important roles.

Curr Microbiol, 2003 Sep, 47(3), 208 - 13
Effect of glutamate synthase (GOGAT) activity on Bacillus subtilis spore properties; Ruzal SM et al.; The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway . A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected . Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT) . The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity . Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media . However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium . In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP) . The content of these proteins was, in fact, reduced, in particular the SASP-gamma type . The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance . Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired . Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain . Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.

Exp Appl Acarol, 2002, 28(1-4), 127 - 34
Expression of defensin-like peptides in tick hemolymph and midgut in response to challenge with Borrelia burgdorferi, Escherichia coli and Bacillus subtilis; Sonenshine DE et al.; Challenge of Dermacentor variabilis by hemocoel injection with Borrelia burgdorferi but not Bacillus subtilis or Escherichia coli provoked secretion of two low molecular weight peptides into the hemolymph plasma; the lower band co-migrated with a band previously identified as varisin (a tick defensin) . These findings are consistent with reports that D . variabilis controls B . burgdorferi but not B . subtilis or E . coli by defensin-dependent bacteriolysis . Challenge of the tick midgut by capillary artificial feeding with bacteria also provoked expression of multiple low molecular weight peptides . In this case, however, all three bacteria elicited the response . Two bands, including the defensin-like peptide were expressed following challenge with B . subtilis and E . coli, but only the upper band following challenge with B . burgdorferi . Although they appeared intact, these spirochetes were no longer viable suggesting that borreliae in the midgut are controlled by a different method than the lytic response of the D . variabilis hemolymph . DD-RT-PCR revealed multiple mRNAs in the midgut of D . variabilis following challenge with B . burgdorferi, E . coli and Rickettsia montana . Although their identification remains to be determined, the large number of genes expressed in response to bacterial challenge presents intriguing possibilities for explaining the ability of the tick midgut to destroy invading microbes at the cellular level.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 149 - 56
Characterisation of preYvaY export reveals differences in the substrate specificities of Bacillus subtilis and Escherichia coli leader peptidases; Linde D et al.; Translocation, processing and secretion of YvaY, a Bacillus subtilis protein of unknown function, were characterised both in B . subtilis and in Escherichia coli . In its natural host B . subtilis, YvaY was transiently synthesised at the end of the exponential growth phase . It was efficiently secreted into the culture supernatant in spite of a calculated membrane spanning domain in the mature part of the protein . In E . coli, despite the high conservation of Sec-dependent transport components, processing of preYvaY was strongly impaired . To uncover which elements of E . coli and B . subtilis translocation systems are responsible for the observed substrate specificity, components of the B . subtilis Sec-system were co-expressed besides yvaY in E . coli . Expression of B . subtilis secA or secYEG genes did not affect processing, but expression of B . subtilis signal peptidase genes significantly enhanced processing of preYvaY in E . coli . While the major signal peptidases SipS or SipT had a strong stimulatory effect on preYvaY processing, the minor signal peptidases SipU, SipV or SipW had a far less stimulatory effect in E . coli . These results reveal that targeting and translocation of preYvaY is mediated by the E . coli Sec proteins but processing of preYvaY is not performed by E . coli signal peptidase LepB . Thus, differences in substrate specificities of E . coli LepB and the B . subtilis Sip proteins provide the bottleneck for export of YvaY in E . coli . Significant slower processing of preYvaY in absence of SecB indicated that SecB mediates targeting of the B . subtilis precursor.

Biochemistry, 2003 Oct 28, 42(42), 12430 - 8
Biosynthesis of the thiazole moiety of thiamin pyrophosphate (vitamin B1); Park JH et al.; While most of the proteins required for the biosynthesis of thiamin pyrophosphate have been known for more than a decade, the reconstitution of this biosynthesis in a defined biochemical system has been difficult due to the novelty of the chemistry involved . Here we demonstrate the first successful enzymatic synthesis of the thiazole moiety of thiamin from glycine, cysteine, and deoxy-D-xylulose-5-phosphate using overexpressed Bacillus subtilis ThiF, ThiS, ThiO, ThiG, and a NifS-like protein . This has facilitated the identification of the biochemical function of each of the proteins involved: ThiF catalyzes the adenylation of ThiS; NifS catalyzes the transfer of sulfur from cysteine to the acyl adenylate of ThiS; ThiO catalyzes the oxidation of glycine to the corresponding imine; and ThiG catalyzes the formation of the thiazole phosphate ring . The complex oxidative cyclization reaction involved in the biosynthesis of the thiamin thiazole has been greatly simplified by replacing ThiF, ThiS, ThiO, and NifS with defined biosynthetic intermediates in a reaction where ThiG is the only required enzyme.

J Zhejiang Univ Sci, 2003 Nov-Dec, 4(6), 719 - 26
Optimization of cultural conditions for thermostable beta-1,3-1,4-glucanase production by Bacillus subtilis ZJF-1A5; He GQ et al.; The optimization of cultural conditions for Beta-glucanase production by Bacillus subtilis ZJF-1A5 was investigated in flask trials . Temperature had great effect on Beta-glucanase production which maximized at optimal temperature of 37 degrees C and decreased significantly when temperature was over 37 degrees C.Charge quantity affected Beta-glucanase production significantly . Adding oxygen vector N-dodecane or acetic ether benefited Beta-glucanase production, but it depended on the concentration and charge quantity . The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting Beta-glucanase production and the cultivation time span to reach the highest Beta-glucanase activity . The optimal cultural conditions for Beta-glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 210 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37 degrees C for 50 h . Repeated experimental results accorded with those predicted by a second-order polynomial model . The amount of Beta-glucanase, Alpha-amylase and neutral protease produced by B subtilis ZJF-1A5 was associated partially with cell growth . Those three enzymes' activities increased following the cell growth and increased significantly when cells entered the stationary phase.

Nucleosides Nucleotides Nucleic Acids, 2003 May-Aug, 22(5-8), 1535 - 8
Intersubunit interactions in human cytidine deaminase; Vincenzetti S et al.; In order to design new efficient cytidine based drugs, an intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F . F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions . In presence of the dissociating agent SDS, wild-type human CDA dissociate into enzymatically inactive monomers without intermediate forms via a non-cooperative transition . Extensive dialysis or dilution of the inactivated monomers restores completely the activity . The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavour dissociation of the tetramer into subunits in the wild-type CDA but not in mutant enzyme F137W/W113F.

J Bacteriol, 2003 Nov, 185(21), 6425 - 33
Isolation and characterization of mutants of the Bacillus subtilis oligopeptide permease with altered specificity of oligopeptide transport; Solomon J et al.; Bacterial oligopeptide permeases are members of the large family of ATP binding cassette transporters and typically import peptides of 3 to 5 amino acids, apparently independently of sequence . Oligopeptide permeases are needed for bacteria to utilize peptides as nutrient sources and are sometimes involved in signal transduction pathways . The Bacillus subtilis oligopeptide permease stimulates competence development and the initiation of sporulation, at least in part, by importing specific signaling peptides . We isolated rare, partly functional mutations in B . subtilis opp . The mutants were resistant to a toxic tripeptide but still retained the ability to sporulate and/or become competent . The mutations, mostly in the oligopeptide binding protein located on the cell surface, affected residues whose alteration appears to change the specificity of oligopeptide transport.

J Bacteriol, 2003 Nov, 185(21), 6358 - 70
Genome-wide transcriptional profiling analysis of adaptation of Bacillus subtilis to high salinity; Steil L et al.; The gram-positive soil bacterium Bacillus subtilis often faces increases in the salinity in its natural habitats . A transcriptional profiling approach was utilized to investigate both the initial reaction to a sudden increase in salinity elicited by the addition of 0.4 M NaCl and the cellular adaptation reactions to prolonged growth at high salinity (1.2 M NaCl) . Following salt shock, a sigB mutant displayed immediate and transient induction and repression of 75 and 51 genes, respectively . Continuous propagation of this strain in the presence of 1.2 M NaCl triggered the induction of 123 genes and led to the repression of 101 genes . In summary, our studies revealed (i) an immediate and transient induction of the SigW regulon following salt shock, (ii) a role of the DegS/DegU two-component system in sensing high salinity, (iii) a high-salinity-mediated iron limitation, and (iv) a repression of chemotaxis and motility genes by high salinity, causing severe impairment of the swarming capability of B . subtilis cells . Initial adaptation to salt shock and continuous growth at high salinity share only a limited set of induced and repressed genes . This finding strongly suggests that these two phases of adaptation require distinctively different physiological adaptation reactions by the B . subtilis cell . The large portion of genes with unassigned functions among the high-salinity-induced or -repressed genes demonstrates that major aspects of the cellular adaptation of B . subtilis to high salinity are unexplored so far.

J Bacteriol, 2003 Nov, 185(21), 6348 - 57
Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis; Fuangthong M et al.; Bacillus subtilis contains three Fur homologs: Fur, PerR, and Zur . Despite significant sequence similarities, they respond to different stimuli and regulate different sets of genes . DNA target site comparisons indicate that all three paralogs recognize operators with a core 7-1-7 inverted repeat . The corresponding consensus sequences are identical at five or more of the seven defined positions . Using site-directed mutagenesis, the Per box at the mrgA promoter was altered to mimic the core 7-1-7 motif of the Fur and Zur boxes . In vitro, the mrgA promoter containing a Zur box was only recognized by Zur, as demonstrated by DNase I footprinting assays . In contrast, both Fur and PerR bound to the mrgA promoter region containing a consensus Fur box . Expression analysis of these promoters is consistent with the in vitro data demonstrating as few as 1 or 2 base changes per half-site are sufficient to alter regulation . Similarly, the Fur box at the feuA promoter can be converted into a Per or a Zur box by appropriate mutations . While both Fur and PerR could recognize some of the same synthetic operator sequences, no naturally occurring sites are known that are subject to dual regulation . However, the PerR-regulated zosA gene is controlled from a regulatory region that contains both Per and Fur boxes . Although purified Fur protein bound to the candidate Fur boxes, Fur has little effect on zosA expression-possibly due to the location of the Fur boxes relative to the zosA promoter . Together, our results identify two nucleotide positions that are important for the ability of PerR, Fur, and Zur to distinguish among the many closely related operator sites present in the B . subtilis genome.

J Bacteriol, 2003 Nov, 185(21), 6316 - 24
Increasing the ratio of Soj to Spo0J promotes replication initiation in Bacillus subtilis; Ogura Y et al.; The ParA and ParB protein families are well conserved in bacteria . However, their functions are still unclear . In Bacillus subtilis, Soj and Spo0J are members of these two protein families, respectively . A previous report revealed that replication initiated early and asynchronously in spo0J null mutant cells, as determined by flow cytometry . In this study, we examined the cause of this promotion of replication initiation . Deletion of both the soj and spo0J genes restored the frequency of replication initiation to almost the wild-type level, suggesting that production of Soj in the absence of Spo0J leads to early and asynchronous initiation of replication . Consistent with this suggestion, overproduction of Soj in wild-type cells had the same effect on replication initiation as in the spo0J null mutant, and overproduction of both Soj and Spo0J did not . These results indicate that when the ratio of Soj to Spo0J increases, Soj interferes with tight control of replication initiation and causes early and asynchronous initiation . Whereas replication initiation also occurred significantly earlier in the two spo0J mutants, spo0J14 and spo0J17, it occurred only slightly early in the sojK16Q mutant and was delayed in the sojG12V mutant . Although Soj localized to nucleoids in the spo0J mutants, the two Soj mutant proteins were distributed throughout the cell or localized to cell poles . Thus, interestingly, the promotion of replication initiation seems to correlate with localization of Soj to nucleoids . This may suggest that Soj inhibits transcription of some cell cycle genes and leads to early and asynchronous initiation of replication . In wild-type cells Spo0J counteracts this Soj function.

Biochem Biophys Res Commun, 2003 Oct 31, 310(4), 1148 - 54
The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco; Yip W et al.; Phytate, the main form of phosphorus storage in plant seeds, is well known to be an anti-nutrient and a major source of phosphorus pollution in animal manure . To improve phosphorus bio-availability, we introduced a recently characterized phytase from Bacillus subtilis into the cytoplasm of tobacco cells . Although the introduction of acid fungal phytase from Aspergillus niger in previous studies did not result in any phenotypic changes in tobacco, here we show that a tobacco line transformed with a neutral phytase exhibited phenotypic changes in flowering, seed development, and response to phosphate deficiency . The transgenic line showed an increase in flower and fruit numbers, small seed syndrome, lower seed IP6/IP5 ratio, and enhanced growth under phosphate-starvation conditions compared with the wildtype . The results suggest that the over-expression of Bacillus phytase in the cytoplasm of tobacco cells shifts the equilibrium of the inositol phosphate biosynthesis pathway, thereby making more phosphate available for primary metabolism . The approach presented here can be applied as a strategy for boosting productivity in agriculture and horticulture.

Biochem Biophys Res Commun, 2003 Oct 31, 310(4), 1096 - 103
The impact of single cysteine residue mutations on the replication terminator protein; Vivian JP et al.; We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies . The cysteine residue 110 was replaced with alanine, valine or serine . Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP . The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity . The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation . Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation . The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.

J Biol Chem, 2004 Jan 23, 279(4), 2809 - 16 Epub 2003 Oct 13.
The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily; Vincent F et al.; The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides . It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/) . Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A . The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain . The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer . Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis . PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein . We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair . We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism.

J Biol Chem, 2003 Dec 26, 278(52), 52146 - 53 Epub 2003 Oct 10.
Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase; Soundar S et al.; Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma . Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation . A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y . O., Koh, H . J., Kim, S . H., Jo, S . H., Huh, J . W., Jeong, K . S., Lee, I . J., Song, B . J., and Huh, T . L . (1999) J . Biol . Chem . 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E . coli . Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa . Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes . In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis . For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP . In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP . These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation . In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme . This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.

Science, 2003 Oct 10, 302(5643), 286 - 90
A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO; Ashida H et al.; The genomes of several nonphotosynthetic bacteria, such as Bacillus subtilis, and some Archaea include genes for proteins with sequence homology to the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) . We found that such a RuBisCO-like protein (RLP) from B . subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway . A growth-defective mutant, in which the gene for this RLP had been disrupted, was rescued by the gene for RuBisCOfrom the photosynthetic bacterium Rhodospirillum rubrum . Thus, the photosynthetic RuBisCO from R . rubrum retains the ability to function in the methionine salvage pathway in B . subtilis.

Biotechnol Adv, 1992, 10(3), 355 - 78
Transport regulation of recombinant gene expression in E . coli and B . subtilis; Boyer JD et al.; Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions . A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy . The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure . While there are many similarities in the influence of transport on both regulating models, there are also important differences . In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme . Nevertheless, transport regulation is an important feature in both instances.

Bioinformatics . 2003 Oct;19 Suppl 2:II15.
Metabolic economics and microbial proteome evolution; Akashi H; Natural selection is thought to act upon protein structures to optimize biochemical properties related to their specific cellular function . Selection pressures related to efficient synthesis, rather than proper function, of proteins may act globally on the amino acid composition of the proteome, but are less firmly established . A substantial fraction of bacterial energy budgets are devoted to biosynthesis of amino acids, the building blocks of proteins . The fueling reactions of central metabolism provide precursor metabolites for synthesis of amino acids . Thus, synthesis of an amino acid entails a dual cost; energy is lost by diverting chemical intermediates from fueling reactions and additional energy is required to convert precursor metabolites to amino acids . Selection to reduce the energetic costs predicts increases in the abundance of less energetically costly amino acids in highly expressed proteins . Using synonymous codon usage bias as a measure of translation rates, we show that amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis reflects the action of natural selection to enhance metabolic efficiency . The primary structures of proteins may also reflect natural selection to enhance the rate and accuracy of their synthesis . Differences in cellular concentrations of tRNAs could lead to translation selection both within and among synonymous families . In yeast, usage of several amino acids show striking associations with gene expression . These changes in amino acid composition result in stronger correlations between amino acid usage and tRNA abundances in highly expressed genes than in less expressed loci . Proteome-wide patterns of amino acid composition in microbes appears to reflect natural selection to enhance the overall physiology of cells as well as the specific functions of proteins . Contact: akashi@psu.edu http://www.bio.psu.edu/people/faculty/akashi/

J Clin Microbiol, 2003 Oct, 41(10), 4755 - 7
Microwave sterilization of femoral head allograft; Dunsmuir RA et al.; The potential shortage of allograft bone has led to the need to investigate other sources of bone for allografts . Some allograft bone donated from primary total hip arthroplasty recipients must be discarded or treated to become usable as a result of bacterial contamination . Femoral head allografts were contaminated with Staphylococcus aureus and Bacillus subtilis . A domestic microwave oven was used . The contaminated bone was exposed to microwave irradiation for different time periods . The samples were then cultured to attempt to grow the two bacterial species . The contaminated bone samples failed to grow any organisms after 2 min of exposure to microwave irradiation . This study shows that sterilization of femoral head allografts contaminated with S . aureus and B . subtilis can be achieved with microwave irradiation in a domestic microwave oven . This method of sterilization of bone allografts is cheap, easily used, and an effective way to process contaminated bone.

Appl Environ Microbiol, 2003 Oct, 69(10), 6307 - 10
Effects of minerals on resistance of Bacillus subtilis spores to heat and hydrostatic pressure; Igura N et al.; Among Bacillus subtilis IFO13722 spores sporulated at 30, 37, and 44 degrees C, those sporulated at 30 degrees C had the highest resistance to treatments with high hydrostatic pressure (100 to 300 MPa, 55 degrees C, 30 min) . Pressure resistance increased after demineralization of the spores and decreased after remineralization of the spores with Ca(2+) or Mg(2+), whereas the resistance did not change when spores were remineralized with Mn(2+) or K(+), suggesting that former two divalent ions were involved in the activation of cortex-lytic enzymes during germination.

Comb Chem High Throughput Screen, 2003 Sep, 6(6), 557 - 67
"Whole cell"--matrix-assisted laser desorption ionization-time of flight-mass spectrometry, an emerging technique for efficient screening of biocombinatorial libraries of natural compounds-present state of research; Vater J et al.; Whole Cell-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) is an emerging sensitive technique for rapid typing of microorganisms, efficient screening of biocombinatorial libraries of natural compounds and the analysis of complex biological samples, as whole cells, subcellular particles, cell extracts and culture filtrates . It is unique to detect metabolites in-situ without the need to isolate and purify the investigated compounds . In favourite cases it enables in-situ structure analysis on the basis of the fragment pattern generated by postsource MALDI-TOF-mass spectrometry . The state of research of this methodology which has mainly been obtained by investigation of lipopeptides from bacilli and the large spectrum of bioactive peptides produced by cyanobacteria is reviewed . The potential of this innovative technique is demonstrated for the lipopeptides produced by various Bacillus subtilis strains.

J Biol Chem, 2003 Dec 19, 278(51), 51108 - 15 Epub 2003 Oct 03.
Control of the Bacillus subtilis antiterminator protein GlcT by phosphorylation . Elucidation of the phosphorylation chain leading to inactivation of GlcT; Schmalisch MH et al.; Bacillus subtilis transports glucose by the phosphotransferase system (PTS) . The genes for this system are encoded in the ptsGHI operon, which is induced by glucose and depends on a termination/antitermination mechanism involving a riboswitch and the RNA-binding antitermination protein GlcT . In the absence of glucose, GlcT is inactive, and a terminator is formed in the leader region of the ptsG mRNA . If glucose is present, GlcT can bind to its RNA target and prevent transcription termination . The GlcT protein is composed of three domains, an N-terminal RNA binding domain and two PTS regulation domains, PTS regulation domain (PRD) I and PRD-II . In this work, we demonstrate that GlcT can be phosphorylated by two PTS proteins, HPr and the glucose-specific enzyme II (EIIGlc) . HPr-dependent phosphorylation occurs on PRD-II and has a slight stimulatory effect on GlcT activity . In contrast, EIIGlc phosphorylates the PRD-I of GlcT, and this phosphorylation inactivates GlcT . This latter phosphorylation event links the availability of glucose to the expression of the ptsGHI operon via the phosphorylation state of EIIGlc and GlcT . This is the first in vitro demonstration of a direct phosphorylation of an antiterminator of the BglG family by the corresponding PTS permease.

J Bacteriol, 2003 Oct, 185(20), 6095 - 103
Effects of carboxy-terminal modifications and pH on binding of a Bacillus subtilis small, acid-soluble spore protein to DNA; Kosman J et al.; Variants of the wild-type Bacillus subtilis alpha/beta-type small, acid-soluble spore protein (SASP) SspC(wt) were designed to evaluate the contribution of C-terminal residues to these proteins' affinity for DNA . SspC variants lacking one to three C-terminal residues were similar to SspC(wt) in DNA binding, but removal of six C-terminal residues greatly decreased DNA binding . In contrast, a C-terminal extension of three residues increased SspC's affinity for DNA 5- to 10-fold . C-terminal and N-terminal changes that independently caused large increases in SspC-DNA binding affinity were combined and produced an additive effect on DNA binding; the affinity of the resulting variant, SspC(DeltaN11-D13K-C3), for DNA was increased >/==" BORDER="0">20-fold over that of SspC(wt) . For most of the SspC variants tested, lowering the pH from 7 to 6 improved DNA binding two- to sixfold, although the opposite effect was observed with variants having additional C-terminal basic residues . In vitro, the binding of SspC(DeltaN11-D13K-C3) to DNA suppressed the formation of cyclobutane-type thymine dimers and promoted the formation of the spore photoproduct upon UV irradiation to the same degree as the binding of SspC(wt) . However, B . subtilis spores lacking major alpha/beta-type SASP and overexpressing SspC(DeltaN11-D13K-C3) had a 10-fold-lower viability and far less UV and heat resistance than spores overexpressing SspC(wt) . This apparent lack of DNA protection by SspC(DeltaN11-D13K-C3) in vivo is likely due to the twofold-lower level of this protein in spores compared to the level of SspC(wt), perhaps because of effects of SspC(DeltaN11-D13K-C3) on gene expression in the forespore during sporulation . The latter results indicate that only moderately strong binding of alpha/beta-type SASP to DNA is important to balance the potentially conflicting requirements for these proteins in DNA transcription and DNA protection during spore formation, spore dormancy, and spore germination and outgrowth.

J Bacteriol, 2003 Oct, 185(20), 6051 - 6
A second PDZ-containing serine protease contributes to activation of the sporulation transcription factor sigmaK in Bacillus subtilis; Pan Q et al.; Gene expression late during the process of sporulation in Bacillus subtilis is governed by a multistep, signal transduction pathway involving the transcription factor sigma(K), which is derived by regulated proteolysis from the inactive proprotein pro-sigma(K) . Processing of pro-sigma(K) is triggered by a signaling protein known as SpoIVB, a serine protease that contains a region with similarity to the PDZ family of protein-protein interaction domains . Here we report the discovery of a second PDZ-containing serine protease called CtpB that contributes to the activation of the pro-sigma(K) processing pathway . CtpB is a sporulation-specific, carboxyl-terminal processing protease and shares several features with SpoIVB . We propose that CtpB acts to fine-tune the regulation of pro-sigma(K) processing, and we discuss possible models by which CtpB influences the sigma(K) activation pathway.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12015 - 20 Epub 2003 Oct 01.
The near attack conformation approach to the study of the chorismate to prephenate reaction; Hur S et al.; Standard free energies (DeltaGN degree) for formation of near attack conformers, those ground state conformers that can convert directly to the transition state, were calculated for the Claisen rearrangement of chorismate to prephenate in six different environments: water, wild-type enzymes from Bacillus subtilis and Escherichia coli, their Arg90Cit and Glu52Ala mutants, and the 1F7 catalytic antibody . Values of the calculated DeltaGN degrees and the experimentally determined activation energies (DeltaG++) are linearly related with the slope of approximately equal to 1 . This demonstrates that the relative rate of the chorismate --> prephenate reaction is overwhelmingly dependent on the efficiency of formation of near attack conformers in the ground state.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12057 - 62 Epub 2003 Oct 01.
The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes; Grundy FJ et al.; Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available . Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription . In this study we show that lysine directly promotes transcription termination during in vitro transcription with B . subtilis RNA polymerase and causes a structural shift in the lysC leader RNA . We find that B . subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements . By analogy with related regulatory systems, we designate this leader RNA pattern the "L box." Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination . Mutations of B . subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro . The identification of the L box pattern also provides an explanation for previously described mutations in both B . subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine . The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.

Microbiology, 2003 Oct, 149(Pt 10), 3023 - 34
Identification of sporulation genes by genome-wide analysis of the sigmaE regulon of Bacillus subtilis; Feucht A et al.; Differentiation in the spore-forming bacterium Bacillus subtilis is governed by the sequential activation of five sporulation-specific transcription factors . The early mother-cell-specific transcription factor, sigma(E), directs the transcription of many genes that contribute to the formation of mature, dormant spores . In this study, DNA microarrays were used to identify genes belonging to the sigma(E) regulon . In total, 171 genes were found to be under the control of sigma(E) . Of these, 101 genes had not previously been described as being sigma(E) dependent . Disruption of some of the previously unknown genes (ydcC, yhaL, yhbH, yjaV and yqfD) resulted in a defect in sporulation.

Microbiology, 2003 Oct, 149(Pt 10), 3011 - 21
Bacillus subtilis spoVIF (yjcC) gene, involved in coat assembly and spore resistance; Kuwana R et al.; In systematic screening four sporulation-specific genes, yjcA, yjcB, yjcZ and yjcC, of unknown function were found in Bacillus subtilis . These genes are located just upstream of the cotVWXYZ gene cluster oriented in the opposite direction . Northern blot analysis showed that yjcA was transcribed by the SigE RNA polymerase beginning 2 h (t(2)) after the onset of sporulation, and yjcB, yjcZ and yjcC were transcribed by the SigK RNA polymerase beginning at t(4) of sporulation . The transcription of yjcZ was dependent on SigK and GerE . The consensus sequences of the appropriate sigma factors were found upstream of each gene . There were putative GerE-binding sites upstream of yjcZ . Insertional inactivation of the yjcC gene resulted in a reduction in resistance of the mutant spores to lysozyme and heat . Transmission electron microscopic examination of yjcC spores revealed a defect of sporulation at stage VI, resulting in loss of spore coats . These results suggest that YjcC is involved in assembly of spore coat proteins that have roles in lysozyme resistance . It is proposed that yjcC should be renamed as spoVIF.

Microbiology, 2003 Oct, 149(Pt 10), 3001 - 9
The regulatory link between carbon and nitrogen metabolism in Bacillus subtilis: regulation of the gltAB operon by the catabolite control protein CcpA; Wacker I et al.; Bacillus subtilis assimilates ammonium by the concerted action of glutamine synthetase and glutamate synthase . The expression of the gltAB operon encoding the latter enzyme is impaired in B . subtilis ccpA mutant strains . CcpA is a pleiotropic transcriptional regulator that is the key factor in the regulation of carbon metabolism . However, in addition to their defect in catabolite repression ccpA mutants are unable to grow on minimal media with glucose and ammonium as the single sources of carbon and nitrogen, respectively . In this work, the expression of the gltAB operon was analysed and its role in growth on minimal sugar/ammonium media was studied . Expression of gltAB requires induction by glucose or other glycolytically catabolized carbon sources . In ccpA mutants, gltAB cannot be induced by glucose due to the low activity of the phosphotransferase sugar transport system in these mutants . A mutation that allowed phosphotransferase system activity in a ccpA background simultaneously restored glucose induction of gltAB and growth on glucose/ammonium medium . Moreover, artificial induction of the gltAB operon in the ccpA mutant allowed the mutant strain to grow on minimal medium with glucose and ammonium . It may be concluded that expression of the gltAB operon depends on the accumulation of glycolytic intermediates which cannot occur in the ccpA mutant . The lack of gltAB induction is the bottleneck that prevents growth of the ccpA mutant on glucose/ammonium media . The control of expression of the gltAB operon by CcpA provides a major regulatory link between carbon and amino acid metabolism.

Nat Struct Biol, 2003 Nov, 10(11), 935 - 41 Epub 2003 Sep 28.
Structural framework of fructosyl transfer in Bacillus subtilis levansucrase; Meng G et al.; Many bacteria and about 40,000 plant species form primary carbohydrate reserves based on fructan; these polymers of beta-D-fructofuranose are thought to confer tolerance to drought and frost in plants . Microbial fructan, the beta(2,6)-linked levan, is synthesized directly from sucrose by levansucrase, which is able to catalyze both sucrose hydrolysis and levan polymerization . The crystal structure of Bacillus subtilis levansucrase, determined to a resolution of 1.5 A, shows a rare five-fold beta-propeller topology with a deep, negatively charged central pocket . Arg360, a residue essential for polymerase activity, lies in a solvent-exposed site adjacent to the central pocket . Mutagenesis data and the sucrose-bound structure of inactive levansucrase E342A, at a resolution of 2.1 A, strongly suggest that three conserved acidic side chains in the central pocket are critical for catalysis, and presumably function as nucleophile (Asp86) and general acid (Glu342), or stabilize the transition state (Asp247).

J Mol Biol, 2003 Oct 10, 333(1), 21 - 31
Interaction of a putative transcriptional regulatory protein and the thermo-inducible cts-52 mutant repressor in the Bacillus subtilis phage phi105 genome; Chan AY et al.; A 144 amino acid residue cts-52 mutant repressor (mtc phi 105) located in the EcoRI-F immunity region (immF) of Bacillus subtilis phage phi 105 is involved in the control mechanism of a thermo-inducible expression system . Adjacent to the repressor gene, an open-reading frame, designated ORF4, encodes a polypeptide of 90 amino acid residues, which shares a 37% homology with the amino acid sequence of the repressor . On the basis of the protein sequence alignment, a DNA-binding alpha helix-beta turn-alpha helix (HTH) motif was identified in the N-terminal region (residues 18-37) of the repressor as well as in the polypeptide of ORF4 (residues 22-41) . In vivo expression of the mutant repressor and ORF4 were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis . To study their DNA binding properties, the wild-type repressor (wtc phi 105) and the mutant repressor mtc phi 105, which has a Thr17 to Ile substitution, were overexpressed in Escherichia coli and purified for affinity assays . Their affinities towards six operator sites at various temperatures were elucidated by surface plasmon resonance (SPR) . Our data showed that a temperature shift does not influence the wtc phi 105-operators' binding affinity, while the binding of mtc phi 105 to the operators was temperature sensitive . This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression . Interestingly, mtc phi 105 and ORF4 demonstrated a large affinity discrepancy towards individual operators at different temperatures . mRNA levels monitored by real-time RT-PCR indicated a suppression of mtc phi 105 expression, but a stimulation of ORF4 transcription after thermo-induction . Our data suggested that ORF4 might be a counter protein to the phage repressor in the modulation of the two divergent-oriented promoters P(M) and P(R) within the immF region.

J Biol Chem, 2003 Dec 19, 278(51), 50978 - 84 Epub 2003 Sep 26.
A fraction of the BglG transcriptional antiterminator from Escherichia coli exists as a compact monomer; Fux L et al.; Expression of the bgl operon in Escherichia coli, induced by beta-glucosides, is positively regulated by BglG, a transcriptional antiterminator . In the presence of inducer, BglG dimerizes and binds to the bgl transcript to prevent premature termination of transcription . The dimeric state of BglG is determined by BglF, a membrane-bound enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which reversibly phosphorylates BglG according to beta-glucoside availability . BglG is composed of an RNA-binding domain followed by two homologous PTS regulation domains (PRD1 and PRD2) . The predicted structure of dimeric LicT, a BglG homologue from Bacillus subtilis, suggests that the two PRDs adopt a similar structure and that the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2 . We have shown recently that the PRD1 and PRD2 domains of BglG can form a stable heterodimer . We report here, based on in vitro and in vivo cross-linking experiments, that a fraction of BglG is present in the cell in a compact form in which PRD1 and PRD2 are in close proximity . The compact form is present mainly in the BglG monomers . Our results imply that the monomer-dimer transition involves a conformational change . The possible role of the compact form in preventing untimely induction of the bgl operon is discussed.

J Biol Chem, 2003 Dec 12, 278(50), 50506 - 13 Epub 2003 Sep 27.
Structural basis for the function of the N-terminal domain of the ATPase CopA from Bacillus subtilis; Banci L et al.; The solution structure of the N-terminal region (151 amino acids) of a copper ATPase, CopA, from Bacillus subtilis, is reported here . It consists of two domains, CopAa and CopAb, linked by two amino acids . It is found that the two domains, which had already been separately characterized, interact one to the other through a hydrogen bond network and a few hydrophobic interactions, forming a single rigid body . The two metal binding sites are far from one another, and the short link between the domains prevents them from interacting . This and the surface electrostatic potential suggest that each domain receives copper from the copper chaperone, CopZ, independently and transfers it to the membrane binding site of CopA . The affinity constants of silver(I) and copper(I) are similar for the two sites as monitored by NMR . Because the present construct "domain-short link-domain" is shared also by the last two domains of the eukaryotic copper ATPases and several residues at the interface between the two domains are conserved, the conclusions of the present study have general validity for the understanding of the function of copper ATPases.

J Biomol NMR, 2003 Dec, 27(4), 323 - 39
Solid state NMR sequential resonance assignments and conformational analysis of the 2x10.4 kDa dimeric form of the Bacillus subtilis protein Crh; Bockmann A et al.; Solid state NMR sample preparation and resonance assignments of the U-{13C,15N} 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented . Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts . For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR . Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography . For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder . Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

J Insect Physiol, 2003 Oct, 49(10), 945 - 54
Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae); Dani MP et al.; Venom from the endoparasitic wasp, Pimpla hypochondriaca, is composed of a mixture of high and low molecular weight proteins, possesses phenoloxidase activity, has immunosuppressive properties, and induces paralysis in several insect species . In the present study we demonstrate that P . hypochondriaca venom also contains antibacterial and proteolytic activity . Antibacterial activity was detected against the Gram-negative bacteria Escherichia coli and Xanthamonas campestris but not against Pseudomonas syringae nor against two Gram-positive bacteria, Bacillus cereus and Bacillus subtilis . Endopeptidase and aminopeptidase activity in venom was detected using the synthetic fluorogenic substrates N-t-BOC-Phe-Ser-Arg-AMC, Arg-AMC and Leu-Arg . The aminopeptidase activity towards Arg-AMC was sensitive to amastatin (70% inhibition), an aminopeptidase inhibitor . Angiotensin-converting enzyme (ACE)-like enzyme activity was detected, by reverse-phase HPLC using the synthetic tripeptide Hip-His-Leu as a substrate . This activity was sensitive to captopril, an ACE inhibitor (IC(50) 3.8 x 10(-8) M) . Using an antiserum raised against recombinant Drosophila melanogaster ACE-like enzyme, (rAnce), Western blot analysis revealed an immunoreactive protein, with a molecular weight estimate of 74 kDa, in P . hypochondriaca venom . The possibility that the endopeptidase, aminopeptidase and ACE are involved in the processing of peptide precursors in the venom sac is discussed.

J Nat Prod, 2003 Sep, 66(9), 1236 - 8
Hyperatomarin, an antibacterial prenylated phloroglucinol from Hypericumatomarium ssp . degenii; Savikin-Fodulovic K et al.; As shown by quantitative (1)H NMR measurements, a lipophilic extract of the aerial parts of Hypericum atomarium ssp . degenii contained a high percentage (3.1% per weight of dried plant material) of a prenylated phloroglucinol (1) . Compound 1, named hyperatomarin, occurring in two tautomeric forms (1a <==> 1b), was isolated by bioactivity-guided preparative TLC and was identified on the basis of spectral data interpretation . This isolated phloroglucinol exhibited activity against Gram-positive (Staphyloccocus aureus and Microccocus luteus) and Gram-positive spore-forming bacteria (Bacillus subtilis and B . IP 5832).

Nucleic Acids Res Suppl, 2003, (3), 295 - 6
Application of recombination transfer to the cognate Bacillus subtilis genome; Tomita S et al.; Isolation of the designated genome region of Bacillus subtilis was investigated using a B . subtilis recombinational transfer (BReT) system . Two DNA sequences flanking the precise genome region are cloned in the BReT vector . The BReT plasmid recovered the predicted genome sequence as large as 100 kb with high fidelity . The result indicates that the BReT system originally developed to recover the non-cognate segments cloned in the B . subtilis genome vector can be applied to the cognate sequence.

Nucleic Acids Res Suppl, 2003, (3), 293 - 4
Bacterial ribonuclease P reaction is affected by substrate shape and magnesium ion concentration; Ando T et al.; Bacterial RNase P is a ribonucleoprotein enzyme which cleaves 5'-precursor sequence of pre-tRNA for pre-tRNA maturation . The RNA component of bacterial RNase P is ribozyme . It recognizes cloverleaf shaped pre-tRNA and hairpin RNA with a CCA-3' tag sequence as its substrates . Previously, we reported that the substrate recognition of the E . coli RNase P ribozyme depends on the concentration of magnesium ion in vitro . In this report, we examined the substrate shape preference of the Bacillus subtilis RNase P ribozyme and compared it with that of the E . coli ribozyme . The results of the B . subtilis ribozyme displayed same tendency as the E . coli ribozyme . We also examined the effect of the protein component of the E . coli RNase P . Under the conditions tested, magnesium ion concentration dependency to substrate shape recognition was not observed when the holo enzyme was used.

Nucleic Acids Res Suppl, 2003, (3), 287 - 8
In vitro analysis of the initial steps of trans-translation; Takada K et al.; tmRNA has a dual function both as tRNA and mRNA and facilitates trans-translation . We observed the tagging derived from tmRNA from Bacillus subtilis in vivo and in vitro . Our studies give progressive suggestions on the mechanism of trans-translation.

Nucleic Acids Res Suppl, 2003, (3), 207 - 8
Construction of physical maps of Bacillus subtilis (natto) strains; Qiu D et al.; The putative physical map of a Bacillus subtilis (natto) strain has been constructed to demonstrate the difference from the strain B . subtilis Marburg . B . subtilis (natto) strains are closely related to the Marburg 168trpC2 strain whose genome was sequenced . Their genome size and organization are also assumed to be similar . The putative SfiI and I-CeuI physical map of a natto strain BEST195 revealed that structure of most genome region is similar to that of 168 but some highly variable chromosome regions are also identified.

Nucleic Acids Res Suppl, 2003, (3), 199 - 200
Allosteric activation of HutP protein, that regulates transcription of hut operon in Bacillus subtilis, mediated by various analogs of histidine; Kumarevel T et al.; HutP is an RNA-binding protein which regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on the hut mRNA in the presence of L-histidine . In the case of HutP-RNA interactions, L-histidine plays an allosteric activation role i.e . only the activated HutP specifically recognize the hut mRNA . In the present study, we analyzed various analogs of L-histidine to evaluate the important functional groups of L-histidine that is responsible for the activation of HutP . Our analysis clearly suggests that imidazole group of a L-histidine plays a vital role for the activation . Our analysis also revealed efficient analogs of L-histidine for the activation, for example, L-histidine beta-naphthylamide and L-Histidine benzyl ester.

Curr Microbiol, 2003 Aug, 47(2), 146 - 52
Heterospecific expression of the Bacillus subtilis cell shape determination genes mreBCD in Escherichia coli; Lee JC et al.; The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD . TnphoA mutagenesis was utilized to analyze a topological model for MreC . MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane . Expression of the B . subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E . coli . Immunolocalization of the MreC protein in B . subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis.

J Biol Chem, 2003 Dec 19, 278(51), 51863 - 71 Epub 2003 Sep 23.
Glycerol-3-phosphate cytidylyltransferase . Structural changes induced by binding of CDP-glycerol and the role of lysine residues in catalysis; Pattridge KA et al.; The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases . Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates . Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate . Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix . Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B . This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol . Binding of CDP-glycerol also induces smaller movements of residues 92-100 . Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate.

Vaccine, 2003 Oct 1, 21(27-30), 4215 - 24
Germination of the spore in the gastrointestinal tract provides a novel route for heterologous antigen delivery; Duc le H et al.; We have evaluated the potential of endospores of the Gram-positive bacterium Bacillus subtilis as an oral vaccine delivery system . The key features of the B . subtilis spore as a vaccine are, non-pathogenicity, advanced cloning tools, extreme robustness, long-term storage properties and its current use as a probiotic for both humans and animals . We have shown previously that the spore germinates in the small intestine of the mouse and have exploited this attribute for heterologous antigen delivery in this work . The first part of this study was to evaluate the fate of spores in vivo as well as under simulated gut conditions . This showed that spores were extremely robust with most being excreted in the faeces . Using a recombinant gene expressing high levels of beta-galactosidase specifically in the germinated spore (vegetative cell) we showed that spores administered orally could elicit beta-galactosidase-specific local and systemic immune responses . This demonstrated proof of principle that the germinating spore might be effective in the safe delivery of antigens across the stomach barrier . Interestingly, analysis of IgG subclasses suggested a potential bias towards a Th1 response and the involvement of cellular immunity.

J Chromatogr A, 2003 Aug 22, 1010(1), 113 - 21
Analysis of carboxylic acid metabolites from the tricarboxylic acid cycle in Bacillus subtilis cell extract by capillary electrophoresis using an indirect photometric detection method; Markuszewski MJ et al.; With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell . Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection . Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures: glucose and malate . CE with an electrokinetic injection mode achieved limits of detection in the range of 13-54 ppb (1.12-10(-7) - 5.96-10(-7) M) . The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients . The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source . The changes in carboxylic acid metabolites profile were observed depending from used culture medium . Carboxylic acid concentrations ranged: in cell extract from malate culture from 59 to 0.5 microM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 microM for glutamate and citrate, respectively . Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.

Med Hypotheses, 2003 Oct, 61(4), 503 - 8
Facilitation of horizontal transfer of antimicrobial resistance by transformation of antibiotic-induced cell-wall-deficient bacteria; Woo PC et al.; It is universally accepted that the use of antibiotics will lead to antimicrobial resistance . Traditionally, the explanation to this phenomenon was random mutation and horizontal gene transfer and amplification by selective pressure . Subsequently, a second mechanism of antibiotic-induced antimicrobial resistance acquisition was proposed, when Davies et al . discovered that genes encoding antimicrobial resistance are present in bacteria that produce antibiotics, and during the process of antibiotic purification from these antibiotic-producing organisms, remnants of the organisms' DNA that contain antibiotic resistance genes are also co-extracted, and can be recovered in antibiotic preparations . In addition to selective pressure and antimicrobial resistance genes in antibiotic preparations, we hypothesize the third mechanism by which administration of antibiotics leads to antimicrobial resistance . beta-Lactams and glycopeptides damage bacteria by inhibiting cell wall murein synthesis . During the process, cell-wall-deficient forms are generated before the bacteria die . These cell-wall-deficient forms have an increased ability to uptake DNA by transformation . It has been demonstrated that plasmids encoding antimicrobial resistance of Staphylococcus aureus can be transformed to Bacillus subtilis after the B . subtilis was treated with penicillin or lysostaphin, a chemical that damage the cell walls of some Gram-positive bacteria; and that short treatment of Escherichia coli with antibiotics disturbing bacterial cell wall synthesis rendered the cells capable of absorbing foreign DNA . Since bacteria occupying the same ecological niche, such as the lower gastrointestinal tract, is common, bacteria are often incubated with foreign DNA encoding resistance coming from the administration of antibiotics or other bacteria that undergone lysis unrelated to antibiotic-induced killing . As few as a single antibiotic resistant gene is taken up by the cell-wall-deficient form, it will develop into a resistant clone, despite most of the other bacteria are killed by the antibiotic . If the hypothesis is correct, one should reduce the use of antibiotics that perturb bacterial cell wall synthesis, such as beta-lactams, which is the largest group being manufactured, in both humans and animals, in order to reduce the acquisition of antibiotic resistance through this mechanism . In contrast to the old theory that antibiotics only provide selective pressures for the development of antimicrobial resistance, antibiotics by themselves are able to generate the whole chain of events towards the development of antimicrobial resistance . Antibiotics provide a source of antimicrobial resistance genes, facilitate the horizontal transfer of antimicrobial resistance genes through facilitating transformation, and provide selective pressures for amplification of the antimicrobial resistance genes . That is perhaps an important reason why antimicrobial resistance is so difficult to control . Further experiments should be performed to delineate which particular type of beta-lactam antibiotics are associated with increase in transformation efficiencies more than the others, so that we can select those less resistance generating beta-lactam for routine usage.

Phytomedicine, 2003, 10(6-7), 511 - 6
Antimicrobial activity of some Hypericum species; Dall'Agnol R et al.; The crude methanolic extracts of six species of Hypericum {H . caprifoliatum Cham . & Schlecht., H . carinatum Griseb., H . connatum Lam., H . ternum A . St . Hil., H . myrianthum Cham . & Schlecht . and H . polyanthemum Klotzsch ex Reichardt} growing in southern Brazil were analyzed for antimicrobial activity against several microorganisms (bacteria and fungi) . The most active plant was H . caprifoliatum, which showed activity against Staphylococcus aureus . Only H . polyanthemum and H . ternum extracts were active against Bacillus subtilis . None of the crude methanolic extracts showed activity against S . epidermidis, Escherichia coli or Saccharomyces cerevisiae . Extracts from these species were evaluated chemically and tannin, flavonoid and phenolic acids were the prominent compounds . The plants contained quercitrin, hyperoside (except H . connatum) and, less frequently, isoquercitrin and chlorogenic acid . In contrast to H . perforatum, which has high concentrations of rutin, these species do not produce this flavonoid or it appears as traces . The tannin concentration varied between 5.1 and 16.7% in H . myrianthum and H . ternum, respectively.

J Bacteriol, 2003 Oct, 185(19), 5897 - 900
Tethering of the Bacillus subtilis sigma E proprotein to the cell membrane is necessary for its processing but insufficient for its stabilization; Ju J et al.; sigma(E), a sporulation-specific transcription factor of Bacillus subtilis, is synthesized as an inactive proprotein with a 27-amino acid extension at its amino terminus . This "pro" sequence is removed by a developmentally regulated protease, but when present, it blocks sigma(E) activity, tethers sigma(E) to the bacterium's cytoplasmic membrane, and promotes sigma(E) stability . To investigate whether pro-sigma(E) processing and/or stabilization are tied to membrane sequestration, we used fluorescent protein fusions to examine the membrane binding of SigE variants . The results are consistent with membrane association as a prerequisite for pro-sigma(E) processing but not as a sufficient cause for the proprotein's stability.

J Bacteriol, 2003 Oct, 185(19), 5714 - 21
RelA is a component of the nutritional stress activation pathway of the Bacillus subtilis transcription factor sigma B; Zhang S et al.; The general stress regulon of Bacillus subtilis is induced by the activation of the sigma(B) transcription factor . Activation of sigma(B) occurs when one of two phosphatases (RsbU and RsbP), each responding to a unique type of stress, actuates a positive regulator of sigma(B) by dephosphorylation . Nutritional stress triggers the RsbP phosphatase . The mechanism by which RsbP becomes active is unknown; however, its activation coincides with culture conditions that are likely to reduce the cell's levels of high-energy nucleotides . We now present evidence that RelA, a (p)ppGpp synthetase and the key enzyme of the stringent response, plays a role in nutritional stress activation of sigma(B) . An insertion mutation that disrupts relA blocks the activation of sigma(B) in response to PO(4) or glucose limitation and inhibits the drop in ATP/GTP levels that normally accompanies sigma(B) induction under these conditions . In contrast, the activation of sigma(B) by physical stress (e.g., ethanol treatment) is not affected by the loss of RelA . RelA's role in sigma(B) activation appears to be distinct from its participation in the stringent response . Amino acid analogs which induce the stringent response and RelA-dependent (p)ppGpp synthesis do not trigger sigma(B) activity . In addition, neither a missense mutation in relA (relA240GE) nor a null mutation in rplK (rplK54), either of which is sufficient to inhibit the stringent response and RelA-dependent (p)ppGpp synthesis, fails to block sigma(B) activation by PO(4) or glucose limitation.

FEMS Microbiol Lett, 2003 Sep 12, 226(1), 121 - 6
Knockout of the high-coupling cytochrome aa3 oxidase reduces TCA cycle fluxes in Bacillus subtilis; Zamboni N et al.; The metabolic impact of electron rerouting in the respiratory chain of Bacillus subtilis was quantitatively assessed during batch growth of quinol oxidase mutants by (13)C-tracer experiments . While disruption of the low-coupling cytochrome bd oxidase was without any apparent phenotype, deletion of the high-coupling cytochrome aa(3) oxidase caused a severe reduction of tricarboxylic acid cycle fluxes and increased overflow metabolism . Since the product-corrected biomass yields were identical in mutants and parent, the results show that efficient ATP generation is not overly important for exponential growth of B . subtilis in batch culture.

FEMS Microbiol Lett, 2003 Sep 12, 226(1), 93 - 100
Interaction of the Bacillus subtilis chaperone CsaA with the secretory protein YvaY; Linde D et al.; Bacillus subtilis CsaA was previously characterised as a molecular chaperone with export-related activities . In order to elucidate the functionality of CsaA further, interaction with its postulated substrate YvaY was investigated . Similar binding to carrier immobilised mature and preYvaY revealed that the interaction was not mediated via the signal peptide of preYvaY . Higher affinity to denatured peptides compared to native peptides indicated preferred binding to unfolded proteins . To characterise affinity of CsaA more detailed, binding to preYvaY derived peptides was analysed . CsaA showed affinity to multiple peptides in the scan, mainly correlated to a positive net charge . Affinity of export-specific Escherichia coli chaperone SecB to the carrier immobilised peptides indicated partially overlapping binding characteristics of SecB and CsaA.

J Biomol NMR, 2003 Nov, 27(3), 221 - 34
15N relaxation study of the cold shock protein CspB at various solvent viscosities; Zeeb M et al.; For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, we determined (15)N transverse and longitudinal relaxation rates and heteronuclear nuclear Overhauser effects at different solvent viscosities . Up to a relative viscosity of 2, which is equivalent to 27% ethylene glycol (EG), the overall correlation time follows the linear Stokes-Einstein equation . At a relative viscosity of 6 (70% EG) the correlation time deviates from linearity by 30%, indicating that CspB tumbles at a higher rate as expected from the solvent viscosity probably due to a preferential binding of water molecules at the protein surface . The corresponding hydrodynamic radii, determined by NMR diffusion experiments, show no variation with viscosity . The amplitudes of intramolecular motions on a sub-nanosecond time scale revealed by an extended Lipari-Szabo analysis were mainly independent of the solvent viscosity . The lower limit of the NMR 'observation window' for the internal correlation time shifts above 0.5 ns at 70% EG, which is directly reflected in the experimentally derived internal correlation times . Chemical exchange contributions to the transverse relaxation rates derived from the Lipari-Szabo approach coincide with the experimentally determined values from the transverse (1)H-(15)N dipolar/(15)N chemical shift anisotropy relaxation interference . These contributions originate from fast protein folding reactions on a millisecond timescale, which get retarded at increased solvent viscosities.

Biochemistry, 2003 Sep 23, 42(37), 10955 - 64
Clamp-loader-helicase interaction in Bacillus . Leucine 381 is critical for pentamerization and helicase binding of the Bacillus tau protein; Haroniti A et al.; Recently, we revealed the architecture of the clamp-loader-helicase (tau-DnaB) complex in Bacillus by atomic force microscopy imaging and constructed a structural model, whereby a pentameric clamp-loader interacts with the hexameric helicase . Crucial to this model is the assumption that the clamp-loader forms a pentamer in the absence of other components of the clamp-loader complex such as deltadelta' . Here, we show that the Bacillus subtilis tau protein, even in the absence of deltadelta', interacts as a pentamer with the hexameric DnaB and that the L381 of tau is critical for the integrity of the tau oligomer and interaction with DnaB . The effects of the L381A mutation were confirmed by gel filtration, ultracentrifugation, circular dichroism, cross-linking studies, and genetic replacement of the dnaX gene with a mutant L381A dnaX gene in vivo . The L381A protein is able to support growth in vivo only when expressed in high quantities . Finally, despite the fact that a mutation at P465 has been reported to result in a thermosensitive gene in vivo, a P465L mutant protein interacts with DnaB in vitro suggesting that this defect is not a result of a defective tau-DnaB interaction.

Proteomics, 2003 Sep, 3(9), 1738 - 49
Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile; Bunai K et al.; Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins . We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis . After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS . This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time . We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins . We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B . subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots . This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.

J Mol Biol, 2003 Sep 26, 332(4), 767 - 76
Dimerization of Crh by reversible 3D domain swapping induces structural adjustments to its monomeric homologue Hpr; Juy M et al.; The crystal structure of the regulatory protein Crh from Bacillus subtilis was solved at 1.8A resolution and showed an intertwined dimer formed by N-terminal beta1-strand swapping of two monomers . Comparison with the monomeric NMR structure of Crh revealed a domain swap induced conformational rearrangement of the putative interaction site with the repressor CcpA . The resulting conformation closely resembles that observed for the monomeric Crh homologue HPr, indicating that the Crh dimer is the active form binding to CcpA . An analogous dimer of HPr can be constructed without domain swapping, suggesting that HPr may dimerize upon binding to CcpA . Our data suggest that reversible 3D domain swapping of Crh might be an efficient regulatory mechanism to modulate its activity.

EMBO J, 2003 Sep 15, 22(18), 4709 - 18
Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases; Mijakovic I et al.; Protein-tyrosine kinases regulating bacterial exopolysaccharide synthesis autophosphorylate on tyrosines located in a conserved C-terminal region . So far no other substrates have been identified for these kinases . Here we demonstrate that Bacillus subtilis YwqD not only autophosphorylates at Tyr-228, but that it also phosphorylates the two UDP-glucose dehydrogenases (UDP-glucose DHs) YwqF and TuaD at a tyrosine residue . However, phosphorylation of YwqF and TuaD occurs only in the presence of the transmembrane protein YwqC . The presumed intracellular C-terminal part of YwqC (last 50 amino acids) seems to interact with the tyrosine-kinase and to allow YwqD-catalysed phosphorylation of the two UDP-glucose DHs, which are key enzymes for the synthesis of acidic polysaccharides . However, only when phosphorylated by YwqD do the two enzymes exhibit detectable UDP-glucose DH activity . Dephosphorylation of P-Tyr-YwqF and P-Tyr-TuaD by the P-Tyr-protein phosphatase YwqE switched off their UDP-glucose DH activity . YwqE, which is encoded by the fourth gene of the B.subtilis ywqCDEF operon, also dephosphorylates P-Tyr-YwqD.

J Appl Microbiol, 2003, 95(4), 868 - 73
Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala, a fermented African locust bean condiment; Ouoba LI et al.; AIMS: To investigate predominant isolates of Bacillus subtilis and B . pumilus in soumbala, a fermented African locust bean condiment, for their ability to degrade African locust bean oil (ALBO) . METHODS AND RESULTS: Agar diffusion test in tributyrin and ALBO agar was used for screening of the isolates for esterase and lipase activity, respectively . The quantity and the profile of free fatty acids (FFA) during 72 h of degradation of ALBO by the Bacillus isolates were studied by titration and gas chromatography . The degradation of tributyrin and ALBO was variable among the isolates . Two strains of B . subtilis and two strains of B . pumilus showed significantly higher esterase and lipolytic activities than the others . The degradation ALBO was most pronounced in enriched nutrient agar except for one isolate of B . pumilus degrading ALBO to the same extent regardless of the enrichment . The quantity of FFA released from ALBO by the most lipolytic strains of Bacillus increased mainly between 0 and 24 h and differed among the isolates . The profile of FFA was similar for the Bacillus isolates with oleic acid (C18:2) occurring as the major FFA in all the samples except in samples incubated with B . subtilis B9 where stearic acid (C18) was dominant . CONCLUSION: Bacillus isolates from soumbala showed high strain dependent lipolytic activity against ALBO . SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of soumbala.

Protein Eng, 2003 Aug, 16(8), 615 - 21
Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface; Ohkuri T et al.; We have investigated factors affecting stability at the subunit-subunit interface of the dimeric enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Bacillus subtilis . Site-directed mutagenesis was used to replace methionine 256, a key residue in the subunit interaction, with other amino acids . Thermal stability against irreversible inactivation of the mutated enzymes was examined by analyzing the residual activity after heat treatment . The mutations M256V and M256A increased thermostability by 2.0 and 6.0 degrees C, respectively, whereas the mutations M256L and M256I had no effect . Thermostability of the M256F mutated enzyme was 4.0 degrees C lower than that of the wild-type enzyme . To our surprise, increasing the hydrophobicity of residue 256 within the hydrophobic core of the enzyme resulted in a lower thermal stability . The mutated enzymes showed an inverse correlation between thermostability and the volume of the side chain at position 256 . Based on the X-ray crystallographic structure of Escherichia coli IPMDH, the environment around M256 in the B.subtilis homolog is predicted to be sterically crowded . These results suggest that Met256 prevents favorable packing . Introduction of a smaller amino acid at position 256 improves the packing and stabilizes the dimeric structure of IPMDH . The van der Waals volume of the amino acid residue at the hydrophobic subunit interface is an important factor for maintaining the stability of the subunit-subunit interface and is not always optimized in the mesophilic IPMDH enzyme.

J Biol Chem, 2003 Nov 21, 278(47), 47002 - 8 Epub 2003 Sep 10.
The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA; Orelle C et al.; ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member . Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive . In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deleage, G., Di Pietro, A., and Jault, J . M . (2001) Trends Biochem . Sci . 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z . E., Muller, M., Peng, X . H., and Ambudkar, S . V . (2002) Biochemistry, 41, 13989-14000) . The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues . All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-{alpha-32P}ATP . However, and in contrast to the wild-type enzyme, the use of 8-N3-{gamma-32P}ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover . These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.

Biotechnol Lett, 2003 Jul, 25(14), 1137 - 42
Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1; Ahn CY et al.; Of several types of chemical surfactants and biosurfactants, only the culture broth of Bacillus subtilis C1 containing surfactin at 10 mg l(-1) completely inhibited the growth of Microcystis aeruginosa, a bloom-forming cyanobacterium in highly eutrophic lakes . The broth with 10 mg surfactin l(-1) also removed 85% of the maximally grown M . aeruginosa (chlorophyll-a concentration, 1000 microg l(-1)) within 2 d, and the removal efficiency was enhanced by Ca2+ over 1 mM . The growth of Anabaena affinis, another bloom-forming cyanobacterium, was also inhibited about 70% with surfactin at 10 mg l(-1) broth . However, the effect of the broth was delayed over 3 d in the green algae, Chlorella vulgaris and Scenedesmus sp., and was negligible in a diatom, Navicula sp., indicating the potential for the selective control of cyanobacterial blooms.

Br Poult Sci, 2003 Jul, 44(3), 410 - 8
Improvement of the nutritive value of soybean meal by protease and alpha-galactosidase treatment in broiler cockerels and broiler chicks; Ghazi S et al.; 1 . Tube-fed broiler cockerels were used in three experiments to measure the effect of different enzyme treatments on true metabolisable energy (TME) and true nitrogen digestibility (TND) of commercial solvent-extracted, heat-treated soybean meal (SBM) . 2 . In experiment 1, proteases P2 and P3 (from Aspergillus niger) and alpha-galactosidase (from A . oryzae) improved TME and TND while protease P1 (from Bacillus subtilis) had little effect . The effects of enzyme treatment were similar whether treatment was applied by pre-incubation of enzymes (proteases P1, P2 and P3, 1.0 g/kg; alpha-galactosidase, 0.1 g/kg) with SBM for 2 h at 50 degrees C or by simple mixing of enzymes (proteases P1, P2 and P3, 0.25 g/kg; alpha-galactosidase, 0.1 g/kg) with SBM prior to feeding . 3 . In experiment 2, the effects of mixing SBM with each of protease P1 (0 or 0.25 g/kg), protease P3 (0 or 0.25 g/kg) or alpha-galactosidase (0 or 0.1 g/kg) alone or in all possible combinations were studied . Effects of protease P1 were limited, but protease P3 and alpha-galactosidase improved TME and TND . There were significant interactions between protease P3 and alpha-galactosidase for both TME and TND; the response when enzymes were applied together was less than that expected from application of the enzyme preparations individually . 4 . In experiment 3, the effect of varying the concentration of protease P3 (0, 0.1, 0.25 and 1.0 g/kg SBM dry matter) and alpha-galactosidase (0, 0.025, 0.0625 and 0.25 g/kg SBM dry matter) mixed with SBM alone or in all possible combinations of protease P3 and alpha-galactosidase were assessed . Increases in TME and TND for both enzymes were non-linear with the greatest response occurring at the lowest concentration of each enzyme . There were also interactions between the two enzyme preparations . 5 . Finally, either protease P3 (0, 0.1 and 1.0 g/kg SBM dry matter) or alpha-galactosidase (0, 0.025 and 0.25 g/kg SBM dry matter) were mixed with SBM alone or in all possible combinations and treated SBMs incorporated into semi-purified diets containing 450 g SBM/kg as the sole source of dietary N and offered to growing broiler chicks for 21 d . Similar patterns of increases in chick growth rate and diet digestibility to those recorded in experiment 3 were obtained when protease P3 and alpha-galactosidase where included in the diets . 6 . It is concluded that responses measured by tube-feeding SBM treated with protease P3 and alpha-galactosidase were similar to responses obtained with growing broiler chicks . Further, these responses were obtained by simple addition of enzymes to diets and nutritive value of SBM was improved by both protease and alpha-galactosidase treatment.

J Mol Biol, 2003 Sep 19, 332(3), 555 - 74
A Mg2+-dependent RNA tertiary structure forms in the Bacillus subtilis trp operon leader transcript and appears to interfere with trpE translation control by inhibiting TRAP binding; Schaak JE et al.; Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms . In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure . We examined the structure of the trp leader readthrough RNA in the absence of TRAP . Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models . In addition, this RNA was found to adopt a Mg(2+)-dependent, long-range tertiary interaction under physiological monovalent salt conditions . Formation of this tertiary structure does not require significant changes in the preformed secondary structure . Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop . UV cross-linking experiments utilizing 5'-p-azidophenacyl-containing transcripts revealed a Mg(2+)-dependent cross-link . Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop . Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure . Filter-binding studies demonstrated that the addition of Mg(2+) inhibits TRAP binding, which may be partially due to the effect of Mg(2+) on RNA tertiary structure formation . Results from expression studies using trpE'-'lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg(2+)-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.

J Mol Biol, 2003 Sep 19, 332(3), 537 - 53
Hyperthermophilic Thermotoga arginine repressor binding to full-length cognate and heterologous arginine operators and to half-site targets; Morin A et al.; The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar . However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus . A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only . Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets . Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors . We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor . Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth . We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga

Nucleic Acids Res . 2003 Sep 15;31(18):e112.
Conversion of sub-megasized DNA to desired structures using a novel Bacillus subtilis genome vector; Kaneko S et al.; A novel genome vector using the 4215 kb Bacillus subtilis genome provides for precise target cloning and processing of the cloned DNA to the desired structure . Each process highly dependent on homologous recombination in the host B.subtilis is distinguished from the other cloning systems . A 120 kb mouse jumonji (jmj) genomic gene was processed in the genome vector to give a series of truncated sub-megasized DNA . One of these truncated segments containing the first intron was copied in a plasmid by a recombinational transfer method developed for B.subtilis . DNA manipulation previously considered difficult is argued with respect to DNA size and accuracy.

J Am Chem Soc, 2003 Sep 10, 125(36), 10862 - 6
Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by thioesterase domains; Sieber SA et al.; Here we report a new experimental approach to characterize recombinant nonribosomal peptide cyclases that do not show activity with conventional N-acetylcysteamine (SNAC) substrates . To explore the great potential of these domains for the catalysis of cell-free cyclization reactions, the new strategy takes advantage of the direct interaction between the natural substrate where the peptide chain is attached to the phosphopantetheine arm of the peptidyl carrier protein (PCP) and the peptide cyclase . A prerequisite for this reaction is the promiscuity of the Bacillus subtilis phosphopantetheinyl transferase Sfp for loading chemically synthesized peptidyl-coenzyme A substrates instead of the smaller natural substrate coenzyme A (CoASH) onto apoPCP . With this novel method we were able to characterize the regioselectivity of branched-chain cyclization catalyzed by the fengycin cyclase, which displays no activity with peptidyl-SNAC substrates.

Appl Opt, 2003 Aug 20, 42(24), 4887 - 900
Detection, identification, and estimation of biological aerosols and vapors with a Fourier-transform infrared spectrometer; Ben-David A et al.; Two experiments were conducted with a Fourier-transform infrared (FTIR) spectrometer . The purpose of the first experiment was to detect and identify Bacillus subtilis subsp . niger (BG) bioaerosol spores and kaolin dust in an open-air release for which the thermal contrast between the aerosol temperature and background brightness temperature is small . The second experiment estimated the concentration of a small amount of triethyl phosphate (TEP) vapor in a closed chamber in which an external blackbody radiation source was used and where the thermal contrast was large . The deduced BG (TEP) extinction spectrum (identification) showed an excellent match to the library BG (TEP) extinction spectrum . Analysis of the time sequence of the measurements coincided well with the presence (detection) of the BG during the measurements, and the estimated concentration of time-dependent TEP vapor was excellent . The data were analyzed with hyperspectral detection, identification, and estimation algorithms . The algorithms were based on radiative transfer theory and statistical signal-processing methods . A subspace orthogonal projection operator was used to statistically subtract the large thermal background contribution to the measurements, and a robust maximum-likelihood solution was used to deduce the target (aerosol or vapor cloud) spectrum and estimate its mass-column concentration . A Gaussian-mixture probability model for the deduced mass-column concentration was computed with an expectation-maximization algorithm to produce the detection threshold, the probability of detection, and the probability of false alarm . The results of this study are encouraging, as they suggest for the first time to the authors' knowledge the feasibility of detecting biological aerosols with passive FTIR sensors.

Biosci Biotechnol Biochem, 2003 Aug, 67(8), 1825 - 7
Comparative analyses of hairpin substrate recognition by Escherichia coli and Bacillus subtilis ribonuclease P ribozymes; Ando T et al.; Previously, we reported that the substrate shape recognition of the Escherichia coli ribonuclease (RNase) P ribozyme depends on the concentration of magnesium ion in vitro . We additionally examined the Bacillus subtilis RNase P ribozyme and found that the B . subtilis enzyme also required high magnesium ion, above 10 mM, for cleavage of a hairpin substrate . The results of kinetic studies showed that the metal ion concentration affected both the catalysis and the affinity of the ribozymes toward a hairpin RNA substrate.

FEMS Microbiol Lett, 2003 Aug 29, 225(2), 319 - 24
Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH; Eggert T et al.; Bacillus subtilis secretes two lipases LipA and LipB into the culture medium . Both enzyme genes were differentially expressed depending on the growth conditions as determined by activity assays and Western blotting in B . subtilis mutant strains lipA, lipB, and the corresponding lipA/lipB double mutant . In minimal medium, LipA was produced at wild-type level in a lipB mutant, however, no LipB protein was detected in a lipA mutant . Interestingly, LipA was produced and secreted at wild-type level in rich medium, but the enzyme remained enzymatically inactive, presumably being caused by a shift of the growth medium to acid pH . Furthermore, expression of the lipase genes was studied using transcriptional fusions with the lacZ reporter gene . The expression of lipA was repressed by high amino acid concentrations, whereas the lipB gene expression remained unaffected.

Mol Microbiol, 2003 Sep, 49(6), 1685 - 97
Binding of response regulator DegU to the aprE promoter is inhibited by RapG, which is counteracted by extracellular PhrG in Bacillus subtilis; Ogura M et al.; We screened the putative rap-phr (response regulator aspartyl-phosphate phosphatase-phosphatase regulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE (alkaline protease gene) expression by using a multicopy plasmid . We found that rapG was involved in the regulation of aprE, which belongs to the regulon of DegU, the response regulator of the DegS-DegU two-component system . Disruption of rapG and phrG resulted in enhancement and reduction of aprE-lacZ expression, respectively, suggesting that PhrG inhibits RapG activity . Addition of 1-30 nM of a synthetic pentapeptide (PhrG; NH2-EKMIG-COOH) to the phrG disruptant completely rescued aprE-lacZ expression, indicating that the PhrG peptide is indeed involved in aprE-lacZ expression . Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100-300 nM) to the phrG cells decreased aprE-lacZ expression . These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA-lacZ expression directed by ComA, the regulator of the ComP-ComA two-component system . Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of DegU-P (phosphorylated DegU) in vitro . RapG, however, did not affect dephosphorylation of DegU-P under the adopted experimental conditions . Therefore, we hypothesized that RapG might inhibit the binding activity of DegU to the target promoters . We analysed the interaction of DegU and RapG using the aprE promoter and another target, a comK promoter . Gel shift analysis revealed that RapG served as the inhibitor of DegU binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.

Mol Microbiol, 2003 Sep, 49(6), 1657 - 69
A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis; Chen CC et al.; SigmaB, an alternative sigma-factor of Bacillus subtilis, mediates the response of the cell to a variety of physical insults . Within the environmental stress signalling pathway RsbU, a protein phosphatase, is stimulated by its interaction with the protein kinase RsbT . In the absence of stress RsbT is expected to be trapped by an alternative binding partner, RsbS . Here, we have demonstrated that RsbS alone cannot act as an alternative partner for RsbT, but instead requires the presence of RsbR to create a high molecular mass RsbR:RsbS complex (approximately 1 MDa) able to capture RsbT . In this complex the phosphorylation state of RsbS, and not that of RsbR, controlled the binding to RsbT, whose kinase activity towards RsbS could be counterbalanced by the activity of RsbX, the phosphatase for RsbS-P . The RsbR:RsbS complex recruited RsbT from a mixture of RsbT and RsbU . The phosphorylated form of RsbR in the complex enhanced the kinase activity of RsbT towards RsbS . This supramolecular complex thus has the functional properties of an alternative partner for RsbT . Electron micrographs of this complex are presented, and the purification of the RsbR:RsbS complex from cellular extracts provides evidence for the existence of such a complex in vivo.

Mol Microbiol, 2003 Sep, 49(6), 1509 - 22
TPR-mediated interaction of RapC with ComA inhibits response regulator-DNA binding for competence development in Bacillus subtilis; Core L et al.; The Bacillus subtilis Rap family of proteins are characterized by protein-protein interaction modules containing the so-called tetratricopeptide repeats (TPRs) . The six TPR motifs of RapC mediate its interaction with the pentapeptide inhibitor PhrC (ERGMT) or with its target protein ComA, a phosphorylation-dependent response regulator transcription factor for genetic competence . Our results show that RapC interaction with ComA inhibits the response regulator's ability to bind its target DNA promoter but does not affect its phosphorylation state . RapC binds equally well to ComA or to ComA approximately P . The PhrC pentapeptide binds to RapC and inhibits its interaction with ComA . The D195 residue in TPR3 and the P263 residue in TPR5 of RapC are critical for the interaction with PhrC as their mutation to asparagine or leucine, respectively, prevents peptide inhibitory activity . The RapC mechanism of regulating ComA activity is a new example of how TPR motifs and their structural organization have been adapted for different specific functions within the B . subtilis Rap family.

Mol Microbiol, 2003 Sep, 49(6), 1477 - 91
The global transcriptional response of Bacillus subtilis to manganese involves the MntR, Fur, TnrA and sigmaB regulons; Guedon E et al.; We have used DNA microarrays to monitor the global transcriptional response of Bacillus subtilis to changes in manganese availability . Mn(II) leads to the MntR-dependent repression of both the mntH and mntABCD operons encoding Mn(II) uptake systems . Mn(II) also represses the Fur regulon . This repression is unlikely to be a direct effect of Mn(II) on Fur as repression is sensitive to 2,2'-dipyridyl, an iron-selective chelator . We suggest that elevated Mn(II) displaces iron from cellular-binding sites and the resulting rise in free iron levels leads to repression of the Fur regulon . Many of the genes induced by Mn(II) are activated by sigmaB or TnrA . Both of these regulators are controlled by Mn(II)-dependent enzymes . Induction of the sigmaB-dependent general stress response by Mn(II) is largely dependent on RsbU, a Mn(II)-dependent phosphatase that dephosphorylates RsbV, ultimately leading to release of active sigmaB from its antisigma, RsbW . The activity of TnrA is inhibited when it forms an inactive complex with feedback-inhibited glutamine synthetase . Elevated Mn(II) reduces the sensitivity of glutamine synthetase to feedback inhibitors, and we suggest that this leads to the observed increase in TnrA activity . In sum, three distinct mechanisms can account for most of the transcriptional effects elicited by manganese: (i) direct binding of Mn(II) to metalloregulators such as MntR, (ii) perturbation of cellular iron pools leading to increased Fur activity and (iii) altered activity of Mn(II)-dependent enzymes that regulate the activity of sigmaB and TnrA.

Mol Microbiol, 2003 Sep, 49(6), 1463 - 75
RacA and the Soj-Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis; Wu LJ et al.; Sporulating cells of Bacillus subtilis undergo a highly polarized cell division and possess a specialized mechanism to move the oriC region of the chromosome close to the cell pole before septation . DivIVA protein, which localizes to the cell pole, and the Soj and Spo0J proteins, which associate with the chromosome, are part of the mechanism that delivers the chromosome to the cell pole . A sporulation-specific protein, RacA, encodes a third DNA-binding protein, which acts in conjunction with Soj and Spo0J to effect efficient polar chromosome segregation . divIVA mutants and soj racA double mutants have an unexpected phenotype in which specific markers to the left and right of oriC can be captured in the prespore compartment but the central oriC region is efficiently excluded . This 'residual' trapping requires Spo0J protein . We suggest that the Soj RacA DivIVA system is required to extract the oriC region from its position determined by the vegetative chromosome segregation machinery and anchor it to the cell pole.

Microbiology, 2003 Sep, 149(Pt 9), 2501 - 11
Induction of L-form-like cell shape change of Bacillus subtilis under microculture conditions; Shingaki R et al.; A remarkable cell shape change was observed in Bacillus subtilis strain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids) . Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells . The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media . The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects . Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass . The cell shape change of CI cultures was repressed when excess KNO3 was added to the medium . Whole-cell protein analysis of the normal rod-shaped cells grown with 0.1% KNO3 and the shape-changed cells grown without KNO3 revealed that the expression of the branched-chain alpha-keto acid dehydrogenase complex (coded by the bfmB gene locus) was elevated in the shape-changed cells . Inactivation of the bfmB locus resulted in the repression of cell shape change, and cells in which bfmB expression was induced by IPTG did show changes in shape . Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0.1 M sucrose was observed . Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.

Microbiology, 2003 Sep, 149(Pt 9), 2345 - 55
Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis; Inacio JM et al.; The Bacillus subtilis proteins involved in the utilization of L-arabinose are encoded by the araABDLMNPQ-abfA metabolic operon and by the araE/araR divergent unit . Transcription from the ara operon, araE transport gene and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR . Additionally, expression of both the ara operon and the araE gene is regulated at the transcriptional level by glucose repression . Here, by transcriptional fusion analysis in different mutant backgrounds, it is shown that CcpA most probably complexed with HPr-Ser46-P plays the major role in carbon catabolite repression of the ara regulon by glucose and glycerol . Site-directed mutagenesis and deletion analysis indicate that two catabolite responsive elements (cres) present in the ara operon (cre araA and cre araB) and one cre in the araE gene (cre araE) are implicated in this mechanism . Furthermore, cre araA located between the promoter region of the ara operon and the araA gene, and cre araB placed 2 kb downstream within the araB gene are independently functional and both contribute to glucose repression . In Northern blot analysis, in the presence of glucose, a CcpA-dependent transcript consistent with a message stopping at cre araB was detected, suggesting that transcription 'roadblocking' of RNA polymerase elongation is the most likely mechanism operating in this system . Glucose exerts an additional repression of the ara regulon, which requires a functional araR.

Microbiology, 2003 Sep, 149(Pt 9), 2331 - 43
The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate; Doan T et al.; A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed . In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis . Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression . Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene . The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km 102 and 10 s(-1) mM(-1), respectively) . However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth . Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression . Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL . ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.

Microbiology, 2003 Sep, 149(Pt 9), 2317 - 29
The Bacillus subtilis YufLM two-component system regulates the expression of the malate transporters MaeN (YufR) and YflS, and is essential for utilization of malate in minimal medium; Tanaka K et al.; The Gram-positive bacterium Bacillus subtilis has a complete set of enzymes for the tricarboxylic acid (TCA) cycle and can grow aerobically using most of the TCA cycle intermediates (malate, fumarate, succinate and citrate) as a sole carbon source . The B . subtilis genome sequence contains three paralogous two-component regulatory systems, CitST, DctSR and YufLM . CitST and DctSR activate the expression of a transporter of the Mg(2+)-citrate complex (CitM) and a fumarate and succinate transporter (DctP), respectively . These findings prompted an investigation of whether the YufL sensor and its cognate regulator, YufM, play a role in malate uptake . This paper reports that the YufM regulator shows in vitro binding to the promoter region of two malate transporter genes, maeN and yflS, and is responsible for inducing their expression in vivo . It was also found that inactivation of the yufM or maeN genes resulted in bacteria that could not grow in a minimal salts medium containing malate as a sole carbon source, indicating that the induction of the MaeN transporter by the YufM regulator is essential for the utilization of malate as a carbon source . Inactivation of the yufL gene resulted in the constitutive expression of MaeN . This expression was suppressed by reintroduction of the kinase domain of YufL, indicating that the YufL sensor is required for proper signal detection and signalling specificity . The authors propose that a phosphatase activity of YufL plays an important role in the YufLM two-component regulatory system . The studies reported here have revealed that members of a set of paralogous two-component regulatory systems in B . subtilis, CitST, DctSR and YufLM, are involved in a related function--uptake (and metabolism) of the TCA cycle intermediates--but with distinct substrate specificities.

Mol Biol Evol, 2003 Dec, 20(12), 2076 - 90 Epub 2003 Aug 29.
Genome engineering reveals large dispensable regions in Bacillus subtilis; Westers H et al.; Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations . If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches . Notably, approximately 47% of the approximately 4,100 genes of B . subtilis belong to paralogous gene families in which several members have overlapping functions . Thus, essential gene functions will outnumber essential genes . To answer the question to what extent the most recently acquired DNA contributes to the life of B . subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B . subtilis genome by removing prophages and AT-rich islands . Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B . subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes . The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility . We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

J Bacteriol, 2003 Sep, 185(18), 5627 - 31
Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion; Kinsinger RF et al.; Motility on surfaces is an important mechanism for bacterial colonization of new environments . In this report, we describe detection of rapid surface motility in the wild-type Bacillus subtilis Marburg strain, but not in several B . subtilis 168 derivatives . Motility involved formation of rapidly spreading dendritic structures, followed by profuse surface colonies if sufficient potassium ion was present . Potassium ion stimulated surfactin secretion, and the role of surfactin in surface motility was confirmed by deletion of a surfactin synthase gene . Significantly, this motility was independent of flagella . These results demonstrate that wild-type B . subtilis strains can use both swimming and sliding-type mechanisms to move across surfaces.

J Bacteriol, 2003 Sep, 185(18), 5380 - 90
YqfS from Bacillus subtilis is a spore protein and a new functional member of the type IV apurinic/apyrimidinic-endonuclease family; Salas-Pacheco JM et al.; The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here . To this end, a recombinant YqfS protein containing an N-terminal His6 tag was synthesized in Escherichia coli and purified to homogeneity . An anti-His6-YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B . subtilis spores . The His6-YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3'-phosphatases . However, the purified protein lacked both 5'-phosphatase and exonuclease III activities . YqfS showed not only a high level of amino acid identity with E . coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA) . These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules . Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA . The ability of yqfS to genetically complement the DNA repair deficiency of an E . coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents . Thus, we conclude that YqfS of B . subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3' blocking groups of DNA generated during both spore dormancy and germination.

J Bacteriol, 2003 Sep, 185(18), 5372 - 9
Effect of translational signals on mRNA decay in Bacillus subtilis; Sharp JS et al.; A 254-nucleotide model mRNA, designated deltaermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis . DeltaermC mRNA features a strong ribosome-binding site (RBS) and a 62-amino-acid-encoding open reading frame, followed by a transcription terminator structure . Inactivation of the RBS or the start codon resulted in a fourfold decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability . Data for the decay of deltaermC mRNAs with stop codons at positions increasingly proximal to the translational start site showed that actual translation--even the formation of the first peptide bond--was not important for stability . The half-life of an untranslated 3.2-kb deltaermC-lacZ fusion RNA was similar to that of a translated deltaermC-lacZ mRNA, indicating that the translation of even a longer RNA was not required for wild-type stability . The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with a 5'-end-dependent activity, possibly a 5'-binding endonuclease, which is required for the initiation of mRNA decay . This model is supported by the finding that increasing the distance from the 5' end to the start codon resulted in a 2.5-fold decrease in the mRNA half-life . These results underscore the importance of the 5' end to mRNA stability in B . subtilis.

Biophys J, 2003 Sep, 85(3), 1377 - 91
Molecular dynamics simulation of surfactin molecules at the water-hexane interface; Nicolas JP; The dynamics of surfactin, a lipopeptide surfactant from Bacillus subtilis, has been studied by molecular dynamics at different interfacial concentrations in a water-hexane medium reproducing a hydrophilic/hydrophobic biphasic system . The shapes and orientations of surfactin molecules, as hydrogen bonds and Ramachandran angles, have been recorded to investigate the environment effect on the molecular structure . We demonstrate that the peptidic backbone can exhibit a large flexibility and that conformational motions and structural fluctuations depend strongly on the interfacial concentration . Moreover, we have measured the surface activity of this biosurfactant by computing the interfacial tension and lateral and rotational diffusion coefficients.

EMBO J, 2003 Sep 1, 22(17), 4534 - 43
Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis; Pellegrini O et al.; In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems . RNase Z cleaves tRNA precursors 3' to the discriminator base . Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis . Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro . We have thus renamed the gene rnz . A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue . Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.

Bioorg Chem, 2003 Oct, 31(5), 389 - 97
A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen; Jeong E et al.; In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B . subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized . The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole . When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase . The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B . subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

Mol Microbiol, 2003 Sep, 49(5), 1435 - 47
In Bacillus subtilis W23, the duet sigmaXsigmaM, two sigma factors of the extracytoplasmic function subfamily, are required for septum and wall synthesis under batch culture conditions; Minnig K et al.; The synthesis of poly(RboP), the main Bacillus subtilis W23 teichoic acid, is encoded by tarDF-tarABIJKL operons, the latter being controlled by two promoters designated PtarA-int and PtarA-ext . Analysis by lacZ fusions reveals that PtarA-int activity exhibits sharp increases at the beginning and end of the transition between exponential and stationary growth phase . As confirmed by mRNA quantification, these increases are mediated by ECF sigma factors sigmaX and sigmaM respectively . In liquid media, strain W23 sigX sigM double mutants experience serious difficulties in the transition and stationary growth phases . Inactivation of sigmaX- and sigmaM-controlled regulons, which precludes transcription from PtarA-int, leads to (i) delays in chromosome segregation and septation and (ii) a transient loss of up to 30% of the culture OD or lysis . However, specific inactivation of PtarA-int, leading mainly to a shortage of poly(RboP), does not affect growth while, nevertheless, interfering with normal septation, as revealed by electron microscopy . The different sigM transcription in strains W23 and 168 is discussed . In W23, expression of tarA and sigM, which is shown to control divIC, is inversely correlated with growth rate, suggesting that the sigM regulon is involved in the control of cell division.

Mol Microbiol, 2003 Sep, 49(5), 1425 - 34
SpoIVB-mediated cleavage of SpoIVFA could provide the intercellular signal to activate processing of Pro-sigmaK in Bacillus subtilis; Dong TC et al.; SpoIVB is the critical determinant for intercompartmental signalling of pro-sigmaK processing during sporulation in Bacillus subtilis . We show here that the SpoIVB serine peptidase can cleave the SpoIVFA protein, which is one component of the pro-sigmaK processing complex . SpoIVFA has been shown elsewhere (Rudner, D.Z., and Losick, R., 2002, Genes Dev 16: 1007-1018) to tether BofA and SpoIVFB in a membrane-embedded heteroligomeric complex in which BofA directly inhibits the activity of SpoIVFB . Cleavage of SpoIVFA would provide the necessary signal to dissolve this complex and release BofA-mediated inhibition on the zinc metalloprotease, SpoIVFB, that is responsible for cleaving pro-sigmaK to its mature form . We also show that the SpoIVB PDZ domain is required for self-recognition and trans cleavage of SpoIVB and is probably also used to target an internal motif within the C-terminal region of SpoIVFA exposed in the space between the inner and outer forespore membranes . This work reveals the mechanism of intercompartmental signalling and provides a unified model as to how sigmaK-directed gene expression in the mother cell is co-ordinated with events in the forespore chamber.

Adv Biochem Eng Biotechnol, 2003, 83, 57 - 92
A proteomic view of cell physiology of Bacillus subtilis--bringing the genome sequence to life; Hecker M; The genome sequence is the "blue-print of life", and the proteomic approach brings this genome sequence to life . Simple model systems are urgently required to "train" this transformation of the genome sequence into life: why not Bacillus subtilis, the model organism for gram-positive bacteria and of functional genomics? By combination of the highly sensitive 2D protein gel electrophoresis with the identification of the protein spots by microsequencing or mass spectrometry we established a 2D protein index of Bacillus subtilis . In order to depict the entire proteome of a B . subtilis cell, alkaline, cell-wall associated, or extracellular proteins were also included . The proteins of this database (see were allocated to proteins with house-keeping functions typical of growing cells and to proteins synthesized particularly in non-growing cells . A computer-aided evaluation of the 2D gels loaded with radioactively-labeled proteins from growing or stressed/starved cells proved to be a powerful tool for the analysis of global regulation of the expression of the entire genome . This is shown for the analysis of glycolysis/TCA cycle (house keeping proteins) and for the analysis of the heat stress stimulon . For the heat stress stimulon it is demonstrated how the proteomic approach can be used: (i) to define the structure of a stimulon, (ii) to dissect stimulons into regulons, (iii) to analyze the regulation, structure, and function of unknown regulons, (iv) to define overlapping reguIons or modulons, and finally (v) to explore complex adaptational networks . Furthermore, it will be demonstrated how the "dual channel pattern comparison" or "proteomics signature" (R . VanBogelen) can be used for a comprehensive understanding or prediction of the physiological state of growing or starving cell populations . This is shown for glucose-starved cells . In order to describe the structure and function of gene regulation groups it is generally recommended to complement the proteomics approach with DNA array technologies . Further studies will focus on the analysis of the global regulation of gene expression by the proteomic approach that cannot be addressed by the application of DNA array techniques: the phosphoproteome and its implications in signal transduction; the global control of protein stability; protein targeting and protein secretion.

J Food Prot, 2003 Aug, 66(8), 1482 - 5
Sporicidal kinetics of Bacillus subtilis spores by heated scallop shell powder; Sawai J et al.; Scallop shell powder heated at 1,000 degrees C for 1 h exhibited sporicidal action against Bacillus subtilis spores . The sporicidal kinetics of this action were analyzed with the use of a nonlogarithmic model . Apparent death rate constants (k) were obtained under various conditions . The value of k increased with powder concentration but became constant beyond the concentration representing the solubility of Ca(OH)2 . A linear inverse relationship between k and temperature was found, and from this relationship the activation energy required for the death of B . subtilis spores in the heated shell powder slurry could be determined.

Ultrason Sonochem, 2003 Oct, 10(6), 315 - 8
The development and evaluation of ultrasound for the treatment of bacterial suspensions . A study of frequency, power and sonication time on cultured Bacillus species; Joyce E et al.; Some species of bacteria produce colonies and spores which agglomerate in spherical clusters (Bacillus subtilis) and this serves as a protection for the organisms inside against biocidal attack . Flocs of fine particles e.g . clay can entrap bacteria which can also protect them against the biocides . It is because of problems such as these that alternative methods of disinfecting water are under active investigation . One such method is the use of power ultrasound, either alone or in combination with other methods . Ultrasound is able to inactivate bacteria and deagglomerate bacterial clusters or flocs through a number of physical, mechanical and chemical effects arising from acoustic cavitation . The aim of this study was to investigate the effect of power ultrasound at different powers and frequencies on Bacillus subtilis . Viable plate count techniques were used as a measure of microbial activity . Results showed a significant increase in percent kill for Bacillus species with increasing duration of exposure and intensity of ultrasound in the low-kilohertz range (20 and 38 kHz) . Results obtained at two higher frequencies (512 and 850 kHz) indicated a significant increase in bacteria count suggesting declumping . In assessing the bacterial kill with time under different sonication regimes three types of behaviour were characterized: High power ultrasound (lower frequencies) in low volumes of bacterial suspension results in a continuous reduction in bacterial cell numbers i.e . the kill rate predominates . High power ultrasound (lower frequencies) in larger volumes results in an initial rise in cell numbers suggesting declumping of the bacteria but this initial rise then falls as the declumping finishes and the kill rate becomes more important . Low intensity ultrasound (higher frequencies) gives an initial rise in cell numbers as a result of declumping . The kill rate is low and so there is no significant subsequent decrease in bacterial cell numbers.

Org Biomol Chem, 2003 May 7, 1(9), 1443 - 6
Stereochemistry and mechanism of the conversion of 5-aminolaevulinic acid into porphobilinogen catalysed by porphobilinogen synthase; Goodwin CE et al.; (3R)- and (3S)-Deuteriated forms of 5-aminolaevulinic acid have been synthesized and the (3R)-form shows a significantly larger isotope effect when incubated with porphobilinogen synthase from bovine liver and from Bacillus subtilis; based on this and on available crystal structures, a modified mechanism for the enzymic reaction is proposed.

Org Biomol Chem, 2003 Feb 7, 1(3), 463 - 71
The type I rat fatty acid synthase ACP shows structural homology and analogous biochemical properties to type II ACPs; Reed MA et al.; While X-ray and NMR structures are now available for most components of the Type II fatty acid synthase (FAS), there are no structures for Type I FAS domains . A region from the mammalian (rat) FAS, including the putative acyl carrier protein (ACP), has been cloned and over-expressed . Here we report multinuclear, multidimensional NMR studies which show that this isolated ACP domain contains four alpha-helices (residues 8-16 {1}; 41-51 {2}; 58-63 {3} and 66-74 {4}) and an overall global fold characteristic of ACPs from both Type II FAS and polyketide synthases (PKSs) . The overall length of the structured ACP domain (67 residues) is smaller than the structured regions of the Eschericia coli FAS ACP (75 residues), the actinorhodin PKS ACP (78 residues) and the Bacillus subtilis FAS ACP (76 residues) . We further show that the rat FAS ACP is recognised as an efficient substrate by enzymes known to modify Type II ACPs including phosphopantetheinyl and malonyl transferases, but not by the heterologous S . coelicolor minimal polyketide synthase.

Photodermatol Photoimmunol Photomed, 2003 Aug, 19(4), 175 - 81
UV exposure in cars; Moehrle M et al.; BACKGROUND: There is increasing knowledge about the hazards of solar and ultraviolet (UV) radiation to humans . Although people spend a significant time in cars, data on UV exposure during traveling are lacking . The aim of this study was to obtain basic information on personal UV exposure in cars . METHODS: UV transmission of car glass samples, windscreen, side and back windows and sunroof, was determined . UV exposure of passengers was evaluated in seven German middle-class cars, fitted with three different types of car windows . UV doses were measured with open or closed windows/sunroof of Mercedes-Benz E 220 T, E 320, and S 500, and in an open convertible car (Mercedes-Benz CLK) . Bacillus subtilis spore film dosimeters (Viospor) were attached to the front, vertex, cheeks, upper arms, forearms and thighs of 'adult' and 'child' dummies . RESULTS: UV wavelengths longer than >335 nm were transmitted through car windows, and UV irradiation >380 nm was transmitted through compound glass windscreens . There was some variation in the spectral transmission of side windows according to the type of glass . On the arms, UV exposure was 3-4% of ambient radiation when the car windows were shut, and 25-31% of ambient radiation when the windows were open . In the open convertible car, the relative personal doses reached 62% of ambient radiation . CONCLUSIONS: The car glass types examined offer substantial protection against short-wave UV radiation . Professional drivers should keep car windows closed on sunny days to reduce occupational UV exposure . In individuals with polymorphic light eruption, produced by long-wave UVA, additional protection by plastic films, clothes or sunscreens appears necessary.

RNA, 2003 Sep, 9(9), 1148 - 56
tRNA requirements for glyQS antitermination: a new twist on tRNA; Yousef MR et al.; Transcription antitermination of the Bacillus subtilis glyQS gene, a member of the T box gene regulation family, can be induced during in vitro transcription in a minimal system using purified B . subtilis RNA polymerase by the addition of unmodified T7 RNA polymerase-transcribed tRNA(Gly) . Antitermination was previously shown to depend on base-pairing between the glyQS leader and the tRNA at the anticodon and acceptor ends . In this study, variants of tRNA(Gly) were generated to identify additional tRNA elements required for antitermination activity, and to determine the effect of structural changes in the tRNA . We find that additions to the 3' end of the tRNA blocked antitermination, in agreement with the prediction that uncharged tRNA is the effector in vivo, whereas insertion of 1 nucleotide between the acceptor stem and the 3' UCCA residues had no effect . Disruption of the D-loop/T-loop tertiary interaction inhibited antitermination function, as was previously demonstrated for tRNA(Tyr)-directed antitermination of the B . subtilis tyrS gene in vivo . Insertion of a single base pair in the anticodon stem was tolerated, whereas further insertions abolished antitermination . However, we find that major alterations in the length of the acceptor stem are tolerated, and the insertions exhibited a pattern of periodicity suggesting that there is face-of-the-helix dependence in the positioning of the unpaired UCCA residues at the 3' end of the tRNA for interaction with the antiterminator bulge and antitermination.

J Biol Chem, 2003 Nov 21, 278(47), 46203 - 9 Epub 2003 Aug 15.
Interactions between the PTS regulation domains of the BglG transcriptional antiterminator from Escherichia coli; Fux L et al.; The E . coli BglG protein inhibits transcription termination within the bgl operon in the presence of beta-glucosides . BglG represents a family of transcriptional antiterminators that bind to RNA sequences, which partially overlap rho-independent terminators, and prevent termination by stabilizing an alternative structure of the transcript . The activity of BglG is determined by its dimeric state, which is modulated by reversible phosphorylation catalyzed by BglF, a PTS permease . Only the non-phosphorylated BglG dimer binds to RNA and allows read-through of transcription . BglG is composed of three domains: an RNA-binding domain followed by two domains, PRD1 and PRD2 (PTS regulation domains), which are similar in their sequence and folding . Based on the three-dimensional structure of dimeric LicT, a BglG homologue from Bacillus subtilis, the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2 . We have shown before that PRD2 mediates homodimerization very efficiently . Using genetic systems and in vitro techniques that assay and characterize protein-protein interactions, we show here that the PRD1 dimerizes very slowly, but once it does, the homodimers are stable . These results support our model that formation of BglG dimers initiates with PRD2 dimerization followed by zipping up of two BglG monomers to create the active RNA-binding domain . Moreover, our results demonstrate that PRD1 and PRD2 heterodimerize efficiently in vitro and in vivo . The affinity among the PRDs is in the following order: PRD2-PRD2 > PRD1-PRD2 > PRD1-PRD1 . The interaction between PRD1 and PRD2 offers an explanation for the requirement of conserved residues in PRD1 for the phosphorylation of PRD2 by BglF.

J Bacteriol, 2003 Sep, 185(17), 5306 - 9
Bacillus subtilis diacylglycerol kinase (DgkA) enhances efficient sporulation; Amiteye S et al.; The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis . After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores . Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration . In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed . We also found that dgkA is expressed mainly during the vegetative phase . It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

J Bacteriol, 2003 Sep, 185(17), 5275 - 8
Unique degradation signal for ClpCP in Bacillus subtilis; Pan Q et al.; Regulation of the cell-specific transcription factor sigma(F) in the spore-forming bacterium Bacillus subtilis involves the antisigma factor SpoIIAB . Contributing to the activation of sigma(F) is the degradation of SpoIIAB in a manner that depends on the protease ClpCP . Here we show that the three residues (LCN) located at the extreme C terminus of SpoIIAB are both necessary and sufficient for this degradation . We also report that the use of the LCN extension as a degradation signal for ClpCP is unique to SpoIIAB.

J Bacteriol, 2003 Sep, 185(17), 5200 - 9
Definition of a second Bacillus subtilis pur regulon comprising the pur and xpt-pbuX operons plus pbuG, nupG (yxjA), and pbuE (ydhL); Johansen LE et al.; In Bacillus subtilis expression of genes or operons encoding enzymes and other proteins involved in purine synthesis is affected by purine bases and nucleosides in the growth medium . The genes belonging to the PurR regulon (purR, purA, glyA, guaC, pbuO, pbuG, and the pur, yqhZ-folD, and xpt-pbuX operons) are controlled by the PurR repressor, which inhibits transcription initiation . Other genes are regulated by a less-well-described transcription termination mechanism that responds to the presence of hypoxanthine and guanine . The pur operon and the xpt-pbuX operon, which were studied here, are regulated by both mechanisms . We isolated two mutants resistant to 2-fluoroadenine in which the pur operon and the xpt-pbuX operon are expressed at increased levels in a PurR-independent manner . The mutations were caused by deletions that disrupted a potential transcription terminator structure located immediately upstream of the ydhL gene . The 5' part of the ydhL leader region contained a 63-nucleotide (nt) sequence very similar to the 5' ends of the leaders of the pur and xpt-pbuX operons . Transcripts of these regions may form a common tandem stem-loop secondary structure . Two additional genes with potential leader regions containing the 63-nt sequence are pbuG, encoding a hypoxanthine-guanine transporter, and yxjA, which was shown to encode a purine nucleoside transporter and is renamed nupG . Transcriptional lacZ fusions and mutations in the 63-nt sequence encoding the possible secondary structures provided evidence that expression of the pur and xpt-pbuX operons and expression of the ydhL, nupG, and pbuG genes are regulated by a common mechanism . The new pur regulon is designated the XptR regulon . Except for ydhL, the operons and genes were negatively regulated by hypoxanthine and guanine . ydhL was positively regulated . The derived amino acid sequence encoded by ydhL (now called pbuE) is similar to the amino acid sequences of metabolite efflux pumps . When overexpressed, PbuE lowers the sensitivity to purine analogs . Indirect evidence indicated that PbuE decreases the size of the internal pool of hypoxanthine . This explains why the hypoxanthine- and guanine-regulated genes are expressed at elevated levels in a mutant that overexpresses pbuE.

J Biol Chem, 2003 Dec 5, 278(49), 48611 - 6 Epub 2003 Aug 14.
Bacillus subtilis hydrolyzes CheY-P at the location of its action, the flagellar switch; Szurmant H et al.; In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P, also causes hydrolysis of CheY-P to form CheY and Pi . This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch . In particular we have identified the phosphatase as FliY, which resembles E . coli switch protein FliN only in its C-terminal part, while an additional N-terminal domain is homologous to another switch protein FliM and to CheC, a protein found in the archaea and many bacteria but not in E . coli . Previous E . coli studies have localized the CheY-P binding site of the switch to FliM residues 6-15 . These residues are almost identical to the residues 6-15 in both B . subtilis FliM and FliY . We were able to show that both of these proteins are capable of binding CheY-P in vitro . Deletion of this binding region in B . subtilis mutant fliM caused the same phenotype as a cheY mutant (clockwise flagellar rotation), whereas deletion of it in fliY caused the opposite . We showed that FliY increases the rate of CheY-P hydrolysis in vitro . Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.

Biosci Biotechnol Biochem, 2003 Jul, 67(7), 1472 - 8
Either soluble or plastidic expression of recombinant protoporphyrinogen oxidase modulates tetrapyrrole biosynthesis and photosynthetic efficiency in transgenic rice; Jung S et al.; Protoporphyrinogen oxidase (Protox) is the last shared enzyme of the porphyrin pathway . As a continuation of our previous work in which the transgenic rice plants expressing the Bacillus subtilis Protox in the cytoplasm or the plastid showed resistance to diphenyl ether herbicide, this study was undertaken to identify the effects of tertapyrrole biosynthesis in these transgenic rice plants . The transgenic plants either targeted into plastids or expressed in cytoplasm showed higher Protox activity than wild-type plants did . Photosynthetic activity, measured as a quantum yield of photosystem II, was slightly higher in transgenic plants than in wild-type plants, but chlorophyll contents were not significantly different between transgenic and wild-type plants . As for porphyrin biosynthesis, both cytoplasm-expressed and plastid-targeted transgenic plants showed increased synthesis of aminolevulinic acid, Mg-Proto IX, and protoheme in comparison to wild-type plants whereas synthesis of protoporphyrin IX was similar for wild-type and transgenic plants . These results indicate that either cytoplasm or plastid expression of B . subtilis Protox in rice can upregulate the porphyrin pathway leading to increase in photosynthetic efficiency in plants.

J Appl Microbiol, 2003, 95(3), 637 - 48
Germination of spores of Bacillus subtilis with dodecylamine; Setlow B et al.; AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination . METHODS AND RESULTS: Spores of B . subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent . Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores . The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients . As measured by DPA release, dodecylamine readily induced germination of B . subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes . The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not . These latter germinated spores also did not release their pool of free adenine nucleotides . CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane . SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general . New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.

J Appl Microbiol, 2003, 95(3), 545 - 52
Effect of fermented soya beans on diarrhoea and feed efficiency in weaned piglets; Kiers JL et al.; AIMS: To evaluate anti-diarrhoeal and growth enhancing properties of fermented soya beans in weaned piglets . METHODS AND RESULTS: In a first phase piglet diet, toasted full-fat soya beans (20%) were replaced with either cooked soya beans or Rhizopus microsporus or Bacillus subtilis fermented soya beans . The effect on the incidence, severity and duration of diarrhoea in enterotoxigenic Escherichia coli (ETEC)-challenged weaned piglets was determined (pen trial, 24 piglets per treatment) . Severity of diarrhoea was significantly less on the diet with Rhizopus-fermented soya beans compared with the control diet containing toasted soya beans . Piglets fed fermented soya beans showed increased feed intake (13 and 12%), average daily weight gain (18 and 21%) and feed efficiency (3 and 8%) (for Rhizopus and Bacillus-fermented soya beans, respectively) . However, in the treatment groups an unequal mortality and a potential unequal distribution of receptor-positive piglets were observed . CONCLUSIONS: Cooked and fermented soya beans could be beneficial in the control of diarrhoea in ETEC-challenged weaned piglets (particularly Rhizopus fermented) and significantly improved weight gain and feed intake (particularly Bacillus fermented) . SIGNIFICANCE AND IMPACT OF THE STUDY: Fermented soya beans could offer benefits with respect to the control of diarrhoea and feed efficiency in piglets.

Nat Struct Biol, 2003 Sep, 10(9), 701 - 7 Epub 2003 Aug 10.
An mRNA structure that controls gene expression by binding S-adenosylmethionine; Winkler WC et al.; Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs . These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite . Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity . SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis . This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.

J Mol Biol, 2003 Aug 22, 331(4), 941 - 9
Transmembrane organization of the Bacillus subtilis chemoreceptor McpB deduced by cysteine disulfide crosslinking; Bunn MW et al.; The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17-34: TM2, residues 280-302) in each monomer with a 2-fold axis of symmetry . To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains . Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations . On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an alpha-helical interface was identified between TM1 and TM1' (containing residues 32, 36, 39, and 43) and between TM2 and TM2' (containing residues 276, 277, 280, 283 and 286) . Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain . Crosslinking of residues that were 150-180 degrees removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo . All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase) . These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins.

AIHA J (Fairfax, Va), 2003 Jul-Aug, 64(4), 533 - 7
Surface germicidal effects of ozone for microorganisms; Li CS et al.; In this study the influences of microorganism species, relative humidity, and ozone dosage on ozone surface disinfection were evaluated . Bacterial and fungal cultures were spread on agar plates and exposed to ozone . The selected microorganisms included Escherichia coli, Bacillus subtilis, Candida famata, and Penicillium citrinum . Results showed that microorganism survival fraction and ozone dosage (ozone concentration times exposure time) have an exponential relationship . Results also indicated that E . coli was the most sensitive organism to ozone exposure . E . coli required only very low ozone doses of 2-2.5 and 3.5-4 mg to obtain 50 and 80% inactivation, respectively . In addition, P . citrinum was more resistant than E . coli and required ozone doses of 40-60 and 60-120 mg to obtain 50 and 80% inactivation . In addition, spores of B . subtilis were observed to be the most resistant organism, requiring ozone doses of 40-75 and 145-150 mg to obtain 50 and 80% inactivation . Yeast was less resistant than P . citrinum and B . subtilis, requiring ozone doses of 10 and 15-19 mg to obtain 50 and 80% inactivation . It was clearly indicated that the ozone dose differences for 80% microorganism inactivation could be as high as 40 times between B . subtilis and E . coli . Ozone surface germicidal efficiency increased as relative humidity increased, which could be related to more radicals generated from ozone reaction with more water vapor at higher relative humidity . It was concluded that ozone should be highly effective and provide a reliable safety factor in treating contaminated surface . In addition, workers might need to wear suitable respiratory protection at high ozone level operation.

Antonie Van Leeuwenhoek, 2003, 84(1), 69 - 80
Transporters involved in uptake of di- and tricarboxylates in Bacillus subtilis; Krom BP et al.; Di- and tricarboxylates found as intermediates in the tricarboxylic acid cycle can be utilized by many bacteria and serve as carbon and energy source under aerobic and anaerobic conditions . A prerequisite for metabolism is that the carboxylates are transported into the cells across the cytoplasmic membrane . Bacillus subtilis is able to metabolize many di- and tricarboxylates and in this overview the available data on all known and putative di- and tricarboxylate transporters in B . subtilis is summarized . The B . subtilis transporters, that are of the secondary type, are discussed in the context of the protein families to which they belong . Available data on biochemical characterization, regulation of gene expression and the physiological function is summarized . It is concluded that in B . subtilis multiple transporters are present for tricarboxylic acid cycle intermediates.

Nucleic Acids Symp Ser, 2000, (44), 149 - 50
A study of the method to pick up a selenocysteine tRNA in Bacillus subtilis; Matsugi J et al.; In Bacillus subtilis, selenocysteine tRNA has not been identified in a total genome sequence so far (1) . To explore the system of selenocysteine incorporation in B . subtilis, we screened serine-acceptable tRNAs to find an unknown tRNA for selenocysteine by the combined method of specific biotinylation of aa-tRNA (2) and RT-PCR (3) . cDNAs obtained from the serine-acceptable tRNA pool were amplified and cloned into plasmid to read its sequence . This procedure gave cDNA library corresponding known serine tRNAs, but no candidate for selenocysteine has been found . Thus, this result, together with the previous data (4), might reveal that there is no selenocysteine tRNA in B . subtilis and/or metabolism of selenium is considerably different from known one as seen in other bacteria.

Nucleic Acids Symp Ser, 2000, (44), 5 - 6
Analysis of histidine-dependent antitermination in Bacillus subtilis hut operon; Oda M et al.; We have previously shown that a positive regulator, HutP, of Bacillus subtilis hut operon is a RNA binding protein . Here, we report precise binding site of HutP in cis-regulatory region on hut mRNA and the role of HutP in histidine-dependent antitermination of hut expression . Ethylnitrosourea modification interference assay showed that four binding sites of HutP were found in the cis-regulatory sequences and were located at the stem and the internal loop of an antiterminator structure . In vitro transcription assay indicated that HutP suppressed transcription termination in the presence of histidine . These results suggest that HutP function as an antiterminator in response to the presence of histidine.

Nucleic Acids Res Suppl, 2002, (2), 47 - 8
Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis; Kumarevel T et al.; HutP is an RNA-binding protein and regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA . A single point mutant (Val51Ile), which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging drop vapor diffusion method . The space group was P2(1)3 with unit cell dimension a = b = c = 95.60 A resolution, and contain two molecules in the asymmetric unit . A complete MAD data set of 3.0 A resolution were collected at the LIII edge of Hg.

Appl Environ Microbiol, 2003 Aug, 69(8), 4875 - 83
High-level production of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis; Kwon SJ et al.; Due to their spectroscopic properties porphyrins are of special interest for a variety of applications, ranging from drug development or targeting to material sciences and chemical and biological sensors . Since chemical syntheses are limited in terms of regio- and stereoselective functionalization of porphyrins, a biosynthetic approach with tailored enzyme catalysts offers a promising alternative . In this paper, we describe assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host . Without further optimization, this approach yielded remarkable porphyrin production levels, up to 90 micro mol/liter, which is close to industrial vitamin B(12) production levels . Different combinations of the genes were used to produce all major porphyrins that occur as intermediates in heme biosynthesis . All these porphyrin intermediates were obtained in high yields . The product spectrum was analyzed and quantified by using high-performance liquid chromatography . Intriguingly, although protoporphyrin IX could be produced at high levels, overexpressed Bacillus subtilis ferrochelatase could not convert this substrate appreciably into heme . However, further investigation clearly revealed a high level of expression of the ferrochelatase and a high level of activity in vitro . These results may indicate that heme has a regulatory impact on the iron uptake of E . coli or that the ferrochelatase is inactive in vivo due to an incompatible enzyme interaction.

Mikrobiologiia, 2003 May-Jun, 72(3), 400 - 6
{Cellulose decomposition under nitrogen deficiency by bacteria isolated from the intestines of phytophagous vertebrates}; Ushakova NA et al.; A nitrogen-fixing strain identified as Klebsiella pneumonia 402-2 and two endoglucanase-synthesizing Bacillus strains were isolated from the intestines of phytophagous animals . One of the Bacillus strains was identified as Bacillus subtilis GL . Klebsiella pneumoniae 402-2 increased the endoglucanase activity of both Bacillus strains in mixed cultures . The data on the taxonomic position of strains 402-2 and GL and on the nitrogen-fixing capacity of strain 402-2 were confirmed by sequencing and analyzing their 16S rRNA genes and by amplifying the nitrogenase gene nifH.

FEMS Microbiol Lett, 2003 Aug 8, 225(1), 93 - 9
The putative anti-anti-sigma factor BldG is post-translationally modified by phosphorylation in Streptomyces coelicolor; Bignell DR et al.; The Streptomyces coelicolor bldG gene encodes a protein showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors of Bacillus subtilis . Purified maltose binding protein-BldG could be phosphorylated in vitro by wild-type S . coelicolor crude extract, and both the phosphorylated and unphosphorylated forms of BldG could be detected in vivo using isoelectric focusing . ATP was shown to serve as the phosphoryl group donor, and phosphorylation of BldG was abolished when the putative phosphorylation site was changed from a serine to an alanine residue . A bldG mutant strain expressing the non-phosphorylatable BldG protein was unable to undergo morphological differentiation or produce antibiotics even after prolonged incubation, suggesting that phosphorylation of BldG is necessary for proper development in S . coelicolor.

Yeast, 2003 Aug, 20(11), 995 - 1005
Saccharomyces cerevisiae QNS1 codes for NAD(+) synthetase that is functionally conserved in mammals; Suda Y et al.; NAD(+), an essential molecule involved in a variety of cellular processes, is synthesized through de novo and salvage pathways . NAD(+) synthetase catalyses the final step in both pathways . Here we show that this enzyme is encoded by the QNS1 gene in Saccharomyces cerevisiae . Expression of Escherichia coli or Bacillus subtilis NAD(+) synthetases was able to suppress the lethality of a qns1 deletion, while a B . subtilis NAD(+) synthetase mutant with lowered catalytic activity was not . Overexpression of QNS1 tagged with HA led to elevated levels of NAD(+) synthetase activity in yeast extracts, and this activity can be recovered by immunoprecipitation using anti-HA antibody . An allele of QNS1 was constructed that carries a point mutation predicted to reduce the catalytic activity . Overexpression of this allele, qns1(G521E), failed to elevate NAD(+) synthetase levels and qns1(G521E) could not rescue the lethality caused by the depletion of Qns1p . These results demonstrate that NAD(+) synthetase activity is essential for cell viability . A GFP-tagged version of Qns1p displayed a diffuse localization in both the nucleus and the cytosol . Finally, the rat homologue of QNS1 was cloned and shown to functionally replace yeast QNS1, indicating that NAD(+) synthetase is functionally conserved from bacteria to yeast and mammals .

Chembiochem, 2003 Aug 4, 4(8), 768 - 73
CloN6, a novel methyltransferase catalysing the methylation of the pyrrole-2-carboxyl moiety of clorobiocin; Westrich L et al.; The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit . This pyrrole unit is derived from L-proline, and it would be expected that its 5-methyl group should be introduced by a methylation reaction . However, sequence analysis of the clorobiocin biosynthetic gene cluster did not reveal a gene with sequence similarity to the SAM-dependent methyltransferases that could be assigned to this reaction . This study, however, has provided evidence that the gene cloN6 is involved in this methylation reaction . Its gene product CloN6 shares conserved sequence motifs with the recently identified radical SAM protein superfamily, and it has been suggested that members of this family can catalyse methylcobalamin-dependent methylation reactions . cloN6 was inactivated in the clorobiocin producer Streptomyces roseochromogenes var . oscitans DS 12.976 by use of the PCR-targeting method . The cloN6(-) mutants accumulated, instead of clorobiocin, a derivative lacking the 5"'-methyl group of the pyrrole moiety (termed novclobiocin 109) . A structural isomer carrying the pyrrole-2-carboxyl moiety at 2"-OH rather than at the 3"-OH of the deoxysugar (novclobiocin 110), and a derivative completely lacking the pyrrole unit (novclobiocin 104) were also identified . The structures of the metabolites were confirmed by NMR and MS analysis . Antibacterial activity tests against Bacillus subtilis showed that novclobiocin 109 and novclobiocin 110 have antibacterial activities about eight times less than that of clorobiocin, whereas novclobiocin 104 showed no activity under the test conditions.

Can J Microbiol, 2003 Apr, 49(4), 253 - 62
Characterization of an antifungal soil bacterium and its antagonistic activities against Fusarium species; Chan YK et al.; Bacteria were isolated from a cultivated soil and screened for antagonistic activity against Fusarium graminearum, a predominant agent of ear rot and head blight in cereal crops . Based on its in vitro effectiveness, isolate D1/2 was selected for characterization and identified as a strain of Bacillus subtilis by phenotypic tests and comparative analysis of its 16S ribosomal RNA gene (rDNA) sequence . It inhibited the mycelial growth of a collection of common fungal phytopathogens, including eight Fusarium species, three other ascomycetes, and one basidiomycete . The cell-free culture filtrate of D1/2 at different dilutions was active against macroconidium germination and hyphal growth of F . graminearum, depending on the initial macroconidium density . It induced the formation of swollen hyphal cells in liquid cultures of this fungus grown from macroconidia . A bioassay also demonstrated that D1/2 offered in planta protection against the damping-off disease in alfalfa seedlings caused by F . graminearum, while the type strain of B . subtilis was ineffective . Hence, B . subtilis D1/2 or its culture filtrate has potential application in controlling plant diseases caused by Fusarium.

Can J Microbiol, 2003 May, 49(5), 313 - 25
Chromosome DNA fragmentation and excretion caused by defective prophage gene expression in the early-exponential-phase culture of Bacillus subtilis; Shingaki R et al.; Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria-Bertani medium . Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands . Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis . Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome . SDS-PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA . Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome . Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion . Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168 . The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.

J Bacteriol, 2003 Aug, 185(16), 5019 - 22
The pst operon of Bacillus subtilis is specifically induced by alkali stress; Atalla A et al.; To cope with a sudden increase in the external pH value to 8.9, Bacillus subtilis cells induce about 80 genes which can be divided into two classes . Most of these genes are members of the sigma(W) regulon, while some are under the control of so-far-unknown transcriptional regulators . The genes of the pst operon belong to the second class . Here, we attempted to answer the questions of why and how the genes of this operon are induced . Using transcriptional fusions to two of the five genes of this operon, we confirmed their induction after alkali stress . Furthermore, a Northern blot experiment revealed that the complete operon was alkali inducible, that the transcriptional start site used was identical to that used after phosphate starvation, and that induction was prevented in a phoR background . Most interestingly, increasing the phosphate concentration within the medium prevented alkali induction of the pst operon, and phoA, another member of the PhoRP regulon, did not respond to alkali stress . In the end, we showed that alkali treatment completely prevented phosphate uptake . These results are discussed to explain alkali induction of the pst operon.

J Bacteriol, 2003 Aug, 185(16), 4883 - 90
Regulation of the Bacillus subtilis extracytoplasmic function protein sigma(Y) and its target promoters; Cao M et al.; The Bacillus subtilis extracytoplasmic function sigma factor sigma(Y) is of unknown function . We demonstrate that the sigY operon is expressed from an autoregulatory promoter site, P(Y) . We selected for transposon-induced mutations that upregulate P(Y) transcription in an attempt to identify genes involved in sigma(Y) regulation . The resulting insertions disrupted yxlC, the gene immediately downstream of sigY . However, the phenotype of the yxlC::Tn10 insertion was due to polarity on the downstream genes of the sigY operon; a nonpolar insertion in yxlC did not lead to derepression of P(Y) . Further analyses revealed that both yxlD and yxlE encoded proteins important for the negative regulation of sigma(Y) activity . A comparison of the transcriptomes of wild-type and yxlC::Tn10 mutant strains revealed elevated expression of several operons . However, only one additional gene, ybgB, was unambiguously identified as a direct target for sigma(Y) . This was supported by analysis of direct targets for sigma(Y) transcription with whole-genome runoff transcription followed by macroarray analysis.

J Bacteriol, 2003 Aug, 185(16), 4861 - 71
Molecular analysis of Phr peptide processing in Bacillus subtilis; Stephenson S et al.; In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins . Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development . The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F approximately P intermediate response regulator of the sporulation phosphorelay . The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation . The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form . The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme . In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide . This processing event is most likely independent of type I signal peptidase activity . In vivo and in vitro analyses indicate that none of the five signal peptidases of B . subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing . However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

J Bacteriol, 2003 Aug, 185(16), 4844 - 50
Allosteric regulation of Bacillus subtilis NAD kinase by quinolinic acid; Garavaglia S et al.; NADP is essential for biosynthetic pathways, energy, and signal transduction . In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase . We expressed, purified, and characterized Bacillus subtilis NAD kinase . This enzyme represents a new member of the inorganic polyphosphate {poly(P)}/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors . NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis . We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme . Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism.

J Bacteriol, 2003 Aug, 185(16), 4816 - 24
Mannitol-1-phosphate dehydrogenase (MtlD) is required for mannitol and glucitol assimilation in Bacillus subtilis: possible cooperation of mtl and gut operons; Watanabe S et al.; We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in Bacillus subtilis . Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the mtl operon, whereas glucitol induced both mtl and gut operons . The B . subtilis mtl operon consists of mtlA (encoding enzyme IICBA(mt1)) and mtlD, and its transcriptional regulator gene, mtlR, is located 14.4 kb downstream from the mtl operon on the chromosome . The mtlA, mtlD, and mtlR mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol . However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-beta-D-thiogalactopyranoside) . This mutant has an IPTG-inducible promoter (Pspac promoter) located in mtlA, and this site corresponds to the upstream region of mtlD . Insertion mutants of mtlD harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol . In contrast, an insertion mutant of mtlA could grow on glucitol but not on mannitol in the presence or absence of IPTG . MtlR bound to the promoter region of the mtl operon but not to a DNA fragment containing the gut promoter region.

J Bacteriol, 2003 Aug, 185(16), 4764 - 71
Transcriptional pausing in the Bacillus subtilis pyr operon in vitro: a role in transcriptional attenuation?
Zhang H, Switzer RL.
The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (pyr genes) are regulated in Bacillus subtilis and many other bacterial species by transcriptional attenuation . When UMP or UTP is bound to the PyrR regulatory protein, it binds to pyr mRNA at specific sequences and secondary structures in the RNA . Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the pyr genes lying downstream from the terminator . The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of pyr genes to test for formation of paused transcripts in vitro . Using templates with each of the three known B . subtilis pyr attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA . In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop . Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for pyr attenuation in vivo . With two of three pyr templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites . This suggests that NusA, by stabilizing pausing, plays a role in termination of pyr transcription in vivo.

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