Bacillus subtilis

Wei Sheng Wu Xue Bao, 1999 Feb, 39(1), 60 - 3
{Production of neutral beta-mannanase by Bacillus subtilis and its properties}; Cui F et al.; Neutral beta-mannanase was produced by Bacillus subtilis BM9602 isolated from a soil . For this strain the neutral beta-mannanase was produced only when polysaccharides were used as a carbon source . Organic nitrogen source was superior to inorganic nitrogen source on the enzyme production . The optimum liquid medium consisted of 4% konjak powder, 1% each of beef peptone and yeast extract . The optimum culture conditions were initial pH 8.5, temperature 35 degrees C and cultivation time 36 h . Enzyme activity of culture filtrate to 0.5% galactomannan polysaccharide was 96 IU/mL per minute at pH 6.0 and 50 degrees C for 10 min . The optimum pH and temperature for enzyme action were 6.0 and 50 degrees C respectively . The enzyme was stable below 50 degrees C and pH 5.0-10.0 . The enzyme hydrolyzed konjak powder and locust bean gum to form oligosaccharides.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 375 - 7 Epub 2003 Jan 23.
Expression, crystallization and activities of the two family 11 aldo-keto reductases from Bacillus subtilis; Ehrensberger A et al.; Two members of the aldo-keto reductase family 11 from Bacillus subtilis have been crystallized and their oxidoreductase activity confirmed . AKR11A is a protein induced by inositol and repressed by glucose . AKR11B is induced when the cell is stressed by heat, acid, ethanol, starvation or osmotic shock and is therefore classified as a general stress protein . The expected NADPH-dependent sugar reductase activities for both proteins have been confirmed kinetically with several substrates . AKR11B exhibited typical aldo-keto reductase kinetics . However, only trace activity was found in AKR11A . To examine the effects of differences in sequence on the structures and functions of these enzymes, a crystallographic study has been initiated . AKR11A has been crystallized in its apo form and AKR11B crystals were obtained in complex with NADP(+).

J Am Chem Soc, 2003 Feb 5, 125(5), 1244 - 52
Heavy atom isotope effects on the reaction catalyzed by the oxalate decarboxylase from Bacillus subtilis; Reinhardt LA et al.; Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate . Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity . The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated . Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B . subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669) . We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC . At pH 4.2, the OxDC-catalyzed formation of formate and CO(2) have normal (13)C isotope effects of 1.5% +/- 0.1% and 0.5% +/- 0.1%, respectively, while the (18)O isotope effect on the formation of formate is 1.1% +/- 0.2% normal . Similarly at pH 5.7, the production of formate and CO(2) exhibits normal (13)C isotope effects of 1.9% +/- 0.1% and 0.8% +/- 0.1%, respectively, and the (18)O isotope effect on the formation of formate is 1.0% +/- 0.2% normal . The (18)O isotope effect on the formation of CO(2), however, 0.7% +/- 0.2%, is inverse at pH 5.7 . These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion . Subsequent reduction and protonation of this intermediate then gives formate.

Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 499 - 504
{Studies on antimicrobial activity of extracts from thyme}; Fan M et al.; The extracts from thyme by water and ethanol, thyme essential oil, thymol and carvacrol were used as antimicrobial agents in this paper . The results show that all antimicrobial agents used have strong inhibition activity against Staphalococcus aureus, Bacillus subtilis, Escherichia coli.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 815 - 9 Epub 2003 Jan 27.
Microbe forensics: oxygen and hydrogen stable isotope ratios in Bacillus subtilis cells and spores; Kreuzer-Martin HW et al.; Bacillus subtilis, a Gram-positive, endospore-forming soil bacterium, was grown in media made with water of varying oxygen (delta(18)O) and hydrogen (deltaD) stable isotope ratios . Logarithmically growing cells and spores were each harvested from the cultures and their delta(18)O and deltaD values determined . Oxygen and hydrogen stable isotope ratios of organic matter were linearly related with those of the media water . We used the relationships determined in these experiments to calculate the effective whole-cell fractionation factors between water and organic matter for B . subtilis . We then predicted the delta(18)O and deltaD values of spores produced in nutritionally identical media and local water sources for five different locations around the United States . Each of the measured delta(18)O and deltaD values of the spores matched the predicted values within a 95% confidence interval, indicating that stable isotope ratio analyses may be a powerful tool for tracing the geographic point-of-origin for microbial products.

Mol Biol Rep, 2002 Dec, 29(4), 383 - 5
Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I, from Bacillus subtilis . Bsu121I, a type II restriction endonuclease from Bacillus subtilis; Jutur PP et al.; A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified . The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography . SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease . The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp . The endonuclease activity required Mg(+2) as cofactor like other type II endonucleases.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 13 - 9
{Purification and properties of antifungal protein X98III from Bacillus subtilis}; Xie D et al.; Bacillus subtilis BS-98 is one of the antagonistic strains strongly against plant fungal pathogens such as Physalospora piricola Nose . The antifungal protein was purified by ammonium sulphate precipitation and column chromatography on Sephadex G-100 and DEAE-cellulose, and it was named X98III . Molecular weight of X98III is 59,000 by SDS-PAGE and PI value is 4.50 by PAG-IEF, respectively . X98III was demonstrated as glycoprotein and lipoprotein by CAM (cellulose acetate membrane) electrophoresis and special staining . We estimated it contains 6% saccharides by using DNS methods . This protein was also found to be thermostabale and partially sensitive to proteinases . The amino acid analysis of the protein X98III showed that it comprises of 11 different amino acids and Glu, Tyr, Cys are the abundant amino acids . No Asp, Phe and Met were found . Purified X98III has strong inhibiting activity against the pathogens of Physalospora piricola, Phoma asparagi, etc . The antifungal mechanism of X98III was mainly disintegration of the cell wall to make the hyphae abnormal and the spores germinate abnormally or can not germinate at all.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 6 - 12
{Identification of a sulfhydryl-reducing agent-inducible protein highly homologous to protein synthesis elongation factor Tu in Bacillus subtilis}; Wang T et al.; It was examined that the effect of beta-mercaptoethanol and dithiothreitol treatments, which should affect disulfide bond formation of proteins, on cellular protein components of Bacillus subtilis . In LB medium, the treatments induced the synthesis of a 50 kD protein (P50), which is synthesized constitutively under normal growth condition and is a major cytoplasmic protein . P50 was also induced by heat shock, but not by sporulation . In Schaeffer's sporulation medium, however, P50 was not induced by the sulfhydryl-reducing agents . This suggests that the sulfhydryl-reducing agent-inducibility of P50 might depent on specific physiological condition(s) . The amino terminal sequences of two of the four main V8 protease fragments of P50 were determined . A search in databases revealed that P50 was highly homologous to protein synthesis elongation factor Tu of B . subtilis.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 105 - 8
{Expression of alpha-acetolactate decarboxylase gene from Bacillus subtilis in Brewer's yeast}; Guo W et al.; Yeast YIp-type expression recombinant plasmid(pCMA2-1) was constructed . The expression of alpha-acetolactate decarboxylase gene from Bacillus subtilis was controled by CUP1 promoter and its own terminator . The recombinant plasmid pCMA2-1 was introduced into the brewer's yeast PJ3-5 . Transformants were selected using copper resistance as selected marker . The results of activity assay showed that PJ3-5 didn't produce alpha-acetolactate decarboxylase(ALDC), where as the activity of alpha-ALDC in transformants were induced by copper sulfate . The laboratory scale fermentation test confirmed that the total diacetyl concentration was reduced effectively by alpha-ALDC in transformant.

Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 198 - 203
{Purification and properties of neutral phytase form Bacillus subtilis}; Wang Y et al.; A strain Bacillus subtilis producing neutral phytase was screened from soil . The protein of phytase was purified by HPLC . Optimal pH value and temperature of the phytase for its activity were 7.5 and 55 degrees C, respectively . The Km values of the phytase for dodecasodium phytate under 37 degrees C was 0.19 mmol/L . The molecule weight of the phytase protein was determined as about 45 kD by SDS-PAGE . The N-terminal amino acids sequence of the phytase protein was determined as Lys-His-Lys-Leu-Ser-Asp-Pro-Tyr-His-Phe-Thr by amino acids sequence analysis.

Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 420 - 4
{Purification and characterization of an endo-beta-1, 4-mannanase from Bacillus subtilis BM9602}; Li W et al.; An extracellular neutral endo-beta-mannanase(endo-beta-1, 4-D-mannan mannanohydrolase, EC 3.2.1.78)of Bacillus subtilis BM9602 was purified to electrophoretic homogeneity by ammonium sulfate precipitation and DEAE-cellulose DE22 chromatography with 45.5 fold and 5.9% yield . It's molecular weight and pl value were 35 kD by SDS-PAGE and 4.5 by isoelectric focusing, respectively . The enzyme was the most active at pH 5.8 . The optimum temperature of the enzyme activity was 50 degrees C . The enzyme was stable at pH 6.0-8.0 and below 50 degrees C . The activity of the enzyme was inhibited by Hg2+, Ag+ strongly . For various substrates, such as locust bean gum, guar gum, sesbania gum and konjac gum, Km and Vmax value of the enzyme were 3.8, 14.9, 11.3, 2.4 mg/mL, and 24.5, 86.5, 38.4, 19.8 mumol.min-1.mg-1, respectively . The enzyme hydrolize various plant beta-mannans, with valuable oligosaccharides and without monosaccharide.

Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 95 - 9
{Bacillus subtilis undergoes natural genetic transformation on agar plates}; Chen X et al.; This paper conducted preliminary investigation on natural genetic transformation of Bacillus subtilis on agar plates . The results showed that, under same conditions, the natural transformation of the strain on agar plate was much more efficient than that of standard liquid method, and the transformation system could sustain higher DNase concentration . In addition, that the LB culture, which usually did not support the strain acquire competency, could undergo transformation as soon as it being spread on agar plate, suggested that the natural genetic transformation of Bacillus subtilis on solid plates may be different form the standard liquid transformation method . The cell-to-cell transformation between strains having different genetic makers could also be observed on agar plates.

J Mol Biol, 2003 Feb 7, 326(1), 189 - 201
Solution structure of the Bacillus subtilis T-box antiterminator RNA: seven nucleotide bulge characterized by stacking and flexibility; Gerdeman MS et al.; The T-box transcription antitermination regulatory system is an important mechanism for regulation of expression of aminoacyl-tRNA synthetase, amino acid biosynthesis and transporter gene expression in Gram-positive bacteria . Antitermination is dependent on a complex set of interactions between uncharged tRNA and the leader region of the mRNA of the regulated gene . Here, we report the solution structure of a model RNA, based on the Bacillus subtilis tyrS antiterminator, determined to an rmsd of 3.47A for all nine converged structures and 2.66A for the seven structures representing the consensus family . The antiterminator is comprised of two short helices with an intervening 7nt bulge . The bulge region of the antiterminator, which ultimately interacts with the acceptor end of tRNA, exhibits extensive stacking at the 3' end (encompassing the highly conserved ACC residues) and is the site of a pronounced kink between the two flanking helices . The 5' end of the bulge exhibits evidence of conformational flexibility . On the basis of the structural studies, there is no indication that the bases at the 5' end of the bulge that ultimately base-pair with tRNA are pre-organized for binding . Instead, the data are consistent with a model in which the stacking-induced structure at the 3' end of the bulge may facilitate the pre-selection of a set of conformations for the tRNA to sample during binding.

Curr Biol, 2003 Jan 21, 13(2), 129 - 33
Trait-to-gene: a computational method for predicting the function of uncharacterized genes; Levesque M et al.; The function of unknown genes is often inferred from comparisons to well-characterized homologs . In this paper, we show that, even if all of the homologs of a gene are unannotated, its function may be deduced through phylogenetic profiling . We have designed a series of algorithms that make functional predictions of genes based on orthology and set theory, but our approach to predicting gene function requires no previous knowledge of homolog function . With this technique, we successfully identified 94% of the clusters of orthologous groups that are known to be involved in flagella development or function . As a test, we removed the function of three putative flagellar genes that had been previously uncharacterized in Bacillus subtilis . We observed a motility phenotype for two of these three genes . Thus, these algorithms allow for high-throughput functional prediction of genes beyond that provided by simple orthology-based annotation endeavors.

Dev Comp Immunol, 2003 Feb, 27(2), 79 - 85
Enhanced antibacterial activity in Hydra polyps lacking nerve cells; Kasahara S et al.; The nervous system evolved within cnidarians . When assessing antibacterial activity in the freshwater polyp Hydra, we observed a strong correlation between the number of neurons present and the antibacterial activity . Tissue lacking neurons had a drastically enhanced antibacterial activity against Gram-positive (Bacillus subtilis) and Gram-negative (E . coli) bacteria compared to control tissue . The results indicate direct and strong neural influences on immunity in the phylogenetically oldest animals having a nervous system.

Infect Immun, 2003 Feb, 71(2), 1011 - 5
Modulation of J774.1 macrophage L-arginine metabolism by intracellular Mycobacterium bovis BCG; Peteroy-Kelly MA et al.; Using a Mycobacterium bovis BCG mutant (AS1) lacking a Bacillus subtilis L-arginine transporter homolog, we demonstrate here that the interaction between intracellular mycobacteria and the macrophage with respect to L-arginine transport and metabolism is quite complex . Intracellular AS1 stimulates macrophage L-arginine transport and accumulates 2.5-fold more (3)H label derived from L-arginine than does the wild type . These studies suggest that the accumulation of (3)H label reflects the acquisition of metabolites of L-arginine produced by the macrophage.

Protein Eng, 2002 Nov, 15(11), 913 - 21
Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase; Symmank H et al.; The Bacillus subtilis strain ATCC 21332 produces the lipoheptapeptide surfactin, a highly potent biosurfactant synthesized by a large multimodular peptide synthetase . We report the genetic engineering of the surfactin biosynthesis resulting in the production of a novel lipohexapeptide with altered antimicrobial activities . A combination of in vitro and in vivo recombination approaches was used to construct a modified peptide synthetase by eliminating a large internal region of the enzyme containing a complete amino acid incorporating module . The remaining modules adjacent to the deletion were recombined at different highly conserved sequence motifs characteristic of amino acid incorporating modules of peptide synthetases . The primary goal of this work was to identify permissive fusion sites suitable for the engineering of peptide synthetase genes by genetic recombination . Analysis of the rearranged enzymes after purification from B . subtilis and from the heterologous host Escherichia coli revealed that the selection of the recombination site is of crucial importance for a successful engineering . Only the recombination at a specific HHII x DGVS sequence motif resulted in an active peptide synthetase . The expected lipohexapeptide was produced in vivo and first evidence of a reduced toxicity against erythrocytes and an enhanced lysis of Bacillus licheniformis cells was shown.

Prostaglandins Leukot Essent Fatty Acids, 2003 Feb, 68(2), 187 - 90
Regulation of fatty acid desaturation in Bacillus subtilis; Mansilla MC et al.; The Des pathway of Bacillus subtilis regulates the expression of the acyl-lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids from saturated phospholipid precursors . Activation of this pathway takes place when cells are shifted to low growth temperature or when they are grown in minimal media in the absence of isoleucine supplies . The master switch for the Des pathway is a two-component regulatory system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene . We propose that both, a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism, involving the ability of DesK to sense a decrease in membrane fluidity.

J Inorg Biochem, 2003 Jan 1, 93(1-2), 92 - 9
Expression, purification and characterisation of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI; Green AJ et al.; The fer gene from Bacillus subtilis has been subcloned and overexpressed in Escherichia coli and the protein (Fer) purified to homogeneity . N-Terminal sequencing and mass spectrometry indicate that the initiator methionine is removed from the protein and that the molecular mass is 8732 Da consistent with that deduced from the gene sequence . Amino-acid sequence comparisons indicate that Fer is a ferredoxin containing a 4Fe-4S cluster . The electron paramagnetic resonance spectrum of the reduced form of Fer is typical for a {4Fe-4S}(+) cluster showing rhombic signals with g values of 2.07, 1.93 and 1.88 . Reduced Fer also gives rise to a magnetic circular dichroism spectrum typical of a {4Fe-4S}(+) cluster . Potentiometric titrations indicate that Fer has a reduction potential of -385+/-10 mV for the {4Fe-4S}(+)-{4Fe-4S}(2+) redox couple, well within the normal range expected for such a ferredoxin . A proposed physiological role for Fer is as an electron donor to cytochrome P450 BioI . Studies on Fer binding to P450 BioI give rise to a K(d) value of 0.87+/-0.10 microM . Anaerobic experiments using CO-saturated buffer indicate that Fer is indeed capable of transferring electrons to this cytochrome P450 albeit at a fairly low rate.

Mol Microbiol, 2003 Feb, 47(3), 699 - 714
Specialized osmotic stress response systems involve multiple SigB-like sigma factors in Streptomyces coelicolor; Viollier PH et al.; Whereas in Bacillus subtilis, a general stress response stimulon under the control of a single sigma factor (SigB) is induced by different physiological and environmental stresses (heat, salt or ethanol shock), in Streptomyces coelicolor, these environmental stresses induce independent sets of proteins, and its genome encodes nine SigB paralogues . To investigate possible functions of multiple sigB-like genes in S . coelicolor, Pctc, a promoter routinely used to assay SigB activity in vivo, was analysed as a heterologous reporter . The fact that Pctc was activated by osmotic shock, but not by heat or ethanol, confirmed that stress response system(s) could operate independently in S . coelicolor . Pctc was also induced transiently during growth of liquid cultures, presumably by nutritional signals . We purified an RNA polymerase holoenzyme from crude extracts that catalysed specific transcription of Pctc in vitro . Its sigma subunit was identified as a product of the sigH gene, which is co-transcribed downstream of a putative antisigma factor gene (prsH) . Although the sigH function was not needed for normal colony morphology, prsH was conditionally required for both aerial hyphae formation and regulation of antibiotic biosynthesis . Levels of two different sigH-encoded proteins were growth phase dependent but not significantly changed by osmotic stress, implying the important roles of post-translational regulatory elements such as PrsH . In addition, synthesis of three other SigH-like proteins was induced by osmotic stress, but not by ethanol or heat . Two of them were genetically assigned to sigH homologous loci sigI and sigJ and shown to be independently regulated . This family of SigH-like proteins displayed different osmotic response kinetics . Thus, in contrast to many other bacteria, S . coelicolor uses an osmotic sensory system that can co-ordinate the activity of multiple paralogues to control the relative activity of promoters within the same stress stimulon . Such specialized stress response systems may reflect adaptive functions needed for colonial differentiation.

J Appl Microbiol, 2003, 94(2), 184 - 90
The effect of acid shock on sporulating Bacillus subtilis cells; Lee JK et al.; AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently . METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation . The D85-value of B . subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C . ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores . Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation . CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B . subtilis wild type . The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B . subtilis cells . The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.

J Appl Microbiol, 2003, 94(2), 159 - 66
An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores; Chitarra GS et al.; AIMS: To identify and characterize an antifungal compound produced by Bacillus subtilis YM 10-20 which prevents spore germination of Penicillium roqueforti . METHODS AND RESULTS: The antifungal compound was isolated by acid precipitation with HCl . This compound inhibited fungal germination and growth . Identification by HPLC and mass spectrometry analysis showed high similarity to iturin A . Permeabilization and morphological changes in P . roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and SEM, respectively . CONCLUSOINS: The iturin-like compound produced by B . subtilis YM 10-20 permeabilizes fungal spores and blocks germination . SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescence staining in combination with flow cytometry and scanning electron microscopy are efficient tools for assessing the action of antifungal compounds against spores . Iturin-like compounds may permeabilize fungal spores and inhibit their germination.

J Bacteriol, 2003 Feb, 185(3), 973 - 82
The absence of FtsH metalloprotease activity causes overexpression of the sigmaW-controlled pbpE gene, resulting in filamentous growth of Bacillus subtilis; Zellmeier S et al.; FtsH is a membrane-bound and energy-dependent metalloprotease in bacteria which is involved in the posttranslational control of the activity of a variety of important transcription factors and in the degradation of uncomplexed integral membrane proteins . For Bacillus subtilis, little is known about the target proteins of FtsH protease . Its gene is not essential, but knockout strains display a pleiotropic phenotype including sensitivity toward salt and heat stress, defects in sporulation and competence, and largely filamentous growth . Comparison of the intracellular proteomes of wild-type and ftsH knockout strains revealed that at least nine proteins accumulated in the absence of ftsH, four of which could be identified . Two of these proteins turned out to be members of the sigma(W) regulon . Accumulation of one of these sigma(W)-controlled proteins, the penicillin-binding protein PBP4*, was analyzed in more detail . We could show that PBP4* is not a proteolytic substrate of FtsH and that its overproduction is due to the enhanced transcription of its gene (pbpE) in ftsH null mutants . The filamentous growth phenotype of DeltaftsH strains was abolished in a DeltaftsH DeltapbpE double knockout . In ftsH wild-type strains with the pbpE gene under regulatable control, pbpE overexpression caused filamentation of the cells . DNA macroarray analysis revealed that most genes of the sigma(W) regulon are transcribed at elevated levels in an ftsH mutant . The influence of FtsH on sigma(W)-controlled genes is discussed.

J Bacteriol, 2003 Feb, 185(3), 957 - 65
Characterization of the stringent response and rel(Bbu) expression in Borrelia burgdorferi; Bugrysheva J et al.; The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp . We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B . burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B . burgdorferi under these conditions . Here we report that (p)ppGpp levels in B . burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis . Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels . One-half as much of the mRNA of the gene encoding the Rel protein of B . burgdorferi (rel(Bbu)) was produced by B31 as by N40 during in vitro growth (2 +/- 0.5 and 4 +/- 0.8 fg of rel(Bbu) mRNA/ng of total Borrelia RNA, respectively) . Although the amounts of N40 rel(Bbu) mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks . In contrast to the lack of change in rel(Bbu) mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis Rel(Bsu) were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro . Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in rel(Bbu) promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 rel(Bbu) ATG start codon and identical sigma(70)-like promoters were identified by primer extension and sequencing analysis . rel(Bbu) complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced rel(Bbu) mRNA . These results indicate that rel(Bbu) is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31 . They also suggest that in B . burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E . coli and that (p)ppGpp may not be responsible for growth rate control.

J Bacteriol, 2003 Feb, 185(3), 879 - 86
Postdivisional synthesis of the Sporosarcina ureae DNA translocase SpoIIIE either in the mother cell or in the prespore enables Bacillus subtilis to translocate DNA from the mother cell to the prespore; Chary VK et al.; The differentiation of vegetative cells of Bacillus subtilis into spores involves asymmetric cell division, which precedes complete chromosome partitioning . The DNA translocase SpoIIIE is required to translocate the origin distal 70% of the chromosome from the larger mother cell into the smaller prespore, the two cells that result from the division . We have tested the effect of altering the time and location of SpoIIIE synthesis on spore formation . We have expressed the spoIIIE homologue from Sporosarcina ureae in B . subtilis under the control of different promoters . Expression from either a weak mother cell-specific (sigma(E)) promoter or a weak prespore-specific (sigma(F)) promoter partly complemented the sporulation defect of a spoIIIE36 mutant; however, expression from a strong prespore-specific (sigma(F)) promoter did not . DNA translocation from the mother cell to the prespore was assayed using spoIIQ-lacZ inserted at thrC; transcription of spoIIQ occurs only in the prespore . Translocation of thrC::spoIIQ-lacZ into the prespore occurred efficiently when spoIIIE(Su) was expressed from the weak sigma(E)- or sigma(F)-controlled promoters but not when it was expressed from the strong sigma(F)-controlled promoter . It is speculated that the mechanism directing SpoIIIE insertion into the septum in the correct orientation may accommodate slow postseptational, prespore-specific SpoIIIE synthesis but may be swamped by strong prespore-specific synthesis.

J Bacteriol, 2003 Feb, 185(3), 854 - 9
CcpA-independent regulation of expression of the Mg2+ -citrate transporter gene citM by arginine metabolism in Bacillus subtilis; Warner JB et al.; Transcriptional regulation of the Mg(2+)-citrate transporter, CitM, the main citrate uptake system of Bacillus subtilis, was studied during growth in rich medium . Citrate in the growth medium was required for induction under all growth conditions . In Luria-Bertani medium containing citrate, citM expression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase . The repression was relieved when the cells were grown in spent Luria-Bertani medium . The addition of a mixture of 18 amino acids restored repression . L-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor of citM expression . Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression . Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR) . Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth.

Water Res, 2003 Feb, 37(4), 833 - 44
Inactivation of Bacillus subtilis spores with ozone and monochloramine; Larson MA et al.; The inactivation kinetics of Bacillus subtilis spores with ozone and monochloramine was characterized by a lag phase followed by a pseudo-first-order rate of inactivation . The lag phase decreased and the post-lag phase rate constant increased with increasing temperature within the range investigated (1-30 degrees C for ozone, 1-20 degrees C for monochloramine) . The corresponding activation energies were 46820 J/mol for ozone and 79640 J/mol for monochloramine . The CT concept was found to be valid within the concentration range investigated of 0.44-4.8 mg/l for ozone, and 3.8-7.7 mg/l as Cl(2) for monochloramine . The inactivation kinetics of B . subtilis spores with both ozone and monochloramine varied with pH within the range of pH 6-10 investigated . The fastest ozone and monochloramine inactivation rates were observed at pH 10 and 6, respectively . Different stocks of the same strain of B . subtilis spores had different resistance to ozone and monochloramine mainly because of discrepancies in the extent of the lag phase . B . subtilis spores might not be conservative surrogates for C . parvum oocysts for ozone disinfection at relatively low temperature mainly due to the spores having a lower activation energy compared to that for the oocysts . In contrast, the activation energy for monochloramine was comparable for both microorganisms but differences in the extent of the lag phase might result in the spores being overly conservative surrogates for the oocysts at relatively low temperature.

Dev Cell, 2003 Jan, 4(1), 19 - 28
The bacterial cytoskeleton: in vivo dynamics of the actin-like protein Mbl of Bacillus subtilis; Carballido-Lopez R et al.; Mbl is a bacterial actin homolog that controls cell morphogenesis in Bacillus subtilis . A functional GFP-Mbl fusion protein was used to examine the behavior of the helical cables formed by Mbl protein in live B . subtilis cells . The cables undergo dynamic changes during cell cycle progression . They are stable but not rigid while elongating in parallel with cell growth, and they require septum formation to divide/cleave . Fluorescence recovery after photobleaching (FRAP) analysis showed that the cables are continuously remodeled during cell elongation . Turnover occurs along the length of the helical Mbl filaments, with no obvious polarity and a recovery half-time of about 8 min . These findings have important implications for the nature of bacterial cell wall architecture and synthesis.

Mikrobiologiia, 2002 Nov-Dec, 71(6), 801 - 8
{The biosynthesis of new secretory high-molecular-weight ribonucleases in Bacillus intermedius and Bacillus subtilis}; Znamenskaia LV et al.; The investigation of new secretory ribonucleases, the Bacillus intermedius binase II expressed in the recombinant B . subtilis strain 3922 and the native RNase Bsn of B . subtilis, showed that they are synthesized in the growth retardation phase, when inorganic phosphate is exhausted in the medium . The biosynthesis of these ribonucleases was found to be suppressed by the presence of inorganic phosphate in the medium and activated by small amounts of the transcriptional inhibitor actinomycin D . The cultivation media of the producing strains were optimized for the maximum production of the enzymes.

Biochemistry, 2003 Jan 21, 42(2), 467 - 74
Conserved residues R420 and Q428 in a cytoplasmic loop of the citrate/malate transporter CimH of Bacillus subtilis are accessible from the external face of the membrane; Krom BP et al.; CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family . Conserved residues R143, R420, and Q428, located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates . R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude . Conservative replacement of R432 with Lys reduced the activity by 1 order of magnitude, by lowering the affinity for the substrate 10-fold . It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates . Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity . Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner . The membrane impermeable reagents MTSET,(1) which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C . It is suggested that the access pathway is optimized for small, negatively charged substrates . Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.

Biochemistry, 2003 Jan 21, 42(2), 257 - 64
LytG of Bacillus subtilis is a novel peptidoglycan hydrolase: the major active glucosaminidase; Horsburgh GJ et al.; LytG (YubE) of Bacillus subtilis is a novel 32 kDa autolysin produced during vegetative growth under the control of Esigma(A) RNA polymerase . Muropeptide analysis of vegetative cells of B . subtilis revealed LytG to be the major glucosaminidase responsible for peptidoglycan structural determination during vegetative growth . Overexpression and purification of LytG allowed its biochemical characterization . Despite sequence homology suggesting muramidase activity, LytG is a novel glucosaminidase with exoenzyme activity and may form part of a novel family of autolysins . It is involved in cell division, lysis, and motility on swarm plates.

J Biotechnol, 2003 Feb 27, 101(1), 19 - 28
Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor; Droge MJ et al.; Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants . Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds . The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence . Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p . The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively . Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor . The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min . Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p . The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.

Life Sci Space Res, 1973, 11, 33 - 9
Survival of bacterial isolates exposed to simulated Jovian trapped radiation belt electrons and solar wind protons; Taylor DM et al.; With missions to Jupiter, the spacecraft will be exposed for extended durations to solar wind radiation and the Jovian trapped radiation belt . This study is designed to determine the effect of these radiation environments on spacecraft bacterial isolates . The information can be used in the probability of contamination analysis for these missions . A bacterial subpopulation from Mariner Mars 1971 spacecraft (nine spore-forming and three non-spore-forming isolates) plus two comparative organisms, Staphylococcus epidermidis ATCC 17917 and a strain of Bacillus subtilis var . niger, were exposed to 2, 12, and 25 MeV electrons at different doses with simultaneous exposure to a vacuum of 1.3 x 10(-4) N m-2 at 20 and -20 degrees C . The radioresistance of the subpopulation was dependent on the isolate, dose and energy of electrons . Temperature affected the radioresistance of only the spore-forming isolates . Survival data indicated that spores were reduced approximately 1 log/1500 J kg-1 (10 J kg-1=1 krad), while non-spore-forming isolates (micrococci) were reduced 1.5-2 logs/1500 J kg-1 with the exception of an apparent radioresistant isolate whose resistance approached that of the spores . The subpopulation was found to be less resistant to lower energy than to higher energy electrons . The bacterial isolates were exposed to 3 keV protons under the same conditions as the electrons with a total fluence of 1.5 x 10(13) p cm-2 and a dose rate of 8.6 x 10(9) p cm-2 s-1 . The results showed that only 20% of S . epidermidis and 45% of B . subtilis populations survived exposure to the 3 keV protons, while the mean survival of the spacecraft subpopulation was 45% with a range from 31.8% (non-spore-former) to 64.8% (non-spore-former) . No significant difference existed between spore-forming and non-spore-forming isolates.

Mol Cells, 2002 Dec 31, 14(3), 374 - 81
Expression, purification, and crystallization of glutamyl-tRNA(Gln) specific amidotransferase from Bacillus stearothermophilus; Kwak JH et al.; Although the genes that encode the glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) specific amidotransferase (Glu-AdTase) from various bacteria and eukaryotic organelles are known, the precise mechanism of the enzyme is still unclear . One of the reasons is that there is no information on the three-dimensional structure of the complex, the Glu-AdTase:Glu-tRNA(Gln):ATP:amino group donor . To obtain the crystals of Glu-AdTase, the Glu-AdTase of Bacillus stearothermophilus was overexpressed and purified after cloning of the gene that encodes the enzyme . The cloned DNA contained the full-length gene cluster that represented the Glu-AdTase of B . stearothermophilus, and was organized as an operon that consisted of three open-reading frames (ORFs) . The order of the genes was gatCAB, as shown in Bacillus subtilis . The ORFs showed a high amino-acid homology to those of B . subtilis (A subunit, 73.2%; B subunit, 81.6%; C subunit, 69.5%) and Staphylococcus aureus (A subunit, 61.9%; B subunit, 71.8%; C subunit, 45.9%) . The ORFs were re-cloned on the overexpression vector, pTrc99a, and a recombinant pTrcgatCABBST was obtained . The Glu-AdTase that was overexpressed with pTrcgatCABBST in Escherichia coli retained transamidation activity on the mischarged glutamic acid on the tRNA(Gln) . It also produced correctly-charged Gln-tRNA(Gln) at 37, 42, and 50 degrees C . Although Glu-AdTases from both B . subtilis and B . stearothermophilus were subjected to crystallization, the micro-crystals were only obtained from the B . stearothermophilus enzyme.

J Biol Chem, 2003 Mar 14, 278(11), 9761 - 7 Epub 2003 Jan 07.
Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis . Crystallographic, spectroscopic, and mutational studies; Lee DS et al.; Cytochrome P450 isolated from Bacillus subtilis (P450(BSbeta); molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g . myristic acid) at the alpha- and beta-positions using hydrogen peroxide as an oxidant . We report here on the crystal structure of ferric P450(BSbeta) in the substrate-bound form, determined at a resolution of 2.1 A . P450(BSbeta) exhibits a typical P450 fold . The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg(242) guanidium group . These interactions are responsible for the site specificity of the hydroxylation site in which the alpha- and beta-positions of the fatty acid come into close proximity to the heme iron sixth site . The fatty acid carboxylate group interacts with Arg(242) in the same fashion as has been reported for the active site of chloroperoxidase, His(105)-Glu(183), which is an acid-base catalyst in the peroxidation reactions . On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450(BSbeta) is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.

Bioorg Med Chem, 2003 Feb 6, 11(3), 407 - 19
Synthesis, antimicrobial and antifungal activity of a new class of spiro pyrrolidines; Raj AA et al.; A new class of spiro pyrrolidines, dispiro{oxindole-cyclohexanone}pyrrolidines, dispiro{oxindole-tetrahydronaphthalen-1-one}pyrrolidines, dispiro{oxindole-arylidene-cyclohexanone}pyrrolidines, dispiro{oxindole-hexahydro-indazole}pyrrolidines, and spiro{butenolide}pyrrolidines, have been screened for their antibacterial and antifungal activity against ten human pathogenic bacteria and four dermatophytic fungi . They were found to have antimicrobial and antifungal activity compounds against various pathogens except Bacillus subtilis . The spiro pyrrolidinines were synthesized by the regioselective 1,3-dipolar cycloaddition reaction of azomethine ylides generated either from isatin and sarcosine or from aziridine . The azomethine ylide so generated reacted with various dipolarophiles such as 2-arylidene-cyclohexanones, 2-arylidene-tetrahydronapthalen-1-ones, 2,6-bis(arylmethylidene)cyclohexanones and 3-arylidene-5-phenyl- butenolides.

Int J Cancer, 2003 Mar 1, 103(6), 775 - 8
Continuous long-term monitoring of UV radiation in professional mountain guides reveals extremely high exposure; Moehrle M et al.; Ultraviolet radiation (UVR) is estimated to be one of the most important risk factors for nonmelanoma and melanoma skin cancers . High occupational UV exposure is assumed to be associated with skin cancer . Mountain guides receive considerable UV doses due to altitude-related increase of UVR and reflection from snow- and ice-covered surfaces . The aim of our study was to assess the annual occupational UV exposure of mountain guides . Spore film test chambers containing spores of Bacillus subtilis (VioSpor) were used as UV dosimeters with a spectral sensitivity profile similar to erythema-weighted data calculated from spectroradiometric measurements . Nine mountain guide instructors carried dosimeters on the sides of their heads on a total of 1,406 working days during one year (July 1999-June 2000) . Dosimeters were changed monthly . Measurements of 92 months could be evaluated (4-12 months/mountain guide) . The mean individual monthly UV exposure was 107 standard erythema doses (SED) (median 71 SED; range 10-505 SED) . The mean annual cumulative UV exposure was 1,097 SED (median 1,273 SED; range 312-1,770 SED) per mountain guide . The mean UV dose per day (4-10 hr) was 6.6 SED (median 5.7 SED; range 0.6-24.2 SED) . This is the second study of continuous annual UV dosimetry in a cohort of outdoor workers . Our study showed that it is not sufficient to interpolate annual UV exposure from a few days' measurements . Only long-term dosimetry can give reliable yearly information of UVR load . Median daily UV exposure exceeded limits for UV radiation (e.g., ACGIH effective dose 30 J/m(2) per 8 hr period corresponding to 1.08 SED/day) 6-fold; maximal exposure exceeded these limits 23-fold . These extremely high exposure values are suggestive for an increased risk of skin cancer and thorough epidemiologic studies in the collectives of professional and recreational mountaineering are required .

Biochemistry, 2003 Jan 14, 42(1), 80 - 9
Solution structure and functional ligand screening of HI0719, a highly conserved protein from bacteria to humans in the YjgF/YER057c/UK114 family; Parsons L et al.; HI0719 belongs to a large family of highly conserved proteins with no definitive molecular function and is found in organisms ranging from bacteria to humans . We describe the NMR structure of HI0719, the first solution structure for a member of this family . The overall fold is similar to the crystal structures of two homologues, YabJ from Bacillus subtilis and YjgF from Escherichia coli, and all three structures are similar to that of chorismate mutase, although there is little sequence homology and no apparent functional connection . HI0719 is a homotrimer with a distinct cavity located at the subunit interface . Six of the seven invariant residues in the high identity group of proteins are located in this cavity, suggesting that this may be a binding site for small molecules . Using previously published observations about the biological role of HI0719 family members as a guide, over 100 naturally occurring small molecules or structural analogues were screened for ligand binding using NMR spectroscopy . The targeted screening approach identified six compounds that bind to HI0719 at the putative active site . Five of these compounds are either alpha-keto acids or alpha,beta-unsaturated acids, while the sixth compound is structurally similar . Previous studies have proposed that some HI0719 homologues may act on small molecules in the isoleucine biosynthetic path and, if this is correct, the ligand screening results presented here suggest that the interaction most likely occurs with 2-ketobutyrate and/or its unstable enamine precursor.

Appl Environ Microbiol, 2003 Jan, 69(1), 683 - 5
Comparison of UV inactivation of spores of three encephalitozoon species with that of spores of two DNA repair-deficient Bacillus subtilis biodosimetry strains; Marshall MM et al.; When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m(2), respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates . The results indicate that spores of Encephalitozoon spp . are readily inactivated at low UV fluences and that spores of UV-sensitive B . subtilis strains can be useful surrogates in evaluating UV reactor performance.

Appl Environ Microbiol, 2003 Jan, 69(1), 154 - 61
Role of ctc from Listeria monocytogenes in osmotolerance; Gardan R et al.; Listeria monocytogenes is a food-borne pathogen with the ability to grow under conditions of high osmolarity . In a previous study, we reported the identification of 12 proteins showing high induction after salt stress . One of these proteins is highly similar to the general stress protein Ctc of Bacillus subtilis . In this study, induction of Ctc after salt stress was confirmed at the transcriptional level by using RNA slot blot experiments . To explore the role of the ctc gene product in resistance to stresses, we constructed a ctc insertional mutant . No difference in growth was observed between the wild-type strain LO28 and the ctc mutant either in rich medium after osmotic or heat stress or in minimal medium after heat stress . However, in minimal medium after osmotic stress, the growth rate of the mutant was increased by a factor of 2 . Moreover, electron microscopy analysis showed impaired morphology of the mutant grown under osmotic stress conditions in minimal medium . Addition of the osmoprotectant glycine betaine to the medium completely abolished the osmotic sensitivity phenotype of the ctc mutant . Altogether, these results suggest that the Ctc protein of L . monocytogenes is involved in osmotic stress tolerance in the absence of any osmoprotectant in the medium.

Chembiochem, 2003 Jan 3, 4(1), 34 - 9
Assembly of designed oligonucleotides as an efficient method for gene recombination: a new tool in directed evolution; Zha D et al.; A new and practical method for gene recombination with formation of libraries of mutant genes is presented . The method is based on the assembly of appropriately prepared oligonucleotides whose design is guided by sequence information . High recombination frequency with formation of full-length products is achieved by controlled overlapping of the designed oligomers . This process (ADO) minimizes self-hybridization of parental genes, which constitutes a significant advantage over conventional family shuffling as used in the directed evolution of functional enzymes . ADO was applied to the recombination of two lipase family genes from Bacillus subtilis (LipA and LipB) . In a library of 3000 lipase variants created by this method, several were found that display increased enantioselectivity in a model reaction involving the hydrolysis of a meso-diacetate.

J Biol Chem, 2003 Apr 4, 278(14), 12344 - 55 Epub 2003 Jan 02.
Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions; Artsimovitch I et al.; The study of mutant enzymes can reveal important details about the fundamental mechanism and regulation of RNA polymerase, the central enzyme of gene expression . However, such studies are complicated by the multisubunit structure of RNA polymerase and by its indispensability for cell growth . Previously, mutant RNA polymerases have been produced by in vitro assembly from isolated subunits or by in vivo assembly upon overexpression of a single mutant subunit . Both approaches can fail if the mutant subunit is toxic or incorrectly folded . Here we describe an alternative strategy, co-overexpression and in vivo assembly of RNA polymerase subunits, and apply this method to characterize the role of sequence insertions present in the Escherichia coli enzyme . We find that co-overexpression of its subunits allows assembly of an RNA polymerase lacking a 188-amino acid insertion in the beta' subunit . Based on experiments with this and other mutant E . coli enzymes with precisely excised sequence insertions, we report that the beta' sequence insertion and, to a lesser extent, an N-terminal beta sequence insertion confer characteristic stability to the open initiation complex, frequency of abortive initiation, and pausing during transcript elongation relative to RNA polymerases, such as that from Bacillus subtilis, that lack the sequence insertions.

J Bacteriol, 2003 Jan, 185(2), 693 - 7
Polar targeting of DivIVA in Bacillus subtilis is not directly dependent on FtsZ or PBP 2B; Hamoen LW et al.; DivIVA is involved in Bacillus subtilis cell division and is located at the cell poles . Previous experiments suggested that the cell division proteins FtsZ and PBP 2B are required for polar targeting of DivIVA . By using outgrowing spores, we show that DivIVA accumulates at the cell poles independent of the presence of FtsZ or PBP 2B.

J Bacteriol, 2003 Jan, 185(2), 466 - 74
Regulation of the Bacillus subtilis heat shock gene htpG is under positive control; Versteeg S et al.; The heat shock genes of Bacillus subtilis are assigned to four classes on the basis of their regulation mechanisms . While classes I and III are negatively controlled by two different transcriptional repressors, class II is regulated by the alternative sigma factor sigma(B) . All heat shock genes with unidentified regulatory mechanisms, among them htpG, constitute class IV . Here, we show that expression of htpG is under positive control . We identified a DNA sequence (GAAAAGG) located downstream of the sigma(A)-dependent promoter of htpG . The heat inducibility of the promoter could be destroyed by inversion, nucleotide replacements, or removal of this DNA sequence . Fusion of this sequence to the vegetative lepA promoter conferred heat inducibility . Furthermore, we were able to show that the heat induction factor is dependent on the absolute temperature rather than the temperature increment and that nonnative proteins within the cytoplasm fail to induce htpG.

Anal Chem, 2002 Dec 15, 74(24), 6224 - 9
Pressure-assisted capillary electrophoresis electrospray ionization mass spectrometry for analysis of multivalent anions; Soga T et al.; We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamide-adenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds . Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall . It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle . Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions . Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface . The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas . The detection limits for these species were between 0.4 and 3.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3 . The utility of the method was demonstrated by analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.

J Mol Biol, 2003 Jan 24, 325(4), 661 - 75
Molecular modeling of the three-dimensional structure of the bacterial RNase P holoenzyme; Tsai HY et al.; Bacterial ribonuclease P (RNase P), an enzyme involved in tRNA maturation, consists of a catalytic RNA subunit and a protein cofactor . Comparative phylogenetic analysis and molecular modeling have been employed to derive secondary and tertiary structure models of the RNA subunits from Escherichia coli (type A) and Bacillus subtilis (type B) RNase P . The tertiary structure of the protein subunit of B.subtilis and Staphylococcus aureus RNase P has recently been determined . However, an understanding of the structure of the RNase P holoenzyme (i.e . the ribonucleoprotein complex) is lacking . We have now used an EDTA-Fe-based footprinting approach to generate information about RNA-protein contact sites in E.coli RNase P . The footprinting data, together with results from other biochemical and biophysical studies, have furnished distance constraints, which in turn have enabled us to build three-dimensional models of both type A and B versions of the bacterial RNase P holoenzyme in the absence and presence of its precursor tRNA substrate . These models are consistent with results from previous studies and provide both structural and mechanistic insights into the functioning of this unique catalytic RNP complex.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 75 - 7
{Study of the effectiveness of methods of purification of metalloprotease produced by Bacillus subtilis}; Khaziev AF et al.; The effectiveness of the purification of proteolytic enzyme produced by B . subtilis production strain 3H in different nutrient media was evaluated . As revealed in this study, the use of the initial material obtained in peptone nutrient medium made it possible to obtain proteolytic enzyme with the highest specific activity by the methods of salting out, gel and ion-exchange chromatography, but the use of these methods on a production scale led to the deterioration of purity characteristics . Changes in the nutrient medium composition used for the cultivation of the production strain resulted in greater effectiveness of the purification methods . The highly purified preparation of metalloprotease with specific activity exceeding that of available commercial preparations more than twofold was obtained.

Arch Pharm (Weinheim), 2002 Dec, 335(10), 495 - 9
Synthesis and in vitro antimicrobial evaluation of 5-(1-methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles; Shafiee A et al.; The increasing clinical importance of drug-resistant pathogens has lent additional urgency to antimicrobial research . Various 5-(1-methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles (4a-6f) were synthesized and tested in vitro for their antibacterial and antifungal activities . Compounds 4a and 4b exhibited significant effects against Bacillus subtilis at MIC ranges of 0.5-1 microg/mL and moderate effects against Staphylococcus aureus.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Feb 5, 784(2), 315 - 22
High-performance thin-layer chromatography-bioautography for multiple antibiotic residues in cow's milk; Ramirez A et al.; An analytical method to identify and quantify multiple antibiotic residues (chloramphenicol, ampicillin, benzylpenicillin, dicloxacillin and erythromycin) in cow's milk by high-performance thin-layer chromatography (HPTLC) combined with bioautography was developed . The test microorganism used for bioautography was Bacillus subtilis ATCC 6633 . Antibiotic residues were extracted with acetonitrile, fat eliminated with petroleum ether and residues isolated with dichloromethane The sensitivity of the method guarantees the detection of the above-mentioned antibiotics at levels below maximum residue limits (MRL) allowed for milk . Percentage recoveries ranged between 90 and 100%, with coefficients of variation between 7.2 and 21.3% . Some advantages of this methodology over thin-layer chromatography (TLC)/bioautography are also discussed.

Genes Dev, 2002 Dec 15, 16(24), 3253 - 64
A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore; Abanes-De Mello A et al.; A hallmark of bacterial endospore formation is engulfment, during which the membrane of one cell (the mother cell) migrates around the future spore, enclosing it in the mother cell cytoplasm . Bacteria lack proteins required for eukaryotic phagocytosis, and previously proteins required for membrane migration remained unidentified . Here we provide cell biological and genetic evidence that three membrane proteins synthesized in the mother cell are required for membrane migration as well as for earlier steps in engulfment . Biochemical studies demonstrate that one of these proteins, SpoIID, is a cell wall hydrolase, suggesting that membrane migration in bacteria can be driven by membrane-anchored cell wall hydrolases . We propose that the bacterial cell wall plays a role analogous to that of the actin and tubulin network of eukaryotic cells, providing a scaffold along which proteins can move.

J Gen Appl Microbiol, 1997 Jun, 43(3), 139 - 143
Chemical analysis of poly-gamma-glutamic acid produced by plasmid-free Bacillus subtilis (natto): Evidence that plasmids are not involved in poly-gamma-glutamic acid production; Nagai T et al.; It has been postulated that the psf gene on a small plasmid, pUH1 (5.8 kb), regulates positively the synthesis of capsular poly-gamma-glutamic acid (gammaPGA) in Bacillus subtilis (natto) Asahikawa . We found that this strain harbored a second plasmid, named pNAGL1 (ca . 50 kb), in addition to pUH1 . The growth conditions that cure pUH1 or pNAGL1 were established . The plasmid-free NAF4 strain derived from B . subtilis (natto) Asahikawa was found to produce gammaPGA which was the same as the parent strain in terms of quantity and chemical properties having the same molecular mass and content of D-glutamic acid . Furthermore, as in the case of the parent cells, the D-glutamic acid in gammaPGA, which is known to increase up to ca . 80% of the total glutamic acid as Mn(2+) ion concentration increases in growth medium, was found to make up 80% of the total glutamic acid of the gammaPGA produced by NAF4 cells grown in the presence of 0.1 mm MnCl(2) . Thus, these results led us to conclude that the plasmids do not encode any gene important for gammaPGA production.

J Gen Appl Microbiol, 1998 Feb, 44(1), 85 - 91
Transglutaminase in sporulating cells of Bacillus subtilis; Kobayashi K et al.; We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein . As a result, we detected TGase activity in sporulating cells of B . subtilis, B . cereus, B . alvei and B . aneurinolyticus, and found TGase activity related to sporulation . TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth . TGase was found to be localized on spores . TGase was preliminarily purified by gel filtration chromatography for characterization . Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa . TGase could cross-link and polymerize a certain protein . The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B . subtilis . The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants . It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.

J Gen Appl Microbiol, 1998 Feb, 44(1), 49 - 55
Production of sound waves by bacterial cells and the response of bacterial cells to sound; Matsuhashi M et al.; Bacterial cells enhance the proliferation of neighboring cells under stress conditions by emitting a physical signal . Continuous single sine sound waves produced by a speaker at frequencies of 6-10, 18-22, and 28-38 kHz promoted colony formation by Bacillus carboniphilus under non-permissive stress conditions of high KCl concentration and high temperature . Furthermore, sound waves emitted from cells of Bacillus subtilis at frequencies between 8 and 43 kHz with broad peaks at approximately 8.5, 19, 29, and 37 kHz were detected using a sensitive microphone system . The similarity between the frequency of the sound produced by B . subtilis and the frequencies that induced a response in B . carboniphilus and the previously observed growth-promoting effect of B . subtilis cells upon B . carboniphilus through iron barriers, suggest that the detected sound waves function as a growth-regulatory signal between cells.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 191 - 3 Epub 2002 Dec 19.
Crystallization and preliminary crystallographic analysis of the kinase-recruitment domain of the PP2C-type phosphatase RsbU; Dutta S et al.; The general stress response of Bacillus subtilis provides a protective resistance to a variety of pressures . The key molecule is a subunit of RNA polymerase, sigma(B), which confers promoter specificity and is regulated by two signalling modules . Each module comprises protein kinases and phosphatases and 'switch' protein substrates for the kinase and phosphatase . The phosphorylation state of the switch molecules indirectly controls the activity of sigma(B) . The binding of the kinase RsbT to the phosphatase RsbU stimulates its enzymatic activity towards its substrate, phosphorylated RsbV . To understand how these enzymes interact, thus regulating transcription, crystallization of the kinase-recruitment domain of RsbU in a form suitable for high-resolution structure determination is reported.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 155 - 7 Epub 2002 Dec 19.
Crystallization of the GTP-dependent transcriptional regulator CodY from Bacillus subtilis; Blagova EV et al.; CodY is a GTP sensor that represses transcription of early stationary phase and sporulation genes in Bacillus subtilis . As nutrients become limiting, GTP levels fall and CodY-mediated repression is relieved . Crystals of CodY have been grown in the presence and absence of GTP from sodium citrate buffered solutions containing lithium sulfate and diffraction data have been collected extending to 3.5 A spacing.

Antimicrob Agents Chemother, 2003 Jan, 47(1), 432 - 5
Lincomycin resistance mutations in two regions immediately downstream of the -10 region of lmr promoter cause overexpression of a putative multidrug efflux pump in Bacillus subtilis mutants; Kumano M et al.; We isolated 19 lincomycin-resistant Bacillus subtilis mutants by expressing lmrB encoding a putative multidrug efflux protein . Eighteen of the mutants altered at two regions (-3 to -1 and +15) immediately downstream of the -10 region of the lmr promoter increased lmr transcription in vivo and in vitro.

Planta Med, 2002 Dec, 68(12), 1137 - 9
Antibacterial activity of a stearic acid derivative from Stemodia foliosa; Dantas da Silva LL et al.; From the hexane-soluble fraction of an ethanol extract from leaves and stems of Stemodia foliosa (Scrophulariaceae), the new stearic acid 4-{(n-pentoxy)phenethyl} ester (1) was isolated . This compound exhibited antibacterial properties at 10 microg/mL concentration by using disc diffusion method against Gram-positive bacteria Bacillus cereus and Bacillus subtilis and fast-acid bacterium Mycobacterium fortuitum . The structure of the new compound was elucidated by spectroscopic methods and by chemical conversion.

Protein Sci, 2003 Jan, 12(1), 112 - 23
Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization; Zeeb M et al.; Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy . Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks . Eight residues belong to the earlier proposed RNP binding motifs . A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding . These residues are located in loop beta3-beta4 and loopbeta4-beta5, and include Ile18 . The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants . Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity . Unrestricted possible backbone conformations of loop beta3-beta4 also markedly contribute to binding . Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged . This might be of importance for the "RNA chaperone" activity of CspB.

Science, 2003 Jan 24, 299(5606), 532 - 6 Epub 2002 Dec 19.
RacA, a bacterial protein that anchors chromosomes to the cell poles; Ben-Yehuda S et al.; Eukaryotic chromosomes are anchored to a spindle apparatus during mitosis, but no such structure is known during chromosome segregation in bacteria . When sister chromosomes are segregated during sporulation in Bacillus subtilis, the replication origin regions migrate to opposite poles of the cell . If and how origin regions are fastened at the poles has not been determined . Here we describe a developmental protein, RacA, that acts as a bridge between the origin region and the cell poles . We propose that RacA assembles into an adhesive patch at a centromere-like element near the origin, causing chromosomes to stick at the poles.

Mol Microbiol, 2003 Jan, 47(1), 159 - 69
A role for division-site-selection protein MinD in regulation of internucleoid jumping of Soj (ParA) protein in Bacillus subtilis; Autret S et al.; The Bacillus subtilis soj-spo0J locus encodes two proteins belonging to a family of proteins (the ParAB proteins) with dual roles in plasmid or chromosome segregation and transcriptional regulation . Soj protein was previously shown to be capable of abrupt subcellular relocation . The movement was highly co-operative and at any moment, most of the Soj in any cell formed a single large 'patch' covering all or part of one nucleoid . Movement, and co-operativity, in the sense of formation of a single patch, was dependent on Spo0J . Movement, but not co-operativity, was also shown to be dependent, directly or indirectly, on FtsZ protein . We now report that the ftsZ effect arises because jumping onto a nucleoid is promoted by proximity to a cell pole . Studies of other mutants affected in cell division suggest that the attraction to the cell pole is mediated by the division-site-selection protein, MinD (which localizes at the cell poles) . It does not require MinC, the main effector of the division site selection system . A mutant form of Soj, putatively locked in the ATP form of the protein, interacts with the cell pole (dependent on MinD) but not with the nucleoid . These results identify a novel function for MinD and demonstrate an intriguing link between proteins involved in the cell division and chromosome segregation machineries.

Mol Microbiol, 2003 Jan, 47(1), 37 - 48
Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site; Harry EJ et al.; The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring . In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell . The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions . All three proteins localize to the division site at mid-cell and to cell poles . Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins . We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B . subtilis spores . We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division . We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell . Furthermore, these proteins were found to be present in mature, dormant spores . These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.

Eur J Biochem, 2003 Jan, 270(1), 155 - 62
Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase; Leemhuis H et al.; Cyclodextrin glycosyltransferase (CGTase) uses an alpha-retaining double displacement mechanism to catalyze three distinct transglycosylation reactions . To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced (8 mg.L(-1) culture) using Bacillus subtilis as a host . Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms . The cyclization reaction (cyclodextrin formation from starch) is a one-substrate reaction, whereas the other two transglycosylation reactions are two-substrate reactions, which obey substituted enzyme mechanism kinetics (disproportionation reaction) or ternary complex mechanism kinetics (coupling reaction) . Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors, whereas acarbose is a mixed type of inhibitor . Our results show that one molecule of acarbose binds either in the active site of the free enzyme, or at a secondary site of the enzyme-substrate complex . The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T . thermosulfurigenes CGTase.

Biotechnol Bioeng, 2003 Feb 20, 81(4), 421 - 9
Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems; Wang JJ et al.; Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties . Conventional purification followed by chemical immobilization is a laborious and costly process . A new genetic construct was developed to produce the keratinase-streptavidin fusion protein . Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step . The method was tested in both the Bacillus subtilis and Escherichia coli systems . In B . subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium . In E . coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract . The overall efficiencies were approximately the same, 24-28%, using both systems .

Nucleic Acids Res, 2002 Dec 15, 30(24), 5517 - 28
Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach; Hamoen LW et al.; Generally, the presence of a consensus sequence in the promoter of a gene is taken as indication for regulation by the transcription factor that binds to this sequence . In light of the recent developments in genome research, we were interested to what extent this supposition is valid . We examined the relationship between the presence of a binding site for ComK, the competence transcription factor of Bacillus subtilis, and actual transcriptional activation by ComK . Bacillus subtilis contains 1062 putative ComK-binding sites (K-boxes) in its genome . We employed DNA macroarrays to identify ComK-activated genes, and found that the presence of a K-box is an unreliable predictor for regulation . Only approximately 8% of the genes containing a K-box in the putative promoter region are regulated by ComK . The predictive value of a K-box could be improved by taking into consideration the degree of deviation from the K-box consensus sequence, the presence of extra ComK-binding motifs and the positions of RNA polymerase-binding sites . Finally, many of the ComK-activated genes show no apparent function related to the competence process . Based on our findings, we propose that the ComK-dependent activation of several genes might serve no biological purpose and can be considered 'evolutionary noise'.

Extremophiles, 2002 Dec, 6(6), 499 - 506 Epub 2002 Sep 13.
Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides: bacillomycin L, plipastatin, and surfactin; Roongsawang N et al.; Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand . One of the strains designated BBK-1 produced the biosurfactants with the highest activity . BBK-1 was isolated from fermented foods and was identified as B . subtilis based on its physiological characteristics and 16S rRNA gene sequence . We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8% . We found that B . subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously . By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin . In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced . The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B . subtilis strain RB14, which co-produces iturin A and surfactin.

J Bacteriol, 2003 Jan, 185(1), 340 - 8
Forespore-specific expression of Bacillus subtilis yqfS, which encodes type IV apurinic/apyrimidinic endonuclease, a component of the base excision repair pathway; Urtiz-Estrada N et al.; The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied . A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment . In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation . The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the Esigma(G) regulon . A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS . Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells . Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the sigma(G) regulon . Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.

J Bacteriol, 2003 Jan, 185(1), 262 - 73
Residue R113 is essential for PhoP dimerization and function: a residue buried in the asymmetric PhoP dimer interface determined in the PhoPN three-dimensional crystal structure; Chen Y et al.; Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which P(i) is growth limiting . Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes . DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3' of PhoP-binding consensus repeats in PhoP-repressed promoters . The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization . A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction . We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon . In vivo expression of either PhoP(R113E), PhoP(R113A), or PhoP(D60K) resulted in a Pho-negative phenotype . In vitro analysis showed that PhoP(R113E) was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize . Monomeric PhoP(R113E) approximately P was deficient in DNA binding, contributing to the PhoP(R113E) in vivo Pho-negative phenotype . While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential . Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

J Bacteriol, 2003 Jan, 185(1), 254 - 61
The crystal structure of the phosphorylation domain in PhoP reveals a functional tandem association mediated by an asymmetric interface; Birck C et al.; PhoP from Bacillus subtilis belongs to the OmpR subfamily of response regulators . It regulates the transcription of several operons and participates in a signal transduction network that controls adaptation of the bacteria to phosphate deficiency . The receiver domains of two members of this subfamily, PhoB from Escherichia coli and DrrD from Thermotoga maritima, have been structurally characterized . These modules have similar overall folds but display remarkable differences in the conformation of the beta4-alpha4 and alpha4 regions . The crystal structure of the receiver domain of PhoP (PhoPN) described in this paper illustrates yet another geometry in this region . Another major issue of the structure determination is the dimeric state of the protein and the novel mode of association between receiver domains . The protein-protein interface is provided by two different surfaces from each protomer, and the tandem unit formed through this asymmetric interface leaves free interaction surfaces . This design is well suited for further association of PhoP dimers to form oligomeric structures . The interprotein interface buries 970 A(2) from solvent and mostly involves interactions between charged residues . As described in the accompanying paper, mutations of a single residue in one salt bridge shielded from solvent prevented dimerization of the unphosphorylated and phosphorylated response regulator and had drastic functional consequences . The three structurally documented members of the OmpR family (PhoB, DrrD, and PhoP) provide a framework to consider possible relationships between structural features and sequence signatures in critical regions of the receiver domains.

J Bacteriol, 2003 Jan, 185(1), 243 - 53
The global transcriptional response of Bacillus subtilis to peroxide stress is coordinated by three transcription factors; Helmann JD et al.; Bacillus subtilis exhibits a complex adaptive response to low levels of peroxides . We used global transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA population after exposure to either hydrogen peroxide (H(2)O(2)) or tert-butyl peroxide (t-buOOH) . The peroxide stimulons could be largely accounted for by three regulons controlled by the PerR, sigma(B), and OhrR transcription factors . Three members of the PerR regulon (katA, mrgA, and zosA) were strongly induced by H(2)O(2) and weakly induced by t-buOOH . The remaining members of the PerR regulon were only modestly up-regulated by peroxide treatment . Overall, the magnitude of peroxide induction of PerR regulon genes corresponded well with the extent of derepression in a perR mutant strain . The sigma(B) regulon was activated by 58 micro M H(2)O(2) but not by 8 micro M H(2)O(2) and was strongly activated by either t-buOOH or, in a control experiment, tert-butyl alcohol . Apart from the sigma(B) regulon there was a single gene, ohrA, that was strongly and rapidly induced by t-buOOH exposure . This gene, controlled by the peroxide-sensing repressor OhrR, was not induced by any of the other conditions tested.

J Bacteriol, 2003 Jan, 185(1), 51 - 9
A bacitracin-resistant Bacillus subtilis gene encodes a homologue of the membrane-spanning subunit of the Bacillus licheniformis ABC transporter; Ohki R et al.; Bacitracin is a peptide antibiotic nonribosomally produced by Bacillus licheniformis . The bcrABC genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells . Bacillus subtilis 168, which has no bacitracin synthesizing operon, has several genes homologous to bcrABC . It was found that the disruption of ywoA, a gene homologous to bcrC, resulted in hypersensitivity to bacitracin . Resistance to other drugs such as surfactin, iturin A, vancomycin, tunicamycin, gramicidin D, valinomycin and several cationic dyes were not changed in the ywoA disruptant . Spontaneous bacitracin-resistant mutants (Bcr-1 and -2) isolated in the presence of bacitracin have a single base substitution from A to G in the ribosome binding region . Northern hybridization analysis and determination of the expression of ywoA-LacZ transcriptional fusion gene revealed that the transcription of the ywoA gene was dependent on extracytoplasmic function (ECF) sigma factors sigma(M) and sigma(X) . Preincubation of wild-type cells in the presence of a low concentration of bacitracin induced increased resistance to bacitracin about two- to threefold, although the mechanism of this induction has not yet been elucidated . It has been reported that a commercially available bacitracin is a mixture of several components and also contains impurity . Bacitracin A was purified by reverse phase high-performance liquid chromatography (HPLC) . Similar results were obtained with bacitracin A as those with crude bacitracin, indicating that contaminating substances were not responsible for the results obtained in this study.

J Bacteriol, 2003 Jan, 185(1), 35 - 40
Binding of sigma(A) and sigma(B) to core RNA polymerase after environmental stress in Bacillus subtilis; Rollenhagen C et al.; In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environmental stress or energy depletion . The general stress regulon under the control of sigma(B) provides the cell with multiple stress resistance . Experiments were designed to determine how activated sigma(B) replaces sigma(A) as a constituent of the RNA polymerase holoenzyme . Studies of the transcription of the sigma(A)-dependent stress gene clpE under sigma(B)-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type . The relative affinities of sigma(A) and sigma(B) for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance . The results showed that the affinity of sigma(B) for E was 60-fold lower than that of sigma(A) . Western blot analyses with antibodies against sigma(A), sigma(B), and E showed that, after exposure to ethanol stress, the concentration of sigma(B) was only twofold higher than those of sigma(A) and E . Thus, the concentration of sigma(B) after stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of sigma(B) to E after stress.

Lett Appl Microbiol, 2003, 36(1), 46 - 9
Augmenting effect of acetic acid for acidification on bactericidal activity of hypochlorite solution; Kuroiwa K et al.; AIMS: Bactericidal activity of chlorine solution is enhanced by weak acidification . We compared the effects of various acids on the bactericidal activity of hypochlorite solution to establish a method for safe and effective use of an acidic hypochlorite solution . METHODS AND RESULTS: The bactericidal activities of acidic hypochlorite solutions that had been adjusted to pH 5.0 with hydrochloric acid, acetic acid, citric acid, lactic acid, formic acid, phosphoric acid or sulphuric acid against Bacillus subtilis spores were compared . The acidic solutions prepared with hydrochloric acid and acetic acid showed the highest bactericidal activity, and all of the spores (5 x 106 cfu ml(-1)) were killed within 10 min . On the other hand, the solutions prepared with citric acid and lactic acid showed no bactericidal activity against any bacterial strains tested in this study despite the low pH . The amount of chlorine gas produced by the preparation using acetic acid was sixfold less than that produced from the preparation using hydrochloric acid . CONCLUSIONS: Acetic acid is the most suitable and safe acid for the preparation of an acidic hypochlorite solution . SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for establishing a method for safe and effective use of an acidic hypochlorite solution.

Lett Appl Microbiol, 2003, 36(1), 15 - 9
Inducible beta-glucosidase synthesis during germination and outgrowth of Bacillus subtilis ATCC 9372 spores; Chandrapati S et al.; AIMS: To investigate the role of germination processes in the expression of the beta-glucosidase enzyme in Bacillus subtilis ATCC 9372 spores . METHODS AND RESULTS: Enzyme activity was monitored in germinating and non-germinating spores of B . subtilis ATCC 9372 . The expression of beta-glucosidase by spores of B . subtilis was further investigated in the presence of a germination inhibitor, d-alanine, and a transcription inhibitor, novobiocin . Detection of enzyme activity required the presence of the germinant, l-alanine, as well as the inducer, 4-methylumbelliferryl-beta-d-glucoside . Furthermore, beta-glucosidase synthesis was abolished in the presence of d-alanine or novobiocin . CONCLUSIONS: The data obtained in this study indicated that beta-glucosidase was not pre-existing, or merely attached to the spore, but was synthesized de novo during spore germination . SIGNIFICANCE AND IMPACT OF THE STUDY: The requirement of functional germination processes for the detection of beta-glucosidase activity makes this enzyme a good candidate for detection of spore viability.

J Gen Appl Microbiol, 2000 Feb, 46(1), 19 - 27
Purification and some properties of a novel chitosanase from Bacillus subtilis KH1; Omumasaba CA et al.; One of at least two chitosanases secreted in the culture filtrate of Bacillus subtilis KH1 was purified by two sequential DEAE Sepharose CL-6B chromatographies, followed by Sephacryl S-100 HR gel chromatography . The purified enzyme was homogenous as judged by SDS-PAGE . It showed an estimated molecular weight and pI of 28,000 and 8.3, respectively . The enzyme drastically reduced the viscosity of highly deacetylated chitosan substrates, with the subsequent formation of chitooligosaccharides {(GlcN)(n), n=2-6} . No activity toward carboxymethylcellulose (CMC), chitobiose (GlcN)(2), or chitotriose (GlcN)(3) was detected . Separation and quantification of products of hydrolysis of 10% (w/v) solutions of chitooligosaccharides, (GlcN)(n), n=2-6, by HPLC showed the splitting of (GlcN) (n), n=4-6, in an endo-splitting manner . Oligomers comprising higher units than the starting substrate were also detected, indicating transglycosylation activity . The amino terminal sequence of this enzyme (A-G-L-N-K-D-Q-K-R-R) is identical to that of the chitosanase derived from Bacillus pumilus BN262 and to the deduced amino terminal sequences of Bacillus subtilis 168 and Bacillus amyloliquefaciens UTK chitosanases.

J Biol Chem, 2003 Feb 28, 278(9), 6921 - 7 Epub 2002 Dec 13.
Kinetic studies of the uracil phosphoribosyltransferase reaction catalyzed by the Bacillus subtilis pyrimidine attenuation regulatory protein PyrR; Grabner GK et al.; The PyrR protein from Bacillus subtilis and many other bacteria is a bifunctional protein . Its primary function is the regulation of expression of pyrimidine biosynthetic (pyr) genes by binding to specific sites on pyr mRNA in a uridine nucleotide-dependent manner and altering the folding of downstream RNA to promote termination of transcription . PyrR also catalyzes the uracil phosphoribosyltransferase (UPRTase) reaction even though it bears little amino acid sequence similarity to other bacterial UPRTases . The PyrR-catalyzed UPRTase reaction obeyed a Ping Pong steady state kinetic pattern under all conditions examined, but no catalysis of {(14)C}uracil-UMP and {(32)P}PP(i)-phosphoribosylpyrophosphate exchange reactions could be detected . Steady state equations for Ordered Bi Bi mechanisms for PyrR that include a kinetically irreversible conformational change after binding of PRPP but before uracil binding were shown to account for the Ping Pong pattern of the enzyme . This mechanism was supported by the following experimental observations . The reverse reaction was extremely slow with a catalytic rate constant 3300 times smaller than for the forward reaction . Patterns of product inhibition of the forward reaction were consistent with a version of the irreversible conformational change model in which PyrR returns to the unliganded conformation before dissociation of UMP and were inconsistent with several other kinetic mechanisms . UMP and phosphoribosylpyrophosphate were shown by equilibrium dialysis to bind to free PyrR (dissociation constants of 27 +/- 3 and 18 +/- 2 microm, respectively), but uracil and PP(i) did not bind at equilibrium concentrations up to 750 microm . We propose that the conformational change kinetic model developed for PyrR can also account for numerous other reports of Ping Pong kinetics for various phosphoribosyltransferases that do not form the phosphoribosyl-enzyme intermediate predicted by classic Ping Pong kinetics.

Phytochemistry, 2003 Jan, 62(2), 239 - 43
5H-furan-2-ones from fungal cultures of Aporpium caryae; Levy LM et al.; Four furanones 1-4 with an unusual skeleton containing an acetylene unit, named aporpinones, were isolated from the culture of the basidiomycete Aporpium caryae and their structures were elucidated by spectroscopic methods . Compounds 3 and 4 showed weak to moderate antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Escherichia coli.

J Biol Chem, 2003 Feb 14, 278(7), 4906 - 11 Epub 2002 Dec 11.
Bacteriophage phi 29 early protein p17 . Self-association and hetero-association with the viral histone-like protein p6; Crucitti P et al.; Gene 17 of the Bacillus subtilis phage Phi29 is expressed early after infection, and it has been shown to be required at the very beginning of phage replication under conditions of low but not high multiplicity of infection . It has been proposed that, at the beginning of the infection, protein p17 could be recruiting limiting amounts of initiation factors at the viral origins . Once the infection process is established and the replication proteins reach optimal concentration, protein p17 becomes dispensable . In this paper we focused, on the one hand, on the study of protein p17 dimerization and the role of a putative coiled-coil region . On the other hand, we focused on its interaction with the viral origin-binding protein p6 . Based on our results we propose that protein p17 function is to optimize binding of protein p6 at the viral DNA ends, thus favoring the initiation of replication and negatively modulating its own transcription.

Microbiology, 2002 Dec, 148(Pt 12), 3971 - 82
Proteomics characterization of novel spore proteins of Bacillus subtilis; Kuwana R et al.; The spores of Bacillus subtilis have characteristic properties and consist of complex structures including various types of proteins . To perform comprehensive analysis of the protein composition of the spores, the proteins extracted from the spore were analysed by a combination of one-dimensional PAGE and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using Turboquest SEQUEST software interfaced with the DNA sequence database of B . subtilis . A total of 154 proteins were identified, and 69 of them were novel . The remaining 85 proteins have been previously reported as sporulation-specific proteins or as proteins that are synthesized in vegetative cells . The expression pattern of each gene deduced to encode novel spore proteins was analysed using a series of strains carrying a lacZ reporter gene . The results revealed that the expression of 26 genes was dependent on sporulation-specific sigma factors, namely sigma(F), sigma(E), sigma(G) and sigma(K) . In this study, it is demonstrated that the combination of the techniques of SDS-PAGE and LC-MS/MS, with the mutant library of B . subtilis, is an effective tool for the analysis of complicated cellular structures.

FEMS Microbiol Lett, 2002 Dec 17, 217(2), 237 - 42
Aerotactic responses in bacteria to photoreleased oxygen; Yu HS et al.; Bacterial aerotaxis is a rapid response towards or away from oxygen . Here we report on the use of computer-assisted motion analysis coupled to flash photolysis of caged oxygen to quantify aerotactic responses in bacteria . The caged compound (mu-peroxo)(mu-hydroxo)bis{bis(bipyridyl) cobalt(III)} perchlorate liberates molecular oxygen upon irradiation with near-UV light . A mixture of cells and the caged oxygen compound was placed in a capillary tube and challenged by discrete stimuli of molecular oxygen produced by photolysis . We then recorded the rate of change of direction (rcd) as an estimate of tumble frequency in response to liberated oxygen and measured the signal processing (excitation) times in Bacillus subtilis, Bacillus halodurans and Escherichia coli . This computer-assisted caged oxygen assay gives a unique physiological profile of different aerotaxis transducers in bacteria .

Arch Microbiol, 2002 Dec, 179(1), 26 - 32 Epub 2002 Nov 09.
Biotin synthase of Bacillus subtilis shows less reactivity than that of Escherichia coli in in vitro reaction systems; Kiyasu T et al.; The biotin synthases of Bacillus subtilis and Escherichia coli were compared in a physiological reduction system using cell-free extracts and in a artificial reduction system using photo-reduced deazariboflavin . The biotin synthase of B . subtilis was less active than that of E . coli in both reaction systems and showed at least ten-fold less biotin-forming activity than that of E . coli in the artificial reduction system . The physiological reduction system using the biotin synthases and cell-free extracts of B . subtilis and E . coli showed species specificity . The results suggest that the activity of the physiological reduction system of B . subtilisis weaker than that of E . coli . Addition of excess dethiobiotin inhibited biotin formation by growing cells of B . subtilis, but not by E . coli.

Mol Genet Genomics, 2002 Dec, 268(4), 455 - 67 Epub 2002 Nov 16.
Bacillus subtilis functional genomics: genome-wide analysis of the DegS-DegU regulon by transcriptomics and proteomics; Mader U et al.; The DegS-DegU two-component regulatory system of Bacillus subtilis controls various processes that characterize the transition from the exponential to the stationary growth phase, including the induction of extracellular degradative enzymes, expression of late competence genes and down-regulation of the sigma(D) regulon . The degU32(Hy) mutation stabilizes the phosphorylated form of DegU (DegU-P), resulting in overproduction of several extracellular degradative enzymes . In this study, the pleiotropic DegS-DegU regulon was characterized by combining proteomic and transcriptomic approaches . A comparative analysis of wild-type B . subtilis and the degU32(Hy) mutant grown in complex medium was performed during the exponential and in the stationary growth phase . Besides genes already known to be under the control of DegU-P, novel putative members of this regulon were identified . Although the degU32(Hy) mutant is assumed to contain high levels of phosphorylated DegU in the exponential as well as in the stationary growth phase, many genes known to be positively regulated by DegU-P did not show enhanced expression in the mutant strain during exponential growth . This is consistent with the fact that most genes belonging to the DegS-DegU regulon are subject to multiple regulation; this is also reflected in the strong stationary-phase induction of these genes in the mutant strain . As expected, during the exponential growth phase, the sigma(D) regulon was expressed at significantly lower levels in the degU32(Hy) mutant than in the wild type.

Chem Rec, 2002, 2(6), 397 - 404
High-sensitivity analyses of metabolites in biological samples by capillary electrophoresis using dynamic pH junction-sweeping; Britz-McKibbin P et al.; Emerging fields of biochemical research, such as metabolomics, present challenges to current separation technologies because of the large number of metabolites present in a cell and their often low (submicromolar) concentration . Although capillary electrophoresis (CE) holds great promise as the method of choice for high-resolution separations of biological samples, it suffers from poor concentration sensitivity, especially with the use of UV detection . In CE, sweeping and dynamic pH junction represent two complementary on-line focusing techniques that have been used for sensitivity enhancement of hydrophobic and weakly acidic analytes, respectively . However, the application of either the sweeping or dynamic pH junction technique alone might, in some cases, be less effective for the analysis of certain sample mixtures . Recent work in the development of a hyphenated dynamic pH junction-sweeping technique is presented as an effective on-line method of preconcentration suitable for both hydrophilic (anionic) and hydrophobic (neutral) analytes . Sensitive analyses of flavin metabolites by CE with laser-induced fluorescence (LIF) detection is demonstrated in various biological matrixes, including cell extracts of Bacillus subtilis, pooled human plasma, as well as heat-deproteinized flavoenzymes . Enhanced analyte band narrowing and improved sensitivity is achieved for flavins using dynamic pH junction-sweeping compared to either sweeping or dynamic pH junction alone . This results in over a 1200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (LOD, defined as S/N = 3) of about 4.0 x 10(-12) M . Strategies for sensitive and more comprehensive analyses of other cell metabolites, including nucleotides, coenzymes, and steroids, are also discussed when using on-line focusing techniques in conjunction with multiplexed CE and UV detection .

Proteomics, 2002 Dec, 2(12), 1724 - 34
Differential proteomic analysis of Bacillus subtilis nitrate respiration and fermentation in defined medium; Clements LD et al.; A comparative investigation of protein expression by two-dimensional gel electrophoresis was conducted between Bacillus subtilis cultures grown in defined medium under aerobic, anaerobic nitrate respiration, or fermentation conditions . Defined medium specific for either nitrate respiration or fermentation allowed distinction between proteins induced by each individual growth process . Our differential protein profiling analysis between aerobic and anaerobic conditions showed that anaerobic fermentation induced at least 44 proteins and nitrate respiration induced at least 19 proteins compared to aerobic controls . Certain proteins were specifically induced during nitrate respiration or fermentation, while others were induced by both anaerobic processes . Eleven proteins induced by nitrate respiration and/or fermentation were identified by peptide mass matching using matrix-assisted laser desorption/ionization-time of flight mass spectrometry . Proteins encoded by feuA, hmp, and ytkD were induced by nitrate respiration . Proteins encoded by pyrR, sucD, trpC, and ywjH were induced by fermentation . Proteins encoded by acuB, pdhC, ydjL, and yvyD were induced by nitrate respiration and fermentation . This proteomic analysis has provided a more complete characterization of B . subtilis anaerobic growth and increased our understanding of its metabolic pathways of nitrate respiration and fermentation.

Gene, 2002 Oct 30, 300(1-2), 105 - 15
Study of statistical correlations in DNA sequences; Bernaola-Galvan P et al.; Here we present a study of statistical correlations among different positions in DNA sequences and their implications by directly using the autocorrelation function . Such an analysis is possible now because of the availability of large sequences or even complete genomes of many organisms . After describing the way in which the autocorrelation function can be applied to DNA-sequence analysis, we show that long-range correlations, implying scale independence, appear in several bacterial genomes as well as in long human chromosome contigs . The source for such correlations in bacteria, which may extend up to 60 kb in Bacillus subtilis, may be related to massive lateral transfer of compositionally biased genes from other genomes . In the human genome, correlations extend for more than five decades and may be related to the evolution of the 'neogenome', a modern evolutionary acquisition composed by GC-rich isochores displaying long-range correlations and scale invariance.

Nucleic Acids Res, 2002 Dec 1, 30(23), 5056 - 64
Bacillus subtilis tau subunit of DNA polymerase III interacts with bacteriophage SPP1 replicative DNA helicase G40P; Martinez-Jimenez MI et al.; Genetic evidence suggests that the Bacillus subtilis dnaX gene only encodes for the tau subunit of both DNA polymerases III (Pol IIIs) . The B.subtilis full-length protein and their mutant derivatives tau(373- 563) (lacking the N-terminal, domains I-III or amino acid residues 1-372) and tau(1-372) (lacking the C-terminal region or amino acids 373-563) have been purified . The tau protein forms tetramers, tau(373- 563) forms dimers, whereas tau(1-372), depending on the ionic strength, forms trimers or tetramers in solution . In the absence of single-stranded (ss) DNA and a nucleotide cofactor, tau interacts with the SPP1 hexameric replicative G40P DNA helicase in solution or with G40P-ATP bound to ssDNA, with a 1:1 stoichiometry . G40P(109-442), lacking the N-terminal amino acid residues 1-108, interacts with the C-terminal moiety of tau . The data indicate that the interaction of G40P with the tau subunit of Pol III, is relevant for the loading of the Pol IIIs into the SPP1 G38P-promoted open complex.

J Biol Chem, 2003 Feb 14, 278(7), 5235 - 41 Epub 2002 Dec 03.
Bimodal protection of DNA by Mycobacterium smegmatis DNA-binding protein from stationary phase cells; Gupta S et al.; Some members of the DNA-binding protein from stationary phase cells (Dps) family of proteins have been shown to play an important role in protecting microorganisms from oxidative or nutritional stress . Dps homologs have been identified in various bacteria such as Escherichia coli, Bacillus subtilis, and Listeria innocua . Recently we have reported the presence of a Dps homolog, Ms-Dps, in Mycobacterium smegmatis . Ms-Dps was found to have a nonspecific DNA binding ability . Here we have detected two stable oligomeric forms of Ms-Dps in vitro, a trimeric and a dodecameric form . Interestingly, the conversion of Dps from a trimeric to a dodecameric form takes place upon incubation at 37 degrees C for 12 h . These two oligomeric forms differ in their DNA binding properties . The dodecameric form is capable of DNA binding and forming large crystalline arrays with DNA, whereas the trimeric form cannot do so . However, even in the absence of DNA binding, the trimeric form has the capacity to protect the DNA against Fenton's-mediated damage . The protection is afforded by the ferroxidase activity of the trimer . However, the trimeric form cannot protect DNA from DNaseI attack, for which a direct physical shielding of DNA by the dodecamer is required . Thus we suggest that Ms-Dps provides a bimodal protection of DNA by its two different oligomeric forms.

Arch Biochem Biophys, 2002 Dec 15, 408(2), 220 - 8
The effect of osmotic stress on the biophysical behavior of the Bacillus subtilis membrane studied by dynamic and steady-state fluorescence anisotropy; Lopez CS et al.; The thermotropic behavior of intact bacterial membranes and vesicles prepared from total and polar lipids isolated from Bacillus subtilis cultures grown at 37 degrees C in normal (LB) and hyperosmotic (LBN) conditions was studied using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), and 2-diethylamino-6-lauroyl-naphthalene (Laurdan) as fluorescent probes . No phase transition of bulk lipids was observed in these preparations at the range of temperature studied . The anisotropy values (r(s)) for DPH and TMA-DPH in purified membranes showed significant differences between the LB and LBN conditions, suggesting that there was an increase in membrane packing during the adaptation to osmotic stress . Furthermore, generalized polarization (GP) parameters for Laurdan indicated small but significant changes in water relaxation at the membrane hydrophobic/hydrophilic interface . Membrane preparations showed r(s) higher values than those of lipid vesicles and a higher temperature dependence of the Laurdan GP parameter . This fact indicates that membrane proteins increase the lipid packing and keep the membrane more sensitive to temperature changes.

Protein Expr Purif, 2002 Dec, 26(3), 337 - 42
A dual protein expression system in Bacillus subtilis; Chan AY et al.; We have developed a dual expression system for the simultaneous overexpression of two proteins in Bacillus subtilis . Two candidate genes, xylanase (xynA) and glucanase (bglS) from B . subtilis strain 168, which were engineered with independent Shine-Dalgarno (SD) sequences, were cloned in tandem into a transfer vector, which was then transformed into B . subtilis strain 1A304 (phi105MU331) . The genes were under the transcriptional control of a strong promoter of a bacteriophage, phi105, where transcription was initiated upon thermal induction . Six constructs were made to compare the factors that affected the yields of the gene products . The expression level of each candidate gene was found to correspond to its position relative to the phage promoter, irrespective of the identity of the insert . The lower expression level of the second insert might have been due to limited resources for protein synthesis, a short half-life of the mRNA, or an early termination of the RNA polymerase . Curiously, gene duplications in tandem did not lead to further increase in production.

Regul Toxicol Pharmacol, 2002 Oct, 36(2), 155 - 61
Cytotoxic potential of industrial strains of Bacillus sp; Pedersen PB et al.; The cytotoxic potential of selected strains of Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis, used in the production of industrial enzyme products, has been assessed . Cytotoxicity was determined in Chinese hamster ovary (CHO-K1) cells by measuring total cellular metabolic activity using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) . Initially the MTT assay was validated against toxigenic strains of Bacillus cereus, to define the exact criteria for a toxigenic versus a nontoxigenic response . The assay proved sensitive to culture broths of both a diarrheagenic strain and an emetic strain of B . cereus . The enzyme-producing strains tested were nontoxic to CHO-K1 cells . Additionally it was demonstrated that our industrial strains did not react with antibodies against B . cereus enterotoxins by use of commercial antibody-based kits from Oxoid and Tecra . A short survey of the literature concerning the toxigenic potential of species within the subtilis group is included, as is a database search of known B . cereus enterotoxins against B . subtilis and B . licheniformis DNA sequences .

J Mol Biol, 2002 Dec 6, 324(4), 625 - 36
The carboxy-terminal domain of the DExDH protein YxiN is sufficient to confer specificity for 23S rRNA; Kossen K et al.; DE x DH proteins are believed to modulate the structures of RNAs and ribonucleoprotein complexes by disrupting RNA helices and RNA-protein interactions . All DE x DH proteins contain a two-domain catalytic core that enables their RNA-dependent ATPase and RNA helicase activities . The catalytic core may be flanked by ancillary domains that are proposed to confer substrate specificity and facilitate the unique functions of individual proteins . The Escherichia coli DE x DH protein DbpA and its Bacillus subtilis ortholog YxiN have similar 75aa carboxy-terminal domains, and both proteins are specifically targeted to 23S rRNA . Here we demonstrate that the carboxy-terminal domain of YxiN is sufficient to confer RNA specificity by characterizing a chimera in which this domain is appended to the core domains of E.coli SrmB, a DE x DH protein with no apparent substrate specificity . Both the RNA-dependent ATPase and RNA helicase activities of the chimera are specifically activated by 23S rRNA and abolished by sequence changes within hairpin 92, a critical recognition element for Y x iN . These data support a model in which the carboxy-terminal domain binds hairpin 92 to target the protein to 23S rRNA.

Lett Appl Microbiol, 2002, 35(6), 468 - 72
Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish; Kim KP et al.; AIMS: To examine cholesterol degradation by Bacillus subtilis SFF34 . METHODS AND RESULTS: Cholesterol degradation and cholesterol oxidase production by B . subtilis SFF34 were investigated in a medium containing 0.2% cholesterol . In addition, the oxidized product of cholesterol by the purified cholesterol oxidase was detected using a gas chromatograph . Cholesterol oxidase production reached its maximal level (3.14 U ml(-1) after 24 h of incubation in the cholesterol medium . The residual cholesterol content reduced to 0.98 mg g(-1) after 60 h of cultivation in the cholesterol medium . Two cholesterol oxidases were purified from the culture supernatant fluid and their reaction product against cholesterol was identified as 4-cholesten-3-one . CONCLUSIONS: B . subtilis SFF34 degraded cholesterol and produced a high level of extracellular cholesterol oxidase . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus subtilis will be very useful for the reduction of cholesterol in many fermented foods and as a source of cholesterol oxidase.

Environ Microbiol, 2002 Nov, 4(11), 735 - 43
Label-free detection of 16S ribosomal RNA hybridization on reusable DNA arrays using surface plasmon resonance imaging; Nelson BP et al.; In this paper, we describe the detection of bacterial cell-extracted 16S ribosomal RNA (rRNA) using an emerging technology, surface plasmon resonance (SPR) imaging of DNA arrays . Surface plasmon resonance enables detection of molecular interactions on surfaces in response to changes in the index of refraction, therefore eliminating the need for a fluorescent or radioactive label . A variation of the more common SPR techniques, SPR imaging enables detection from multiple probes in a reusable array format . The arrays developed here contain DNA probes (15-21 bases) designed to be complementary to 16S rRNA gene sequences of Escherichia coli and Bacillus subtilis as well as to a highly conserved sequence found in rRNAs from most members of the domain Bacteria . We report species-specific hybridization of cell-extracted total RNA and in vitro transcribed 16S rRNA to oligonucleotide probes on SPR arrays . We tested multiple probe sequences for each species, and found that success or failure of hybridization was dependent upon probe position in the 16S rRNA molecule . It was also determined that one of the probes intended to bind 16S rRNA also bound an unknown protein . The amount of binding to these probes was quantified with SPR imaging . A detection limit of 2 micro g ml-1 was determined for fragmented E . coli total cellular RNA under the experimental conditions used . These results indicate the feasibility of using SPR imaging for 16S rRNA identification and encourage further development of this method for direct detection of other RNA molecules.

J Biol Chem, 2003 Feb 14, 278(7), 5333 - 42 Epub 2002 Nov 27.
Contribution of DNA conformation and topology in right-handed DNA wrapping by the Bacillus subtilis LrpC protein; Beloin C et al.; The Bacillus subtilis LrpC protein belongs to the Lrp/AsnC family of transcriptional regulators . It binds the upstream region of the lrpC gene and autoregulates its expression . In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy . LrpC is a structure-specific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosome-like structures . Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation . In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy.

Biochimie, 2002 Sep, 84(9), 945 - 51
Implication of the prohead RNA in phage phi29 DNA packaging; Bourassa N et al.; RNA is an important component of many biological processes, including DNA encapsidation of bacteriophage phi29 of Bacillus subtilis . Interestingly, the prohead RNA is involved in this encapsidation, and was found in monomer, dimer, pentamer and hexamer conformations . This article presents and debates current knowledge about the prohead RNA structures, mechanisms, and roles in DNA encapsidation . A new dimer structure is presented, and its specific role in DNA encapsidation is discussed.

J Struct Biol, 2002 Sep, 139(3), 161 - 70
Structure of Bacillus subtilis YXKO--a member of the UPF0031 family and a putative kinase; Zhang RG et al.; We determined the 1.6-A resolution crystal structure of a conserved hypothetical 29.9-kDa protein from the SIGY-CYDD intergenic region encoded by a Bacillus subtilis open reading frame in the YXKO locus . YXKO homologues are broadly distributed and are by and large described as proteins with unknown function . The YXKO protein has an alpha/beta fold and shows high structural homology to the members of a ribokinase-like superfamily . However, YXKO is the only member of this superfamily known to form tetramers . Putative binding sites for adenosine triphosphate (ATP), a substrate, and Mg(2+)-binding sites were revealed in the structure of the protein, based on high structural similarity to ATP-dependent members of the superfamily . Two adjacent monomers contribute residues to the active site . The crystal structure provides valuable information about the YXKO protein's tertiary and quaternary structure, the biochemical function of YXKO and its homologues, and the evolution of its ribokinase-like superfamily.

Microbiol Mol Biol Rev, 2002 Dec, 66(4), 671 - 701, table of contents
Regulation of bacterial drug export systems; Grkovic S et al.; The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs . This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription . Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves . Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump . This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions . In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E . coli mar, expression of which is controlled by the MarA and MarR proteins . However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter . Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.

Front Biosci, 2003 Jan 01, 8, d32 - 45
The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis; Pottathil M et al.; In the field of cell-cell communication, an emerging class of extracellular signaling peptides that function intracellularly has been identified in Gram-positive bacteria . One illustrative member of this group is the Phr family of extracellular signaling peptides of Bacillus subtilis . The Phr signaling peptides are secreted by the bacterium, and then, despite the presence of intracellular peptidases, they are actively transported into the cell where they interact with intracellular receptors to regulate gene expression . The intracellular receptors are members of a family of aspartyl-phosphate phosphatases, the Rap phosphatases . These phosphatases cause the dephosphorylation of response regulator proteins, ubiquitous regulatory proteins in bacteria . Immediately downstream of the genes for the Rap phosphatases are the genes for the Phr peptides, forming rap phr signaling cassettes . There are at least seven rap phr signaling cassettes in B . subtilis, and the genome sequence of other Gram-positive, endospore-forming bacteria suggests that similar cassettes may also function in these bacteria . In B . subtilis, the rap phr cassettes regulate sporulation, genetic competence, and genes comprising the quorum response (i.e . the response to high cell density) . This review will address the mechanism of extracellular Phr signaling peptide production, transport, response, and their role in quorum sensing.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2138 - 40 Epub 2002 Nov 23.
Crystallization and preliminary X-ray analysis of the ytxM gene product from Bacillus subtilis; Mehanni MM et al.; The ytxM gene product from Bacillus subtilis has been cloned, expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant . Multiple-wavelength anomalous dispersive X-ray data have been collected to 2.0 A resolution on a single selenomethionine-incorporated crystal . This crystal belongs to the primitive orthorhombic system, with approximate unit-cell parameters a = 44.3, b = 90.9, c = 136.1 A, alpha = beta = gamma = 90 degrees and two monomers in the asymmetric unit.

Mol Microbiol, 2002 Dec, 46(5), 1223 - 38
A central regulator of morphological differentiation in the multicellular bacterium Streptomyces coelicolor; Nguyen KT et al.; In the multicellular bacterium Streptomyces coelicolor, functions of developmental (bald) genes are required for the biosynthesis of SapB, a hydrophobic peptidic morphogen that facilitates aerial hyphae formation . Here, we show that aerial hyphal growth and SapB biosynthesis could be activated independently from the normal developmental cascade by providing unprogrammed expression of functionally interactive genes within the ram cluster . ramC, ramS and ramR were essential for normal growth of aerial hyphae, and ramR, a response regulator gene, was a key activator of development . The ramR gene restored growth of aerial hyphae and SapB formation in all bald strains tested (albeit only weakly in the bldC mutant), many of which are characterized by physiological defects . Disruption of the ramR gene abolished SapB biosynthesis and severely delayed growth of aerial hyphae . Transcription of ramR was developmentally controlled, and RamR function in vivo depended on its putative phosphorylation site (D53) . We identified and mapped RamR targets immediately upstream of the region encoding ramC and ramS, a putative operon . Overexpression of ramR in the wild-type strain increased SapB levels and caused a distinctive wrinkled surface topology . Based on these results, we propose that phenotypes of bald mutations reflect an early stage in the Streptomyces developmental programme similar to the spo0 mutations in the unicellular bacterium Bacillus subtilis, and that RamR has analogies to Spo0A, the Bacillus response regulator that integrates physiological signals before triggering endospore formation.

Biotechnol Appl Biochem, 2002 Dec, 36(Pt 3), 227 - 34
Purification and characterization of a novel enantioselective hydrolase from Bacillus subtilis; Maqbool QA et al.; Screening of the micro-organisms from an in-house microbial culture repository, identified a bacterial strain bearing membrane-bound, inducible ester hydrolase activity . The strain designated as RRL-BB1 has been identified as Bacillus subtilis by 16 S rRNA typing . Its application in the kinetic resolution of several racemates, including drug intermediates, showed moderate to high enantioselectivity . The enzyme, designated as BBL, exhibited high enantioselectivity (ee approximately 99%) with acyl derivatives of unsubstituted and substituted 1-(phenyl)ethanols and 1-(6-methoxy-2-naphthyl)ethanols . With acyl derivatives of 2-(6-methoxy-2-naphthyl)propan-1-ol, moderate enantioselectivity was observed . The enzyme also showed moderate enantioselectivity with alkyl esters of carboxylic acids i.e . 2-bromopropanoic acid and 2-hydroxy-4-phenylbutanoic acid . The enzyme was purified to >90% purity from cell-free extract of RRL-BB1 with 26% overall yield . The purified enzyme exhibited hydrolase activity without any noticeable decrease in the rate of hydrolysis or the enantioselectivity profile . A specific activity of 450 units/mg protein resulted after at least a 200-fold purification of the crude cell-free extract . The key purification step was the irreversible adsorption of the salt-precipitated crude enzyme on hydrophobic resin, in the presence of a low salt concentration, and desorption of the enzyme with a linear gradient of 1% sodium cholate . The purified enzyme was a 45 kD monomer as shown by SDS/PAGE . The N-terminal amino acid sequence of the purified enzyme was determined as Thr-Lys-Leu-Thr-Val-Gln-Thr-Arg-Asp-Gly-Ala-Leu-Arg-Gly-Thr . The N-terminal sequence did not bear any homology with other known bacterial lipases . BBL is maximally active at 37 degrees C, pH 8.0 and fairly stable up to 40 degrees C, pH 6-10 . The enzyme is insensitive to EDTA but inhibited by serine protease inhibitor PMSF . Its activity (72%) was retained in the presence of the anionic detergent SDS at a concentration of 0.2% (w/v).

J Appl Microbiol, 1997 Feb, 82(2), 267 - 72
Non-variable sources of pure water and the germination and outgrowth of Bacillus subtilis spores; Knott AG et al.; Variable germination and outgrowth occurred when Bacillus subtilis NCTC 8236 spores were inoculated into nutrient broth prepared with distilled water . More reproducible findings were achieved when the medium was prepared with Elgastat water and the greatest reproducibility occurred with Elgastat water as vehicle combined with a rigorous acid-washing of all glassware . This combined procedure also produced optimum and reproducible results for the synchronous growth of two B . subtilis 168 strains in casein medium supplemented with appropriate amino acids, a technique of value in monitoring the development of resistance to antibacterial agents during sporulation . The levels of aluminium in distilled water were higher than those of other elements; however, the incorporation of aluminium sulphate into broth prepared with Elgastat water had no effect on germination, and outgrowth was reduced (but not eliminated) only at high concentrations of this salt.

Zhong Yao Cai, 2002 Sep, 25(9), 651 - 3
{Study on the antibacterial activity of ginkgolic acids}; Yang X et al.; In this article we studied the anti-bacterial activity of the extract from testa of Ginkgo biloba and ginkgolic acids . They can inhibit the growth of Staphylocococus aureus, Escherichia coli, Bacillus subtilis, B . cereus and MRSA . Ginkgolic acids combined with peniciline can enhance their inhibitory activity to MRSA.

Appl Environ Microbiol, 2002 Dec, 68(12), 6210 - 9
Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge; Vater J et al.; An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements . This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants . This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B . subtilis C-1 . This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B . subtilis strains . These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry . The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization . Iturin compounds which contain unusual fatty acid components were detected.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2052 - 9
Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification, characterization, gene cloning, and applications; Yamaguchi F et al.; Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5) . The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP . None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3 . Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2 . The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa . The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively . The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C . The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively . The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues . The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases . We caused the NAD synthetase gene to be expressed in E . coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E . coli was 180-fold that of B . stearothermophilus H-804 . The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.

Mikrobiologiia, 2002 Sep-Oct, 71(5), 705 - 12
{The effect of gamma-radiation and desiccation on the viability of the soil bacteria isolated from the alienated zone around the Chernobyl Nuclear Power Plant}; Romanovskaia VA et al.; Methylobacterium extorquens, M . mesophilicum, and Bacillus subtilis strains were found to be resistant to gamma-radiation, irrespective of whether they were isolated from the alienated zone around the Chernobyl Nuclear Power Plant or outside this zone . The LD90 of Methylobacterium and B . subtilis strains with respect to gamma-radiation was 2.0-3.4 and 3.7-4.4 kGy, respectively, whereas their LD99.99 values were 4.5-6.9 and more than 10 kGy, respectively . The high threshold levels of gamma-radiation for Methylobacterium and B . subtilis imply the efficient functioning of DNA repair systems in these bacteria . Unlike Bacillus polymyxa cells, the cells of M . extorquens, M . mesophilicum, and B . subtilis were also resistant to desiccation . Pseudomonas sp., Nocardia sp., and nocardioform actinomycetes were sensitive to both gamma-radiation and desiccation . Similar results were obtained when the bacteria studied were exposed to hydrogen peroxide and ultraviolet radiation . The results obtained indicate that the bacteria that are resistant to gamma-radiation are also resistant to desiccation, UV radiation, and hydrogen peroxide . The possibility of using simple laboratory tests (such as the determination of bacterial resistance to UV light and desiccation) for the evaluation of bacterial resistance to gamma-radiation is discussed.

Mikrobiologiia, 2002 Sep-Oct, 71(5), 581 - 95
{The Pho regulons of bacteria}; Vershinina OA et al.; Bacterial Pho regulons contain genes whose expression is regulated by a specific two-component signal transduction system . The products of these genes are involved in the transport and assimilation of inorganic phosphate . The paper summarizes data on the organization and function of Pho regulons in gram-negative and gram-positive bacteria, with particular emphasis on the Pho regulons of the best studied bacteria Escherichia coli and Bacillus subtilis.

J Biomol NMR, 2002 Sep, 24(1), 31 - 9
Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme; Eletsky A et al.; A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of HisIO6 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis . The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of 15N-15N and 1H-15N trans-hydrogen bond scalar couplings, (2h)J(NN) and (lh)J(HN), and by transfer of nuclear polarization across the hydrogen bond . The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT) . The observed hydrogen bond might stabilize the scaffold at the active site of BsCM . Because the Arg7-His 106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 27 - 32
Bacterial endospores and their significance in stress resistance; Nicholson WL et al.; In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution . Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure . Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination . Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment . We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components . Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV . The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment.

EMBO Rep, 2002 Dec, 3(12), 1163 - 7 Epub 2002 Nov 21.
The Min system is not required for precise placement of the midcell Z ring in Bacillus subtilis; Migocki MD et al.; In bacteria, the Min system plays a role in positioning the midcell division site by inhibiting the formation of the earliest precursor of cell division, the Z ring, at the cell poles . However, whether the Min system also contributes to establishing the precise placement of the midcell Z ring is unresolved . We show that the Z ring is positioned at midcell with a high degree of precision in Bacillus subtilis, and this is completely maintained in the absence of the Min system . Min is therefore not required for correct midcell Z ring placement in B . subtilis . Our results strongly support the idea that the primary role of the Min system is to block Z ring formation at the cell poles and that a separate mechanism must exist to ensure cell division occurs precisely at midcell.

J Biotechnol, 2003 Feb 13, 100(3), 203 - 8
Enzymatic synthesis of trehalose esters having lipophilicity; Raku T et al.; To improve trehalose lipophilicity, trehalose was regioselectively esterified with vinyl fatty acid esters in dimethyl formamide by protease from Bacillus subtilis to give 6-O-lauroyltrehalose, 6-O-myristoyltrehalose, 6-O-palmitoyltrehalose, 6-O-stearoyltrehalose, 6-O-oleoyltrehalose and 6-O-linoleoyltrehalose . The influence of structural variation by changing fatty acid substitute was examined by measurement of the surface tension and biodegradability.

Environ Pollut, 2002, 120(3), 517 - 20
Stability of green fluorescent protein using luminescence spectroscopy: is GFP applicable to field analysis of contaminants?
Smith CB, Anderson JE, Fischer RL, Webb SR.
Green fluorescent protein (GFP) was first isolated in the early 1970s for experimental use from coelenterates or the Pacific jellyfish . Aequorea victoria (Morin and Hastings, 1971) . GFP has since become a favored biomarker in the photophysical analysis of molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (Creemers et al., 2000) . Here we report using Bacillus subtilis expressing GFP to evaluate the influence of different environmental pH conditions on GFP fluorescence . Emission acquisitions were configured to excite at 471 nm and detect at an emission from 490 to 650 nm at 1-nm increments . Fluorescence intensity was significantly better at pH 7 (4.2 x 105 cps; P-value < 0.01) than at acid or alkaline conditions . GFP is a good biomarker for environments near netural conditions: however, GFP may be unsuitable where soils or waters are below or above pH 7 because of loss in fluorescence intensity . Alternative fluorescent markers and delivery systems must be examined in different environments to optimize responses from bioreporter molecules.

Z Naturforsch {C}, 2002 Sep-Oct, 57(9-10), 874 - 82
Zoosporicidal activities of anacardic acids against Aphanomyces cochlioides; Begum P et al.; The EtOAc soluble constituents of the unripe fruits of Ginkgo biloba showed motility inhibition followed by lysis of zoospores of the phytopathogenic Aphanomyces cochlioides . We purified 22:1-omega7-anacardic acid (1), 24:1-omega9-anacardic acid (2) and 22:0-anacardic acid (3), together with other related compounds, 21:1-omega7-cardol (4) and 21:1-omega7-cardanol (5) from the crude extracts of Ginkgo fruits . Amongst them, compound 1 was a major active agent in quality and quantity, and showed potent motility inhibition (98% in 30 min) followed by lysis (55% in 3 h) of the zoospores at 1 x 10(-7) M . The 2-O-methyl derivative (1-c) of 1 displayed antibacterial activity against Bacillus subtilis, but practically inactive to Escherichia coli . A brief study on structure-activity relationships revealed that a carboxyl group on the aromatic ring and an unsaturated side chain in the anacardic acid derivative are important for strong motility inhibitory and lytic activities against the zoospore.

FEMS Microbiol Lett, 2002 Nov 5, 216(2), 185 - 91
Heterologous expression of nonribosomal peptide synthetases in B . subtilis: construction of a bi-functional B subtilis/E coli shuttle vector system; Doekel S et al.; A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes . Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster . In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B . subtilis shuttle expression vectors . Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B . subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins.

J Mol Microbiol Biotechnol, 2002 Sep, 4(5), 495 - 501
The general stress protein Ctc of Bacillus subtilis is a ribosomal protein; Schmalisch M et al.; Cells respond to stress conditions by synthesizing general or specific stress proteins . The Ctc protein of Bacillus subtilis belongs to the general stress proteins . The synthesis of Ctc is controlled by an alternative sigma factor of RNA polymerase, sigmaB . Sequence analyses revealed that Ctc is composed of two domains, an N-terminal domain similar to the ribosomal protein L25 of Escherichia coli, and a C-terminal domain . The similarity of the N-terminal domain of Ctc to L25 suggested that Ctc might be a ribosomal protein in B . subtilis . The function of the C-terminal domain is unknown . We purified Ctc to homogeneity and used the pure protein to raise antibodies . Western blot analyses demonstrate that Ctc is induced under stress conditions and can be found in ribosomes of B . subtilis . As observed for its E . coli counterpart L25, Ctc is capable of binding 5S ribosomal RNA in a specific manner . The stress-specific localization of Ctc in B . subtilis ribosomes and the sporulation defect of ctc mutants at high temperatures suggest that Ctc might be required for accurate translation under stress conditions.

Structure (Camb), 2002 Nov, 10(11), 1581 - 92
Structural and biochemical analysis of the Obg GTP binding protein; Buglino J et al.; The Obg nucleotide binding protein family has been implicated in stress response, chromosome partitioning, replication initiation, mycelium development, and sporulation . Obg proteins are among a large group of GTP binding proteins conserved from bacteria to man . Members of the family contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain . Structural analysis of Bacillus subtilis Obg revealed respective domain architectures and how they are coupled through the putative switch elements of the C-terminal GTPase domain in apo and nucleotide-bound configurations . Biochemical analysis of bacterial and human Obg proteins combined with the structural observation of the ppGpp nucleotide within the Obg active sight suggest a potential role for ppGpp modulation of Obg function in B . subtilis.

Structure (Camb), 2002 Nov, 10(11), 1471 - 2
Obg, a G domain with a beautiful extension; Wittinghofer A; The structure of Obg, a protein involved in a complicated genetic network that regulates stress response and sporulation in Bacillus subtilis, reveals a completely new type of guanine nucleotide binding protein and provides some hints about its function.

Microbiology, 2002 Nov, 148(Pt 11), 3539 - 52
Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis; Morimoto T et al.; GTP-binding proteins are found in all domains of life and are involved in various essential cellular processes . With the recent explosion of available genome sequence data, a widely distributed bacterial subfamily of GTP-binding proteins was discovered, represented by the Escherichia coli Era and the Bacillus subtilis Obg proteins . Although only a limited number of the GTP-binding proteins belonging to the subfamily have been experimentally characterized, and their function remains unknown, the available data suggests that many of them are essential to bacterial growth . When the complete genomic sequence of B . subtilis was surveyed for genes encoding GTP-binding proteins of the Era/Obg family, nine such genes were identified . As a first step in elucidating the functional networks of those nine GTP-binding proteins, data presented here indicates that six of them are essential for B . subtilis viability . Additionally, it is shown that the six essential proteins are able to specifically bind GTP and GDP in vitro . Experimental depletion of the essential GTP-binding proteins was examined in the context of cell morphology and chromosome replication, and it was found that two proteins, Bex and YqeH, appeared to participate in the regulation of initiation of chromosome replication . Collectively, these results suggest that members of the GTP-binding Era/Obg family are important proteins with precise, yet still not fully understood, roles in bacterial growth and viability.

Microbiology, 2002 Nov, 148(Pt 11), 3441 - 55
Genome-wide transcriptional profiling of the Bacillus subtilis cold-shock response; Kaan T et al.; The transcriptome of Bacillus subtilis was analysed at different time points (30, 60 and 90 min) after a temperature downshift from 37 to 18 degrees C using DNA macroarrays . This approach allowed the identification of around 50 genes exhibiting an increased mRNA level and around 50 genes exhibiting a decreased mRNA level under cold-shock conditions . Many of the repressed genes encode enzymes involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating metabolic adaptation of the cells to the decreased growth rate at the lower temperature . The strongest cold-inducible gene encodes fatty acid desaturase, which forms unsaturated fatty acids from saturated phospholipid precursors, thereby increasing membrane fluidity . The cold-shock-induced increase of mRNA levels of the classical cold-shock genes cspB, cspC and cspD could be verified . Furthermore, besides many genes encoding proteins of unknown function, some genes encoding ribosomal proteins were transcriptionally up-regulated, which points to an adaptive reprogramming of the ribosomes under cold-shock conditions . Interestingly, the amount of mRNA specified by the operon ptb-bcd-buk-lpd-bkdA1-bkdA2-bkdB, which encodes enzymes involved in degradation of branched-chain amino acids, also increases after a temperature downshift . As cells utilize the isoleucine and valine degradation intermediates alpha-methylbutyryl-CoA and isobutyryl-CoA for synthesis of branched-chain fatty acids, this finding reflects the adaptation of membrane lipid composition, ensuring the maintenance of appropriate membrane fluidity at low temperatures . The results of the DNA array analyses were verified for several selected genes by RNA slot-blot analysis and compared with two-dimensional PAGE analyses.

Microbiology, 2002 Nov, 148(Pt 11), 3405 - 12
Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+-citrate transporter CitM; Warner JB et al.; Bacillus subtilis 168 was assayed for its growth on tricarboxylic acid (TCA) cycle intermediates and related compounds as the sole carbon sources . Growth of the organism was supported by citrate, D-isocitrate, succinate, fumarate and L-malate, whereas no growth was observed in the presence of cis-aconitate,2-oxoglutarate, D-malate, oxaloacetate and tricarballylate . Growth of the organism on the tricarboxylates citrate and D-isocitrate required the presence of functional CitM, an Mg(2+)-citrate transporter, whereas its growth on succinate, fumarate and L-malate appeared to be CitM-independent . Interestingly, the naturally occurring enantiomer D-isocitrate was favoured over L-isocitrate by the organism . Like citrate, D-isocitrate was shown to be an inducer of citM expression in B . subtilis . The addition of 1 mM Mg(2+) to the growth medium improved growth of the organism on both citrate and D-isocitrate, suggesting that D-isocitrate was taken up by CitM in complex with divalent metal ions . Subsequently, the ability of CitM to transport D-isocitrate was demonstrated by competition experiments and by heterologous exchange in right-side-out membrane vesicles prepared from E . coli cells expressing citM . None of the other TCA cycle intermediates and related compounds tested were recognized by CitM . Uptake experiments using radioactive (63)Ni(2+) provided direct evidence that D-isocitrate is transported in complex with divalent metal ions.

Biochemistry, 2002 Nov 19, 41(46), 13499 - 506
Metal binding to Saccharomyces cerevisiae ferrochelatase; Karlberg T et al.; Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway . It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX . The crystal structures of ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work . The enzyme is a homodimer, with clear asymmetry between the monomers with regard to the porphyrin binding cleft and the mode of metal binding . The Co(II) and Cd(II) complexes reveal the metal binding site which consists of the invariant amino acids H235, E314, and S275 and solvent molecules . The shortest distance to the metal reveals that amino acid H235 is the primary metal binding residue . A second site with bound Cd(II) was found close to the surface of the molecule, approximately 14 A from H235, with E97, H317, and E326 participating in metal coordination . It is suggested that this site corresponds to the magnesium binding site in Bacillus subtilis ferrochelatase . The latter site is also located at the surface of the molecule and thought to be involved in initial metal binding and regulation.

EMBO J, 2002 Nov 15, 21(22), 6267 - 74
MinCD-dependent regulation of the polarity of SpoIIIE assembly and DNA transfer; Sharp MD et al.; During Bacillus subtilis sporulation, the SpoIIIE DNA translocase moves a trapped chromosome across the sporulation septum into the forespore . The direction of DNA translocation is controlled by the specific assembly of SpoIIIE in the mother cell and subsequent export of DNA into the forespore . We present evidence that the MinCD heterodimer, which spatially regulates cell division during vegetative growth, serves as a forespore-specific inhibitor of SpoIIIE assembly . The deletion of minCD increases the ability of forespore-expressed SpoIIIE to assemble and translocate DNA, and causes otherwise wild-type cells to reverse the direction of DNA transfer, producing anucleate forespores . We propose that two distinct mechanisms ensure the specific assembly of SpoIIIE in the mother cell, the partitioning of more SpoIIIE molecules into the larger mother cell by asymmetric cell division and the MinCD-dependent repression of SpoIIIE assembly in the forespore . Our results suggest that the ability of MinCD to sense positional information is utilized during sporulation to regulate protein assembly differentially on the two faces of the sporulation septum.

EMBO J, 2002 Nov 15, 21(22), 6185 - 94
The phi29 transcriptional regulator contacts the nucleoid protein p6 to organize a repression complex; Calles B et al.; The nucleoid protein p6 of Bacillus subtilis phage phi29 binds to DNA, recognizing a structural feature rather than a specific sequence . Upon binding to the viral DNA ends, p6 generates an extended nucleoprotein complex that activates the initiation of phi29 DNA replication . Protein p6 also participates in transcription regulation, repressing the early C2 promoter and assisting the viral regulatory protein p4 in controlling the switch from early to late transcription . Proteins p6 and p4 bind cooperatively to an approximately 200 bp DNA region located between the late A3 and the early A2c promoters, generating an extended nucleoprotein complex that helps to repress the early A2c promoter and to activate the late A3 promoter . We show that stable assembly of this complex requires interaction between protein p6 and the C-terminus of protein p4 . Therefore, at this DNA region, stable polymerization of protein p6 relies on p4-specified signals in addition to the structural features of the DNA . Protein p4 would define the phase and boundaries of the p6-DNA complex.

J Bacteriol, 2002 Dec, 184(23), 6734 - 8
cis-acting sequences of Bacillus subtilis pyrG mRNA essential for regulation by antitermination; Meng Q et al.; Expression of the Bacillus subtilis pyrG gene, which encodes CTP synthetase, is repressed by cytidine nucleotides . Regulation involves a termination-antitermination mechanism acting at a transcription terminator located within the 5' untranslated pyrG leader sequence . Deletion and substitution mutagenesis of a series of pyrG'-lacZ transcriptional fusions integrated into the B . subtilis chromosome demonstrated that only the terminator stem-loop and two specific 4- to 6-nucleotide RNA sequences were required for derepression of pyrG by starvation for cytidine nucleotides . The first sequence, GGGC/U, comprises the first four nucleotides at the 5' end of the pyrG transcript, and the second, GCUCCC, forms the first six nucleotides of the 5' strand of the terminator stem . All of the nucleotides lying between the two required RNA sequences can be deleted without loss of regulation . We propose that an as-yet-unidentified regulatory protein binds to these two RNA segments and prevents termination of transcription in the pyrG leader region when intracellular CTP levels are low.

J Bacteriol, 2002 Dec, 184(23), 6508 - 14
Functional analysis of the Bacillus subtilis Zur regulon; Gaballa A et al.; The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake . We have used DNA microarrays to identify genes that are derepressed in a zur mutant . In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation . Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation . Zur binds to an approximately 28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA . Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant . Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway . Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation . Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system . Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.

J Bacteriol, 2002 Dec, 184(23), 6424 - 33
Requirement of flhA for swarming differentiation, flagellin export, and secretion of virulence-associated proteins in Bacillus thuringiensis; Ghelardi E et al.; Bacillus thuringiensis is being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen . While phospholipases, hemolysins, and enterotoxins are claimed to be responsible for B . thuringiensis virulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions . This report describes the characterization of a mini-Tn10 mutant of B . thuringiensis that is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C . The mutant strain was determined to carry the transposon insertion in flhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus . Interestingly, the flhA mutant of B . thuringiensis synthesized flagellin but was impaired in flagellin export . Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable in B . thuringiensis, thus suggesting that the flagellar gene expression hierarchy of B . thuringiensis differs from that described for Bacillus subtilis . The flhA mutant of B . thuringiensis was also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant . Since complementation of the mutant with a plasmid harboring the flhA gene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence in B . thuringiensis may be coordinately regulated by flhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.

Mol Microbiol, 2002 Nov, 46(4), 997 - 1009
Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein; Lindow JC et al.; The Bacillus subtilis structural maintenance of chromosomes (SMC) protein is a member of a large family of proteins involved in chromosome organization . We found that SMC is a moderately abundant protein ( approximately 1000 dimers per cell) . In vivo cross-linking and immunoprecipitation assays revealed that SMC binds to many regions on the chromosome . Visualization of SMC in live cells using a fusion to the green fluorescent protein (GFP) and in fixed cells using immunofluorescence microscopy indicated that a portion of SMC localizes as discrete foci in positions similar to that of the DNA replication machinery (replisome) . When visualized simultaneously, SMC and the replisome were often in similar regions of the cell but did not always co-localize . Persistence of SMC foci did not depend on ongoing replication, but did depend on ScpA and ScpB, two proteins thought to interact with SMC . Our results indicate that SMC is bound to many sites on the chromosome and a concentration of SMC is localized near replication forks, perhaps there to bind and organize newly replicated DNA.

EMBO J, 2002 Nov 1, 21(21), 5733 - 44
Hinge-mediated dimerization of SMC protein is essential for its dynamic interaction with DNA; Hirano M et al.; Structural maintenance of chromosomes (SMC) proteins play central roles in regulating higher order chromosome dynamics from bacteria to humans . As judged by electron microscopy, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel, coiled-coil arms with a flexible hinge . Site-directed cross-linking experiments show here that dimerization of BsSMC is mediated by a hinge-hinge interaction between self-folded monomers . This architecture is conserved in the eukaryotic SMC2-SMC4 heterodimer . Analysis of different deletion mutants of BsSMC unexpectedly reveals that the major DNA-binding activity does not reside in the catalytic ATPase domains located at the ends of a dimer . Instead, point mutations in the hinge domain that disturb dimerization of BsSMC drastically reduce its ability to interact with DNA . Proper hinge function is essential for BsSMC to recognize distinct DNA topology, and mutant proteins with altered hinge angles cross-link double-stranded DNA in a nucleotide-dependent manner . We propose that the hinge domain of SMC proteins is not a simple dimerization site, but rather it acts as an essential determinant of dynamic SMC-DNA interactions.

J Biol Chem, 2003 Jan 10, 278(2), 1174 - 85 Epub 2002 Oct 30.
Properties and regulation of the bifunctional enzyme HPr kinase/phosphatase in Bacillus subtilis; Ramstrom H et al.; The bifunctional allosteric enzyme HPr kinase/phosphatase (HPrK/P) from Bacillus subtilis is a key enzyme in the main mechanism of carbon catabolite repression/activation (i.e . a means for the bacteria to adapt rapidly to environmental changes in carbon sources) . In this regulation system, the enzyme can phosphorylate and dephosphorylate two proteins, HPr/HPr(Ser(P)) and Crh/Crh(Ser(P)), sensing the metabolic state of the cell . To acquire further insight into the properties of HPrK/P, electrospray ionization mass spectrometry, dynamic light scattering, and BIACORE were used to determine the oligomeric state of the protein under native conditions, revealing that the enzyme exists as a hexamer at pH 6.8 and as a monomer and dimer at pH 9.5 . Using an in vitro radioactive assay, the influence of divalent cations, pH, temperature, and different glycolytic intermediates on the activity as well as kinetic parameters were investigated . The presence of divalent cations was found to be essential for both opposing activities of the enzyme . Furthermore, pH values equal to the internal pH of vegetative cells seem to favor the kinase activity, whereas lower pH values increased the phosphatase activity . Among the glycolytic intermediates evaluated, fructose 1,6-diphosphate and fructose 2,6-diphosphate were found to be allosteric activators in the kinase assay, whereas high concentrations inhibited the phosphatase activity, except for fructose 1,6-diphosphate in the case of HPr(Ser(P)) . Phosphatase activity was induced by inorganic phosphate as well as acetyl phosphate and glyceraldehyde 3-phosphate . Kinetic parameters indicate a preference for binding of HPr compared with Crh to the enzyme and supported a strong positive cooperativity . This work suggests that the oligomeric state of the enzyme is influenced by several effectors and is correlated to the kinase or phosphatase activity . The phosphatase activity is mainly supported by the hexameric form.

Protein Expr Purif, 2002 Nov, 26(2), 235 - 42
The preparation and characterization of an antimicrobial polypeptide from the loach, Misgurnus anguillicaudatus; Dong XZ et al.; Using Sephadex G-50 gel filtration, DEAE-52 cellulose ion-exchange chromatography, and an improved polyacrylamide gel electrophoresis together with electroelution, a novel polypeptide with antimicrobial activity in vitro was isolated and characterized from loach, Misgurnus anguillicaudatus . The polypeptide, named MAPP, contains about 94 residues containing l0 different amino acids, of which cysteine was the most abundant . No alkaline residue was found in MAPP . MAPP is a single-chain polypeptide with Mw of about 9800Da and pI of about 4.78; the N-terminus of MAPP was CFGWN . MAPP showed good inhibition of various bacteria including Bacillus subtilis, Escherichia coli, and Staphylococcus aureus . MAPP is thermally stable with more than 70% inhibitory bioactivity remaining after treatment at 60 degrees C for 30min . In addition, MAPP could inhibit the autoxidation of pyrogallol with a high efficiency . Similarity searches by comparing amino acid composition, MS-fingerprint, and the N-terminus of MAPP demonstrated that no protein exactly matched MAPP in databases around the world.

PDA J Pharm Sci Technol, 2002 Sep-Oct, 56(5), 242 - 7
Study of sterilizing effectivity of different ethylene oxide gaseous mixtures using CFCs and HCFCs (Oxyfume 12R and 2002R); de Oliveira DC et al.; The use of ethylene oxide as a sterilizing agent has been frequently practiced, especially taking into account the huge variety of medical devices produced with heat sensitive materials . Mixtures of ethylene oxide and inert gases have been widely adopted in order to decrease the undesirable flammable and explosive properties of ethylene oxide . This article provides a study regarding the sterilizing effectiveness of two ethylene oxide blends: Oxyfume 2002R (using HCFCs 22 and 124) and Oxyfume 12R (using CFC 12), at different temperatures (45, 55, and 65 degrees C) . To accomplish this procedure, sub-lethal challenges were performed (0, 3, 6, 9, 12, and 15 minutes) using biological indicators (obtained in the laboratory) made of Bacillus subtilis var . niger ATCC 9372 . The sterilizing efficacy of both mixtures was equivalent at the gas concentration of 600 mg/L . The influence of higher temperatures was thus proved.

Proteins, 2002 Dec 1, 49(4), 432 - 8
Influence of induced fit in the interaction of Bacillus subtilis trp RNA-binding attenuator protein and its RNA antiterminator target oligomer; Flynn PF et al.; In the presence of excess tryptophan, tryptophan-activated TRAP (trp RNA-binding attenuator protein) binds to a specific target in the trp-leader transcript, which induces the formation of a transcription terminator and transcription halts in the leader region . In the absence of tryptophan, TRAP does not bind RNA, an antiterminator forms, and the operon is expressed . Although the ternary complex involving TRAP (Bacillus stearothermophilus), tryptophan, and the RNA target has recently been crystallized, efforts to obtain structural data for the apo-form of TRAP (in any species) have not been successful . We have used multidimensional/multinuclear nuclear magnetic resonance (NMR) spectroscopy to probe the structure-function relationship in the TRAP-activated system, and have obtained high-resolution multidimensional/multinuclear NMR spectra of TRAP in all three of its functional states: tryptophan-free or apo-TRAP, tryptophan-activated TRAP, and tryptophan-activated TRAP-RNA ternary complex . Chemical shift perturbation analysis of the NMR data clarifies the interpretation of results obtained from previous crystal studies . Results presented herein demonstrate that tryptophan binding induces an essential structural change in TRAP that supports high-affinity binding of the RNA target oligonucleotide .

Biochem J, 2003 Feb 1, 369(Pt 3), 731 - 8
Calcium triggers the refolding of Bacillus subtilis chitosanase; Colomer-Pallas A et al.; We characterized the reversible folding-unfolding transition of Bacillus subtilis exocellular chitosanase from either thermal or urea denaturation of the protein . The transitions were monitored in each case by intrinsic fluorescence changes and resistance to proteolysis . Unfolding and refolding kinetics and differential scanning calorimetry analysis suggested a two-state equilibrium . The equilibrium between the folded and unfolded states was rapidly displaced towards the folded state in the presence of a low concentration of calcium (2-20 mM) . The binding titration curve indicated that chitosanase possesses one weak Ca(2+)-binding site (with an equilibrium affinity constant, K (A), of 0.3x10(3) M(-1)) . These results support the hypothesis that this metal ion, which is accumulated in the cell wall environment of B . subtilis, is an effector that influences folding and stability of newly translocated proteins.

Biosci Biotechnol Biochem, 2002 Sep, 66(9), 1994 - 6
Frequency of the insertion sequence IS4Bsu1 among Bacillus subtilis strains isolated from fermented soybean foods in Southeast Asia; Kimura K et al.; Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome . In contrast, none of 49 B . subtilis strains of non-food origin contained IS4Bsu1 . Frequent occurrence of this mobile DNA element in the soybean-fermenting B . subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.

J Bacteriol, 2002 Nov, 184(22), 6395 - 402
Genomewide transcriptional analysis of the cold shock response in Bacillus subtilis; Beckering CL et al.; Previous studies with two-dimensional gel electrophoresis techniques revealed that the cold shock response in Bacillus subtilis is characterized by rapid induction and accumulation of two classes of specific proteins, which have been termed cold-induced proteins (CIPs) and cold acclimatization proteins (CAPs), respectively . Only recently, the B . subtilis two-component system encoded by the desKR operon has been demonstrated to be essential for the cold-induced expression of the lipid-modifying desaturase Des, which is required for efficient cold adaptation of the membrane in the absence of isoleucine . At present, one of the most intriguing questions in this research field is whether DesKR plays a global role in cold signal perception and transduction in B . subtilis . In this report, we present the first genomewide transcriptional analysis of a cold-exposed bacterium and demonstrate that the B . subtilis two-component system DesKR exclusively controls the desaturase gene des and is not the cold-triggered regulatory system of global relevance . In addition to this, we identified a set of genes that might participate as novel players in the cold shock adaptation of B . subtilis . Two cold-induced genes, the elongation factor homolog ylaG and the sigma(L)-dependent transcriptional activator homolog yplP, have been examined by construction and analysis of deletion mutants.

J Bacteriol, 2002 Nov, 184(22), 6389 - 94
Bex, the Bacillus subtilis homolog of the essential Escherichia coli GTPase Era, is required for normal cell division and spore formation; Minkovsky N et al.; The Bacillus subtilis bex gene complemented the defect in an Escherichia coli era mutant . The Bex protein showed 39 percent identity and 67 percent similarity to the E . coli Era GTPase . In contrast to era, bex was not essential in all strains . bex mutant cells were elongated and filled with diffuse nucleoid material . They grew slowly and exhibited severely impaired spore formation.

J Bacteriol, 2002 Nov, 184(22), 6250 - 9
Bacillus subtilis YhaM, a member of a new family of 3'-to-5' exonucleases in gram-positive bacteria; Oussenko IA et al.; A strain of Bacillus subtilis lacking two 3'-to-5' exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3'-to-5' exoribonuclease, which is encoded by the yhaM gene . YhaM was active in the presence of Mn(2+) (or Co(2+)), was inactive in the presence of Mg(2+), and could also degrade single-stranded DNA . The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover . Sequence homologues of YhaM were found only in gram-positive organisms . The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn(2+)-dependent exoribonuclease . YhaM protein has a C-terminal "HD domain," found in metal-dependent phosphohydrolases . By structure modeling, it was shown that YhaM also contains an N-terminal "OB-fold," present in many oligosaccharide- and oligonucleotide-binding proteins . The combination of these two domains is unique . Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.

J Bacteriol, 2002 Nov, 184(22), 6123 - 9
Regulation of the Bacillus subtilis bcrC bacitracin resistance gene by two extracytoplasmic function sigma factors; Cao M et al.; Bacitracin resistance is normally conferred by either of two major mechanisms, the BcrABC transporter, which pumps out bacitracin, or BacA, an undecaprenol kinase that provides C(55)-isoprenyl phosphate by de novo synthesis . We demonstrate that the Bacillus subtilis bcrC (ywoA) gene, encoding a putative bacitracin transport permease, is an important bacitracin resistance determinant . A bcrC mutant strain had an eightfold-higher sensitivity to bacitracin . Expression of bcrC initiated from a single promoter site that could be recognized by either of two extracytoplasmic function (ECF) sigma factors, sigma(X) or sigma(M) . Bacitracin induced expression of bcrC, and this induction was dependent on sigma(M) but not on sigma(X) . Under inducing conditions, expression was primarily dependent on sigma(M) . As a consequence, a sigM mutant was fourfold more sensitive to bacitracin, while the sigX mutant was only slightly sensitive . A sigX sigM double mutant was similar to a bcrC mutant in sensitivity . These results support the suggestion that one function of B . subtilis ECF sigma factors is to coordinate antibiotic stress responses.

J Bacteriol, 2002 Nov, 184(22), 6109 - 14
The PrpC serine-threonine phosphatase and PrkC kinase have opposing physiological roles in stationary-phase Bacillus subtilis cells; Gaidenko TA et al.; Loss of the PrpC serine-threonine phosphatase and the associated PrkC kinase of Bacillus subtilis were shown to have opposite effects on stationary-phase physiology by differentially affecting cell density, cell viability, and accumulation of beta-galactosidase from a general stress reporter fusion . These pleiotropic effects suggest that PrpC and PrkC have important regulatory roles in stationary-phase cells . Elongation factor G (EF-G) was identified as one possible target of the PrpC and PrkC pair in vivo, and purified PrpC and PrkC manifested the predicted phosphatase and kinase activities against EF-G in vitro.

J Biol Chem, 2002 Dec 27, 277(52), 50476 - 81 Epub 2002 Oct 23.
Altering of the metal specificity of Escherichia coli alkaline phosphatase; Wojciechowski CL et al.; Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands . The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328 . Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively . The mutations D153H, K328W, and D153H/K328W were induced in E . coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme . The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis . Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+) . These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis . These studies suggest that the active site residues His and Trp at the E . coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.

Biochemistry, 2002 Oct 29, 41(43), 12986 - 94
Dimeric and monomeric Bacillus subtilis RNase P holoenzyme in the absence and presence of pre-tRNA substrates; Barrera A et al.; Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that catalyzes the 5' maturation of tRNA precursors . The bacterial RNase P holoenzyme is composed of a large, catalytic RNA and a small protein . Our previous work showed that Bacillus subtilis RNase P forms a specific "dimer" that contains two RNase P RNA and two RNase P protein subunits in the absence of substrate . We investigated the equilibrium and the structure of the dimeric and the monomeric holoenzyme in the absence and presence of substrates by synchrotron small-angle X-ray scattering, 3' autolytic processing, and hydroxyl radical protection . In the absence of substrate, the dimer-monomer equilibrium is sensitive to monovalent ions and the total holoenzyme concentration . At 0.1 M NH4Cl, formation of the dimer is strongly favored, whereas at 0.8 M NH4Cl, the holoenzyme is a monomer . Primary hydroxyl radical protection in the dimer is located in the specificity domain, or domain I, of the RNase P RNA . The ES complex with a substrate containing a single tRNA is always monomeric . In contrast, the dominant ES complex with a substrate containing two tRNAs is dimeric at 0.1 M NH4Cl and monomeric at 0.8 M NH4Cl . Our results show that the B . subtilis holoenzyme can be a dimer and a monomer, and the fraction of the dimer is very sensitive to the environment . Under a variety of conditions, both the holoenzyme dimer and monomer can be present in significant amounts . Because the majority of tRNA genes are organized in large operons and because of the lack of RNase E in B . subtilis, a dimeric holoenzyme may be necessary to facilitate the processing of large precursor tRNA transcripts . Alternatively, the presence of two forms of the RNase P holoenzyme may be required for other yet unknown functions.

J Biol Chem, 2002 Dec 13, 277(50), 48574 - 8 Epub 2002 Oct 16.
Zinc is required for assembly and function of the anti-trp RNA-binding attenuation protein, AT; Valbuzzi A et al.; The anti-TRAP protein (AT) of Bacillus subtilis regulates expression of the trp operon and other genes concerned with tryptophan metabolism . AT acts by inhibiting the tryptophan-activated trp RNA-binding attenuation protein (TRAP) . AT is an oligomer of identical 53-residue polypeptides; it is produced in response to the accumulation of uncharged tRNA(Trp) . Each AT polypeptide has two cysteine-rich clusters that correspond to the signature motif of the cysteine-rich zinc-binding domain of the chaperone protein DnaJ . Here we characterize the putative zinc-binding domain of AT and establish the importance of zinc for AT assembly and activity . AT is shown to contain Zn(II) at a ratio of one ion per monomer . Bound zinc is necessary for maintenance of the quaternary structure of AT; the removal of zinc converts the AT complex into inactive monomers . All four cysteine residues in the AT polypeptide are involved in Zn(II) coordination . Chemical cross-linking analyses indicate that the AT functional oligomer is a hexamer composed of two trimers . Substituting alanine for any cysteine residue of AT results in rapid degradation of the mutant protein in vivo . We propose a model for the AT trimer in which three AT chains are held together by three zinc atoms, each coordinated by the N-terminal segment and the C-terminal segment of separate AT polypeptides.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14089 - 94 Epub 2002 Oct 16.
Does RNA polymerase help drive chromosome segregation in bacteria?
Dworkin J, Losick R.
In contrast to eukaryotic cells, bacteria segregate their chromosomes without a conspicuous mitotic apparatus . Replication of bacterial chromosomes initiates bidirectionally from a single site (oriC), and visualization of the region of the chromosome containing oriC in living cells reveals that origins rapidly move apart toward opposite poles of the cell during the cell cycle . The motor that drives this poleward movement is unknown . An attractive candidate is RNA polymerase, which is a powerful and abundant molecular motor . If, as has been suggested for other macromolecular complexes, the movement of RNA polymerase is restricted in the cell, then transcription would translocate the DNA template, thereby providing the motive force to separate replicating chromosomes . A coordinated effect of many transcribing RNA polymerases could result from the widely conserved global bias of gene orientation away from oriC . By using fluorescence microscopy of living Bacillus subtilis cells, we demonstrate that an inhibitor of RNA polymerase acts to inhibit separation of newly duplicated DNAs near the origin of replication . We propose that the force exerted by RNA polymerase contributes to chromosome movement in bacteria, and that this force, coupled with the biased orientation of transcription units, helps to drive chromosome segregation.

Gene, 2002 Aug 21, 296(1-2), 187 - 94
The in vivo function of phage phi29 nucleoid-associated protein p6 requires formation of dimers; Abril AM et al.; The Bacillus subtilis phage phi29 nucleoid-associated protein p6 (103 amino acids) is essential for in vivo viral DNA replication and control of transcription, and it has been proposed to play a role in genome organization and compaction . This protein self-associates in vitro from preformed dimers forming high-molecular-weight oligomers and binds to double-stranded DNA giving rise to multimeric nucleoprotein complexes . Site-directed mutants, p6I8T and p6A44V, were completely or partially inactive, respectively, in an in vitro dimerization assay . In this paper, and by in vivo crosslinking, we have detected dimers of protein p6 either in phage-infected cells or in protein p6 producing B . subtilis or Escherichia coli cells . Therefore, this self-association does not require viral DNA . We also show that mutants p6I8T and p6A44V are deficient in dimer formation, and they do not support phage DNA replication in a trans-complementation assay with phi29sus6 mutant-infected B . subtilis cells . We conclude that dimeric protein p6 is the active form of the protein in vivo, required for viral DNA replication.

Int J Food Microbiol, 2003 Jan 25, 80(2), 131 - 43
Thermal properties of bacterial spores and biopolymers; Leuschner RG et al.; Differential scanning calorimetry (DSC) measurements of dormant bacterial spores is traditionally associated with an endothermic transition at around 50 degrees C . This endothermic transition was described as an indicator for two main physico-chemical states in spores . These were a glassy state in the dormant spore core as a model for spore dormancy and a heat-activated state that generally facilitates spore resuscitation . The idea of a glassy state in dormant spores is based on the observation that a similar transition as in dormant spores was observed in low moisture biopolymers that are in a glassy state . Thermal properties of spores of Bacillus subtilis and B . cereus in a dormant and germinated, resuscitated state and of an outer and an inner coatless spore mutant of B . subtilis were investigated . Biopolymers with low moisture (<15%) and high moisture (>30%) contents such as maize starch, pectin, RNA and DNA were further studied . Critical evaluation of results revealed that the low temperature transition in dormant spores has some similarities to those observed in glassy biopolymers, but also to those of fully hydrated proteins and therefore does not necessarily indicate a glassy low moisture state . Its origin can also be attributed to the outer spore coats and it occurred at a lower temperature and for a shorter duration to be of significance for thermal heat activation of spores.

J Agric Food Chem, 2002 Oct 23, 50(22), 6473 - 84
Enzymatic solubilization of arabinoxylans from isolated rye pentosans and rye flour by different endo-xylanases and other hydrolyzing enzymes . Effect of a fungal caccase on the flour extracts oxidative gelation; Figueroa-Espinoza MC et al.; Water-extractable (WEP) and water-unextractable (WUP) pentosans were isolated from a rye flour . The effect of a commercial enzyme preparation, Grindamyl S 100 (GS100), containing pentosanase activities, was investigated on WEP, WUP, a mix of WEP and WUP, and the rye flour, with the aim to monitor the solubilization and depolymerization of high molecular weight arabinoxylans and the effect on the viscosity of the reaction medium . The effects of other hydrolyzing enzymes were also tested . Three xylanases were used: xylanase 1 (Xyl-1) from Aspergillus niger, the main activity present in GS100; xylanase 2 (Xyl-2) from Talaromyces emersonii; and xylanase 3 (Xyl-3) from Bacillus subtilis . Xyl-3 was used in combination with Xyl-1, (1,4)-beta-D-arabinoxylan arabinofuranohydrolase, endo-beta-D-glucanase, or ferulate esterase from A . niger, but no synergism was observed . GS100 and xylanases increased the arabinoxylan solubilization, Xyl-3 and Xyl-1 being those that presented the best yields of extraction without extensive depolymerization of water-extractable arabinoxylans . Both xylanases were affected by an inhibitor in rye flour . Flour treated with hot ethanol was used to study the oxidative gelation of flour extracts treated with xylanases, in the presence of laccase from Pycnoporus cinnabarinus . Two doses of xylanases were tested (0.5 and 2.5 units) . Only the flour extracts treated with 0.5 unit of Xyl-1 thickened.

Arch Microbiol, 2002 Nov, 178(5), 376 - 86 Epub 2002 Aug 22.
Molecular and biochemical characterization of a novel two-component signal transduction system, amrA- amkA, involved in rifamycin SV production in Amycolatopsis mediterranei U32; Wang W et al.; Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli . Although several model systems, including sporulation in Bacillus subtilis and chemotaxis in Escherichia coli, have been extensively studied, the two-component signal transduction systems in industrially important actinomycetes are not well studied . We report the molecular and biochemical characterization of a novel two-component signal system, amrA-amkA,from the rifamycin-SV-producing Amycolatopsis mediterranei U32 . The deduced sequences of amkAand amrA contain all the structural features that are highly conserved in the typical bacterial histidine kinases and response regulators, respectively . BLAST analyses showed that AmrA and AmkA displayed high similarities to AfsQ1/AfsQ2 of Streptomyces coelicolor and MtrA/MtrB of Mycobacterium tuberculosis . The amrAand amkA genes were over-expressed and the gene products were purified from E . coli . Biochemical studies showed that AmkA is able to autophosphorylate, supporting its functional assignment as a histidine kinase . That AmrA functions as the cognate response regulator for histidine kinase AmkA was demonstrated by in vitro phosphotransfer from {gamma-(32)P}ATP-labeled AmkA to AmrA . Rifamycin SV production was also decreased by 10-20% in amrAor amkA gene disruption mutants under the tested condition . Although the detailed regulatory mechanism is still unknown, this is the first report regarding the involvement of two-component signal systems in rifamycin biosynthesis in the genus Amycolatopsis.

Arch Microbiol, 2002 Nov, 178(5), 370 - 5 Epub 2002 Aug 13.
Impact of the Mg(2+)-citrate transporter CitM on heavy metal toxicity in Bacillus subtilis; Krom BP et al.; Bacillus subtilis possesses a secondary transporter, CitM, that is specific for the complex of citrate and Mg(2+) but is also capable of transporting citrate in complex with the heavy metal ions Zn(2+), Ni(2+) and Co(2+) . We report on the impact of CitM activity on the toxicity of Zn(2+), Ni(2+) and Co(2+) in B . subtilis . In a citM deletion mutant or under conditions in which CitM is not expressed, the toxic effects of the metals were reduced by the presence of citrate in the medium . In contrast, the presence of citrate dramatically enhanced toxicity when the Mg(2+)-citrate transporter was present in the membrane . It is demonstrated that the complex of Ni(2+) and citrate is transported into the cell and that the uptake is responsible for the enhanced toxicity . At toxic concentrations of the metal ions, the cultures adapted by developing tolerance against these ions . Tolerant cells isolated by exposure to one of the metal ions remained tolerant after growth in the absence of toxic metal ions and were cross-tolerant against the other two toxic ions . Tolerant strains were shown to contain point mutations in the citM gene, which resulted in premature termination of translation.

J Bacteriol, 2002 Nov, 184(21), 6073 - 80
A green nonsulfur bacterium, Dehalococcoides ethenogenes, with the LexA binding sequence found in gram-positive organisms; Fernandez de Henestrosa AR et al.; Dehalococcoides ethenogenes is a member of the physiologically diverse division of green nonsulfur bacteria . Using a TBLASTN search, the D . ethenogenes lexA gene has been identified, cloned, and expressed and its protein has been purified . Mobility shift assays revealed that the D . ethenogenes LexA protein specifically binds to both its own promoter and that of the uvrA gene, but not to the recA promoter . Our results demonstrate that the D . ethenogenes LexA binding site is GAACN(4)GTTC, which is identical to that found in gram-positive bacteria . In agreement with this fact, the Bacillus subtilis DinR protein binds specifically to the D . ethenogenes LexA operator . This constitutes the first non-gram-positive bacterium exhibiting a LexA binding site identical to that of B . subtilis.

J Bacteriol, 2002 Nov, 184(21), 6060 - 4
Roles of PucR, GlnR, and TnrA in regulating expression of the Bacillus subtilis ure P3 promoter; Brandenburg JL et al.; Expression of the P3 promoter of the Bacillus subtilis ureABC operon is activated during nitrogen-limited growth by PucR, the transcriptional regulator of the purine-degradative genes . Addition of allantoic acid, a purine-degradative intermediate, to nitrogen-limited cells stimulated transcription of ure P3 twofold . Since urea is produced during purine degradation in B . subtilis, regulation of ureABC expression by PucR allows purines to be completely degraded to ammonia . The nitrogen transcription factor TnrA was found to indirectly regulate ure P3 expression by activating pucR expression . The two consensus GlnR/TnrA binding sites located in the ure P3 promoter region were shown to be required for negative regulation by GlnR . Mutational analysis indicates that a cooperative interaction occurs between GlnR dimers bound at these two sites . B . subtilis is the first example where urease expression is both nitrogen regulated and coordinately regulated with the enzymes involved in purine transport and degradation.

J Bacteriol, 2002 Nov, 184(21), 6007 - 15
A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic delta-lactam residues in the spore cortex of Bacillus subtilis; Fukushima T et al.; The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases . Beta-galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase . pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L-alanine . Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed . In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant . Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam . A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B . subtilis spores . The biosynthetic pathway of muramic delta-lactam is discussed.

J Bacteriol, 2002 Nov, 184(21), 5826 - 32
Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence; Baichoo N et al.; Ferric uptake repressor (Fur) proteins regulate the expression of iron homeostasis genes in response to intracellular iron levels . In general, Fur proteins bind with high affinity to a 19-bp inverted repeat sequence known as the Fur box . An alignment of 19 operator sites recognized by Bacillus subtilis Fur revealed a different conserved 15-bp (7-1-7) inverted repeat present twice within this 19-bp consensus sequence . We demonstrated using electrophoretic mobility shift assays that this 7-1-7 inverted repeat comprises a minimal recognition site for high-affinity binding by Fur . The resulting revised consensus sequence is remarkably similar to a related 7-1-7 inverted repeat sequence recognized by PerR, a Fur paralog . Our analysis of the affinity and stoichiometry of DNA binding by B . subtilis Fur, together with a reinterpretation of previously described studies of Escherichia coli Fur, supports a model in which the 19-bp Fur box represents overlapping recognition sites for two Fur dimers bound to opposite faces of the DNA helix . The resulting recognition complex is reminiscent of that observed for the functionally related protein DtxR . Like Fur, DtxR contains a helix-turn-helix DNA-binding motif, recognizes a 19-bp inverted repeat sequence, and has a typical DNase I footprint of approximately 30 bp . By envisioning a similar mode of DNA recognition for Fur, we can account for the internal symmetries noted previously within the Fur box, the tendency of Fur to extend into adjacent regions of DNA in a sequence-selective manner, and the observed patterns of DNA protection against enzymatic and chemical probes.

J Biol Chem, 2003 Jan 24, 278(4), 2169 - 76 Epub 2002 Oct 07.
Guanine nucleotides guanosine 5'-diphosphate 3'-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis; Inaoka T et al.; We found that a polycistronic operon (ywfBCDEFG) and a monocistronic gene (ywfH) are required for the biosynthesis of bacilysin in Bacillus subtilis . The disruption of these genes by plasmid integration caused loss of the ability to produce bacilysin, accompanied by a lack of bacilysin synthetase activity in the crude extract . We investigated the regulatory mechanism for bacilysin biosynthesis using the transcriptional lacZ fusion system . The transcription of these genes was found to be induced at the transition from exponential to stationary phase . Induction of transcription was accelerated by depleting a required amino acid, which was done by transferring the wild-type (rel(+)) cells to an amino acid-limited medium . In contrast, no enhancement of the gene expression was detected in relA mutant cells . In wild-type (rel(+)) cells, a forced reduction of intracellular GTP, brought about by addition of decoyinine, which is a GMP synthetase inhibitor, enhanced the expression of both the ywfBCDEFG operon and the ywfH gene, resulting in a 2.5-fold increase in bacilysin production . Disruption of the codY gene, which regulates stationary phase genes by detecting the level of GTP, also induced transcription of these genes . In contrast, the expression of ywfBCDEFG in relA cells was not activated either by decoyinine addition or codY disruption, although the expression of ywfH was induced . Moreover, the codY disruption resulted in an increase of bacilysin production only in rel(+) cells . These results indicate that guanosine 5'-diphosphate 3'-diphosphate (ppGpp) plays a crucial role in transcription of the ywfBCDEFG operon and that the transcription of these genes are dependent upon the level of intracellular GTP which is transmitted as a signal via the CodY-mediated repression system . We propose that, unlike antibiotic production in Streptomyces spp., bacilysin production in B . subtilis is controlled by a dual regulation system composed of the guanine nucleotides ppGpp and GTP.

Microbiology, 2002 Oct, 148(Pt 10), 3277 - 84
A novel mode of control of Mycoplasma pneumoniae HPr kinase/phosphatase activity reflects its parasitic lifestyle; Steinhauer K et al.; Among the few regulatory proteins encoded by Mycoplasma pneumoniae is HPr kinase/phosphatase (HPrK/P), the key regulator of carbon metabolism in low-GC Gram-positive bacteria . The corresponding gene, hprK, and the gene encoding the target protein HPr, ptsH, were overexpressed . In vitro analysis of the purified proteins confirmed ATP-dependent phosphorylation of HPr by HPrK/P . In contrast to HPrK/P of Bacillus subtilis, which is by default a phosphatase and needs high ATP concentrations for kinase activity, the M . pneumoniae enzyme exhibits kinase activity at very low ATP concentrations and depends on P(i) for phosphatase activity . This inverted control of enzymic activity may result from the adaptation to very different ecological niches . While the standard activities of HPrK/P from M . pneumoniae and other Gram-positive bacteria differ, they are both modulated by the concentration of ATP, P(i) and glycolytic intermediates . Site-directed mutagenesis of a potential ATP-binding site and of the HPrK/P signature sequence resulted in four different activity classes: (i) inactive proteins, (ii) enzymes with reduced kinase and phosphatase activities, (iii) enzymes that had lost phosphatase, but not kinase activity, and (iv) enzymes that exhibited increased phosphatase activity.

Genes Dev, 2002 Oct 1, 16(19), 2544 - 56
A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ; Gueiros-Filho FJ et al.; Cell division in bacteria is mediated by the tubulin-like protein FtsZ, which assembles into a structure known as the Z ring at the future site of cytokinesis . We report the discovery of a Z-ring-associated protein in Bacillus subtilis called ZapA . ZapA was found to colocalize with the Z ring in vivo and was capable of binding to FtsZ and stimulating the formation of higher-order assemblies of the cytokinetic protein in vitro . The absence of ZapA alone did not impair cell viability, but the absence of ZapA in combination with the absence of a second, dispensable division protein EzrA caused a severe block in cytokinesis . The absence of ZapA also caused lethality in cells producing lower than normal levels of FtsZ or lacking the division-site-selection protein DivIVA . Conversely, overproduction of ZapA reversed the toxicity of excess levels of the division inhibitor MinD . In toto, the evidence indicates that ZapA is part of the cytokinetic machinery of the cell and acts by promoting Z-ring formation . Finally, ZapA is widely conserved among bacteria with apparent orthologs in many species, including Escherichia coli, in which the orthologous protein exhibited a strikingly similar pattern of subcellular localization to that of ZapA . Members of the ZapA family of proteins are likely to be a common feature of the cytokinetic machinery in bacteria.

Mol Microbiol, 2002 Oct, 46(1), 25 - 36
A new mutation delivery system for genome-scale approaches in Bacillus subtilis; Fabret C et al.; In Bacillus subtilis, although many genetic tools have been developed, gene replacement remains labour-intensive and not compatible with large-scale approaches . We have developed a new one-step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B . subtilis without altering the chromosome in any other way . This novel approach relies on the use of upp, which encodes uracil phosphoribosyl-transferase, as a counter-selectable marker . We fused the upp gene to an antibiotic-resistance gene to create an 'upp-cassette' . A polymerase chain reaction (PCR)-generated fragment, consisting of the target gene with the desired mutation joined to the upp-cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance . Then, the eviction of the upp-cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter-selection of upp . This procedure was successfully used to deliver a point mutation, to generate in-frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two-component sensor kinases . Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined . This work outlines a strategy for identifying essential functions that could be used at genome scale.

J Enzyme Inhib Med Chem, 2002 Feb, 17(1), 61 - 8
Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms; Gebruers K et al.; An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow . When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L . endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II) . Based on their inhibition activities against a B . subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished . The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B . subtilis endoxylanase.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 1109 - 15
Isoprene formation in Bacillus subtilis: a barometer of central carbon assimilation in a bioreactor?
Shirk MC, Wagner WP, Fall R.
Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon of uncertain function in Bacillus subtilis, and we hypothesized that it is an overflow metabolite produced during excess carbon utilization . Here we tested this idea for phase 2 of isoprene release, a phase that occurs during extracellular acetoin accumulation and its reassimilation . Phase 2 isoprene formation could be disrupted in three different ways, all related to acetoin metabolism . Disruption of a gene essential for acetoin biosynthesis (acetolactic acid synthase, alsS) blocked acetoin formation and led to cessation of phase 2 isoprene formation as well as a variety of pleiotropic effects related to loss of pH control . Growth of the alsS mutant with external pH control reversed most of these effects . Disruption of acetoin catabolism (acetoin dehydrogenase, acoA), also eliminated phase 2 isoprene formation and caused cells to transition directly from phase 1 to phase 3; the latter is attributed to amino acid catabolism . A third alteration of acetoin metabolism was detected in the widely used strain 168 (trpC2) but not in strain MS175, a trpC mutant constructed in the Marburg strain genetic background . Strain 168 exhibited slow acetoin assimilation compared to that of MS175 or the parental strain, with little or no isoprene formation during this growth phase . These findings support the idea that isoprene release occurs primarily when the rate of carbon catabolism exceeds anabolism and that this volatile hydrocarbon is a product of overflow metabolism when precursors are not required for higher isoprenoid biosynthesis . It is suggested that isoprene release might serve as a useful barometer of the rise and fall of central carbon fluxes during the growth of Bacillus strains in industrial bioreactors.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 986 - 93
Exquisite regioselectivity and increased transesterification activity of an immobilized Bacillus subtilis protease; Ferreira L et al.; Commercially available proteases and lipases were screened for their ability to acylate regioselectively sucrose with divinyladipate either in pyridine or dimethylformamide (DMF) . The protease (EC 3.4.21.62) from Bacillus subtilis (Proleather FG-F) exhibited the highest conversion (100% in 24 h of reaction in DMF) yielding sucrose 2-O-vinyladipate as main product . The enzyme preference for a secondary hydroxyl group is a distinct feature of this biocatalyst compared to others described in the literature . Two sets of chemically distinct silica supports were used for Proleather immobilization presenting terminal amino (S(APTES)) or hydroxyl groups (S(TESPM)(-)(pHEMA)) . The percentage of immobilized enzyme was smaller in S(APTES) (7-17%) than in S(TESPM)(-)(pHEMA) (52-56%), yet Proleather immobilized into S(APTES) supports presented higher total and specific hydrolytic activity . The highest total and specific activities were obtained with S(TESPM)(-)(pHEMA) and S(APTES), respectively . Silicas with large pore (bimodal distribution of pores, 130/1200 A, denoted as S(1000)) presented higher specific activities relative to those with smaller pore sizes . Furthermore, the synthetic specific activity of S(1000)S(APTES) immobilized protease was ca . 10-fold higher than that of the free enzyme . In addition to sucrose, the immobilized protease was used to acylate methyl alpha-D-glucopyranoside, trehalose, and maltose in nearly anhydrous DMF . Finally, immobilized Proleather was reasonably stable, retaining ca . 55% activity after six reaction cycles.

Farmaco, 2002 Aug, 57(8), 657 - 61
Antimicrobial activity of some N-alkyl substituted of (E)-4-azachalconium and (E)-3'-hydroxy-4-azachalconium bromides; Nowakowska Z et al.; Twelve new N-substituted (E)-azachalconium bromides were synthesized and tested for antimicrobial and antifungal activities . Compounds 5c, 5d and 5h-5l showed very good antimicrobial activity against Staphylococcus aureus, Enterococcusfaecalis as well as Bacillus subtilis and 5h-5j showed moderate activity against Escherichia coli . In particular, (E)-N-dodecyl-4-azachalconium bromide (5i) and (E)-N-tetradecyl-4-azachalconium bromide (5j) showed the most intensive activity against all tested microorganisms.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13442 - 7 Epub 2002 Oct 01.
Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life?
Mijakovic I, Poncet S, Galinier A, Monedero V, Fieulaine S, Janin J, Nessler S, Marquez JA, Scheffzek K, Hasenbein S, Hengstenberg W, Deutscher J.
In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes ( approximately 10% of their genome) by acting as catabolite corepressor . HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate . We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate . HprK/P, which binds phosphate at the same site as the beta phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr . HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho-phosphorolysis mechanism . This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr . Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations ( approximately 6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation . To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM . In B . subtilis, this might be achieved by YvoE . This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.

J Agric Food Chem, 2002 Oct 9, 50(21), 6199 - 204
Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers; Yeh CM et al.; A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins . Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104 . The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers . The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates . Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility . The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C . Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.

FEBS Lett, 2002 Oct 2, 529(1), 6 - 10
AAA+ proteins and substrate recognition, it all depends on their partner in crime; Dougan DA et al.; Members of the AAA+ superfamily have been identified in all organisms studied to date . They are involved in a wide range of cellular events . In bacteria, representatives of this superfamily are involved in functions as diverse as transcription and protein degradation and play an important role in the protein quality control network . Often they employ a common mechanism to mediate an ATP-dependent unfolding/disassembly of protein-protein or DNA-protein complexes . In an increasing number of examples it appears that the activities of these AAA+ proteins may be modulated by a group of otherwise unrelated proteins, called adaptor proteins . These usually small proteins specifically modify the substrate recognition of their AAA+ partner protein . The occurrence of such adaptor proteins are widespread; representatives have been identified not only in Escherichia coli but also in Bacillus subtilis, not to mention yeast and other eukaryotic organisms . Interestingly, from the currently known examples, it appears that the N domain of AAA+ proteins (the most divergent region of the protein within the family) provides a common platform for the recognition of these diverse adaptor proteins . Finally, the use of adaptor proteins to modulate AAA+ activity is, in some cases, an elegant way to redirect the activity of an AAA+ protein towards a particular substrate without necessarily affecting other activities of that AAA+ protein while, in other cases, the adaptor protein triggers a complete switch in AAA+ activity.

Mol Microbiol, 2002 Sep, 45(6), 1613 - 29
Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon; Baichoo N et al.; The Bacillus subtilis ferric uptake repressor (Fur) protein coordinates a global transcriptional response to iron starvation . We have used DNA microarrays to define the Fur regulon and the iron starvation stimulon . We identify 20 operons (containing 39 genes) that are derepressed both by mutation of fur and by treatment of cells with the iron chelator 2,2'-dipyridyl . These operons are direct targets of Fur regulation as judged by DNase I footprinting . Analyses of lacZ reporter fusions to six Fur-regulated promoter regions reveal that repression is highly selective for iron . In addition to the Fur regulon, iron starvation induces members of the PerR regulon and leads to reduced expression of cytochromes . However, we did not find any evidence for genes that are directly activated by Fur or repressed by Fur under iron-limiting conditions . Although genome searches using the 19 bp Fur box consensus are useful in identifying candidate Fur-regulated genes, some genes associated with Fur boxes are not demonstrably regulated by Fur, whereas other genes are regulated from sites with little apparent similarity to the conventional Fur consensus.

J Org Chem, 2002 Oct 4, 67(20), 6871 - 7
Incorporation of an amide into 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors results in an unexpected reversal of selectivity for riboflavin synthase vs lumazine synthase; Cushman M et al.; Several analogues of a hypothetical intermediate in the reaction catalyzed by lumazine synthase were synthesized and tested as inhibitors of both Bacillus subtilis lumazine synthase and Escherichia coli riboflavin synthase . The new compounds were designed by replacement of a two-carbon fragment of several 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors with an amide linkage that was envisioned as an analogue of a Schiff base moiety of a hypothetical intermediate in the enzyme-catalyzed reaction . The incorporation of the amide group led to an unexpected reversal in selectivity for inhibition of lumazine synthase vs riboflavin synthase . Whereas the parent 5-phosphonoalkyl-6-D-ribitylaminopyrimidinediones were lumazine synthase inhibitors and did not inhibit riboflavin synthase, the amide-containing derivatives inhibited riboflavin synthase and were only very weak or inactive as lumazine synthase inhibitors . Molecular modeling of inhibitor-lumazine synthase complexes did not reveal a structural basis for these unexpected findings . However, molecular modeling of one of the inhibitors with E . coli riboflavin synthase demonstrated that the active site of the enzyme could readily accommodate two ligand molecules.

Syst Appl Microbiol, 2002 Aug, 25(2), 284 - 6
Comparative growth analysis of the facultative anaerobes Bacillus subtilis, Bacillus licheniformis, and Escherichia coli; Clements LD et al.; Bacillus subtilis anaerobic respiration and fermentative growth capabilities were compared to two other facultative anaerobes, Bacillus licheniformis and Escherichia coli . In glycerol defined medium, B . subtilis grew with nitrate, but not nitrite or fumarate, while B . licheniformis grew with nitrate or fumarate, but not nitrite . Growth of E . coli occurred in glycerol defined medium with either nitrate, nitrite, or fumarate . In order to grow by fermentation, B . subtilis required both glucose and pyruvate, while B . licheniformis and E . coli were capable of using either glucose or pyruvate.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 1), 1542 - 3 Epub 2002 Sep 26.
Bacillus subtilis division protein DivIVA - screen for stable oligomer state conditions; Muchova K et al.; The cell division protein DivIVA is predicted to be a coiled-coil, tropomyosin-like protein, that self-associates both in vivo and in vitro into oligomers of up to 10-12 monomers . A simple and quick screen for conditions supporting the stable oligomer structure has been developed revealing that DivIVA forms a homogeneous oligomer in the presence of PEGs (PEG 4 K or PEG 8K and PEG 1K).

J Biol Chem, 2002 Dec 13, 277(50), 48099 - 106 Epub 2002 Sep 24.
Membrane topology of the acyl-lipid desaturase from Bacillus subtilis; Diaz AR et al.; The Bacillus subtilis acyl-lipid desaturase (Delta5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids . Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase . The topology of Delta5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (Delta5-Des-PhoA) . The alkaline phosphatase activities of cells expressing the Delta5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane . These data, together with surface Lys biotinylation experiments, support a model for Delta5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif . This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase . On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.

J Bacteriol, 2002 Oct, 184(20), 5661 - 71
A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis; Darmon E et al.; Bacteria need dedicated systems that allow appropriate adaptation to the perpetual changes in their environments . In Bacillus subtilis, two HtrA-like proteases, HtrA and HtrB, play critical roles in the cellular response to secretion and heat stresses . Transcription of these genes is induced by the high-level production of a secreted protein or by a temperature upshift . The CssR-CssS two-component regulatory system plays an essential role in this transcriptional activation . Transcription of the cssRS operon is autoregulated and can be induced by secretion stress, by the absence of either HtrA or HtrB, and by heat stress in a HtrA null mutant strain . Two start sites are used for cssRS transcription, only one of which is responsive to heat and secretion stress . The divergently transcribed htrB and cssRS genes share a regulatory region through which their secretion and heat stress-induced expression is linked . This study shows that CssRS-regulated genes represent a novel class of heat-inducible genes, which is referred to as class V and currently includes two genes: htrA and htrB.

J Bacteriol, 2002 Oct, 184(20), 5609 - 18
Characterization of the depletion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis; Campbell TL et al.; The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis . In this work, the E . coli gene ispF and its Bacillus subtilis orthologue, yacN, were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms . In E . coli, complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B . subtilis, yacN was placed at amyE under control of the xylose-inducible xylA promoter . In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival . E . coli cells depleted of MEC synthase revealed a filamentous phenotype . This was in contrast to the depletion of MEC synthase in B . subtilis, which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls . To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics . Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics.

J Bacteriol, 2002 Oct, 184(20), 5583 - 9
Protein-protein interactions that regulate the energy stress activation of sigma(B) in Bacillus subtilis; Delumeau O et al.; Sigma(B) is an alternative sigma factor that controls the general stress response in Bacillus subtilis . In the absence of stress, sigma(B) is negatively regulated by anti-sigma factor RsbW . RsbW is also a protein kinase which can phosphorylate RsbV . When cells are stressed, RsbW binds to unphosphorylated RsbV, produced from the phosphorylated form of RsbV by two phosphatases (RsbU and RsbP) which are activated by stress . We now report the values of the K(m) for ATP and the K(i) for ADP of RsbW (0.9 and 0.19 mM, respectively), which reinforce the idea that the kinase activity of RsbW is directly regulated in vivo by the ratio of these nucleotides . RsbW, purified as a dimer, forms complexes with RsbV and sigma(B) with different stoichiometries, i.e., RsbW(2)-RsbV(2) and RsbW(2)-sigma(B)(1) . As determined by surface plasmon resonance, the dissociation constants of the RsbW-RsbV and RsbW-sigma(B) interactions were found to be similar (63 and 92 nM, respectively) . Nonetheless, an analysis of the complexes by nondenaturing polyacrylamide gel electrophoresis in competition assays suggested that the affinity of RsbW(2) for RsbV is much higher than that for sigma(B) . The intracellular concentrations of RsbV, RsbW (as a monomer), and sigma(B) measured before stress were similar (1.5, 2.6, and 0.9 micro M, respectively) . After ethanol stress they all increased . The increase was greatest for RsbV, whose concentration reached 13 micro M, while those of RsbW (as a monomer) and sigma(B) reached 11.8 and 4.9 micro M, respectively . We conclude that the higher affinity of RsbW for RsbV than for sigma(B), rather than a difference in the concentrations of RsbV and sigma(B), is the driving force that is responsible for the switch of RsbW to unphosphorylated RsbV.

J Bacteriol, 2002 Oct, 184(20), 5545 - 53
Mutation in yaaT leads to significant inhibition of phosphorelay during sporulation in Bacillus subtilis; Hosoya S et al.; In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect . Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage . Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation . However, neither the level of transcription of spo0A, the activity of sigma(H), which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT . Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases . We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant . These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation . Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

Biochemistry, 2002 Oct 1, 41(39), 11670 - 80
Role of entropy in protein thermostability: folding kinetics of a hyperthermophilic cold shock protein at high temperatures using 19F NMR; Schuler B et al.; We used (19)F NMR to extend the temperature range accessible to detailed kinetic and equilibrium studies of a hyperthermophilic protein . Employing an optimized incorporation strategy, the small cold shock protein from the bacterium Thermotoga maritima (TmCsp) was labeled with 5-fluorotryptophan . Although chaotropically induced unfolding transitions revealed a significant decrease in the stabilization free energy upon fluorine labeling, the protein's kinetic folding mechanism is conserved . Temperature- and guanidinium chloride-dependent equilibrium unfolding transitions monitored by (19)F NMR agree well with the results from optical spectroscopy, and provide a stringent test of the two-state folding character of TmCsp . Folding and unfolding rate constants at high temperatures were determined from the (19)F NMR spectra close to the midpoint of thermal unfolding by global line shape analysis . In combination with results from stopped-flow experiments at lower temperatures, they show that the folding rate constant of TmCsp and its temperature dependence closely resemble those of its mesophilic homologue from Bacillus subtilis, BsCspB . However, the unfolding rate constant of TmCsp is two orders of magnitude lower over the entire temperature range that was investigated . Consequently, the difference in conformational stability between the two proteins is solely due to the unfolding rate constant over a wide temperature range . A thermodynamic analysis points to an important role of entropic factors in the stabilization of TmCsp relative to its mesophilic homologues.

EPI Newsl, 1984 Dec, 6(6), 5 - 8
Using a pressure cooker as an autoclave; Copper-mediated dimerization of CopZ et al.; Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UKUnderstanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer . Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif . We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers . The nature of the copper(I)-CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein . The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)-CopZ occurs mainly as a monomer species . Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein . We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer.

Shokuhin Eiseigaku Zasshi, 2002 Jun, 43(3), 155 - 9
New estimation methods of bacterial concentration by measuring ATP changes during incubation; Fujikawa H et al.; New estimation methods of bacterial cell concentration in samples by measurement of the increase in bacterial adenosine-triphosphate (ATP) content during incubation using a conventional firefly luminometer were established . When an Escherichia coli cell suspension was incubated in nutrient broth, the increase in the ATP content of the suspension during the incubation period followed a sigmoidal curve . The increase ratio of the ATP content of the suspension at a given period of incubation (5 hours in this study) to the initial ATP content was greater at higher initial cell concentrations . With this relationship, the initial cell concentration of a test suspension could be predicted from the measured ratio; this was called the end point method . On the other hand, the lag period in the ATP increase curve was longer at lower initial cell concentrations . A highly linear relationship was observed between the lag period and the logarithm of the initial cell concentrations . Based on this relationship, a delay method was developed for prediction . The two relationships were also observed for bacterial suspensions of Klebsiella sp., Staphylococcus aureus, Bacillus subtilis, and Pseudomonas sp . These results suggested that the two methods have the potential to estimate the bacterial cell concentration of a sample suspension.

Yeast, 2002 Sep 30, 19(13), 1153 - 63
A cell surface display system using novel GPI-anchored proteins in Hansenula polymorpha; Kim SY et al.; A cell surface display system was developed in yeast Hansenula polymorpha.The four genes HpSED1, HpGAS1, HpTIP1and HpCWP1, encoding glycosylphosphatidyl-inositol (GPI)-anchored cell surface proteins from H . polymorpha, were cloned, characterized and evaluated for their efficacies as cell surface display motifs of reporter proteins . Sequence analysis of these genes revealed that each encodes a typical GPI-anchored protein that is structurally similar to a counterpart gene in S . cerevisiae . The genes showed a high content of serine-threonine (alanine) and harboured a putative secretion signal in the N-terminus and the GPI-attachment signal in the C-terminus . The surface anchoring efficiency of these putative cell surface proteins was tested by fusion to the C-terminal of carboxymethylcellulase (CMCase) from Bacillus subtilis . In all cases, high CMCase activities were detected in intact cell fraction, indicating anchoring of CMCase to the cell surface . HpCwp1p, HpGas1p and the 40 C-terminal amino acids of HpTip1p from H . polymorpha exhibited a comparatively high CMCase surface anchoring efficiency . When these proteins were used as anchoring motifs for surface display of the glucose oxidase (GOD) from Aspergillus niger, most enzyme activity was detected at the cell surface . Fluorescence activated cell sorter (FACS) analysis of cells displaying GOD on the cell surface demonstrated that GOD was well exposed on the cell surface . HpCwp1p showed the highest anchoring efficiency among others .

Yakugaku Zasshi, 2002 Sep, 122(9), 651 - 71
{New antifungal antibiotics, bacillopeptins and fusaricidins}; Kaneda M et al.; We isolated four strains of bacteria producing antifungal antibiotics from the rhizosphere of garlic with basal rot caused by the plant pathogenic fungal strain Fusarium oxysporum . Among them, Bacillus subtilis FR-2 was found to produce new antifungal antibiotics, named bacillopeptins A, B, and C . Their structures have been determined by 1D and 2D NMR and MS experiments, and amino acid analysis coupled with chiral HPLC, to be cyclic lipopeptides each containing a long-chain beta-amino acid . Another bacterial strain, Bacillus polymyxa KT-8, was shown to produce new antifungal antibiotics named fusaricidins A, B, C, and D which are more potent than bacillopeptins in their antimicrobial activity . The structures of the fusaricidins have been elucidated similarly as bacillopeptins to be cyclic hexadepsipeptides all containing 15-guanidino-3-hydroxypentadecanoic acid as a side chain . Fusaricidins strongly inhibit the growth of various kinds of fungi and moreover surprisingly show strong inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus or Micrococcus luteus.

J Biol Chem, 2002 Nov 8, 277(45), 43454 - 62 Epub 2002 Aug 30.
Crystal structure of porcine mitochondrial NADP+-dependent isocitrate dehydrogenase complexed with Mn2+ and isocitrate . Insights into the enzyme mechanism; Ceccarelli C et al.; The crystal structure of porcine heart mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH) complexed with Mn2+ and isocitrate was solved to a resolution of 1.85 A . The enzyme was expressed in Escherichia coli, purified as a fusion protein with maltose binding protein, and cleaved with thrombin to yield homogeneous enzyme . The structure was determined by multiwavelength anomalous diffraction phasing using selenium substitution in the form of selenomethionine as the anomalous scatterer . The porcine NADP+-IDH enzyme is structurally compared with the previously solved structures of IDH from E . coli and Bacillus subtilis that share 16 and 17% identity, respectively, with the mammalian enzyme . The porcine enzyme has a protein fold similar to the bacterial IDH structures with each monomer folding into two domains . However, considerable differences exist between the bacterial and mammalian forms of IDH in regions connecting core secondary structure . Based on the alignment of sequence and structure among the porcine, E . coli, and B . subtilis IDH, a putative phosphorylation site has been identified for the mammalian enzyme . The active site, including the bound Mn2+-isocitrate complex, is highly ordered and, therefore, mechanistically informative . The consensus IDH mechanism predicts that the Mn2+-bound hydroxyl of isocitrate is deprotonated prior to its NADP+-dependent oxidation . The present crystal structure has an active site water that is well positioned to accept the proton and ultimately transfer the proton to solvent through an additional bound water.

Biochemistry, 2002 Sep 24, 41(38), 11325 - 30
Protection of DNA by alpha/beta-type small, acid-soluble proteins from Bacillus subtilis spores against cytosine deamination; Sohail A et al.; Spores of Bacillus subtilis contain high levels of proteins, termed alpha/beta-type small, acid-soluble proteins (SASP), that protect the spore's DNA against different types of DNA damage . We tested one such protein, SspC, and two of its variants for their ability to protect plasmid DNA against hydrolytic deamination of cytosine to uracil . If unrepaired, such damage to DNA causes C to T mutations . We found that one SspC variant, SspC(Delta 11-D13K), protected DNA against cytosine deamination at two different temperatures (45 and 70 degrees C) and pH values (5.2 and 7.9), reducing the rate of deamination by as much as 10-fold . At 70 degrees C, pH 7.9, the wild-type SspC and its variant, SspC(Delta 11), provided little protection against deamination but were effective in protecting DNA at 45 degrees C, pH 7.9 . Parallel studies of the abilities of these proteins to protect DNA against restriction digestion revealed that there was a good correlation between the abilities of the proteins to protect against restriction endonucleases and reductions in cytosine deaminations . These results show that the binding of SspC variants to DNA can prevent attack on DNA bases by water and suggest a new general mechanism by which DNA-binding proteins in cells may be able to protect chromosomes from endogenous and exogenous reactive chemicals by excluding them from the vicinity of DNA.

J Org Chem, 2002 Sep 20, 67(19), 6725 - 30
Investigation of the enzymatic and nonenzymatic cope rearrangement of carbaprephenate to carbachorismate; Aemissegger A et al.; The dimethyl esters of carbaprephenate and 4-epi-carbaprephenate were prepared by modification of published procedures . In methanol these compounds are converted quantitatively to isomeric 6-hydroxytricyclo{3.3.1.0(2,7)}non-3-en-1,3-dimethyl esters via a two-step sequence involving an initial Cope rearrangement, followed by intramolecular Diels-Alder reaction of the dimethyl carbachorismate or 4-epi-carbachorismate intermediates . Carbaprephenate and its epimer were obtained by alkaline hydrolysis of the corresponding dimethyl esters . These compounds, in contrast to their ester precursors, undergo spontaneous acid-catalyzed decarboxylation in aqueous solution . Only at high pH does the Cope rearrangement compete with decarboxylation . At pH 12 and 90 degrees C, carbaprephenate slowly rearranges to carbachorismate, which rapidly loses water to give 3-(2-carboxyallyl)benzoic acid as the major product . A small amount of the intramolecular Diels-Alder adduct derived from carbachorismate is also observed by NMR as a minor product . Carbaprephenate is not a substrate for the enzyme chorismate mutase from Bacillus subtilis (BsCM), nor does carbaprephenate inhibit the normal chorismate mutase activity of this enzyme, even when present in 200-fold excess over chorismate . Its low affinity for the enzyme-active site is presumably a consequence of placing a methylene group rather than an oxygen atom proximal to the essential cationic residue Arg90 . Nevertheless, BsCM variants that lack this cation (R90G and R90A) do not accelerate the Cope rearrangement of carbaprephenate either, and a catalytic antibody 1F7, which exhibits modest chorismate mutase activity, is similarly inactive . Poor substrate binding and the relatively high barrier for the Cope compared to the Claisen rearrangement presumably account for the lack of detectable catalysis . Acceleration of this sigmatropic rearrangement apparently requires more than an active site that is complementary in shape to the reactive substrate conformer.

Biochim Biophys Acta, 2002 Sep 20, 1565(1), 1 - 5
Highly efficient over-production in E . coli of YvcC, a multidrug-like ATP-binding cassette transporter from Bacillus subtilis; Steinfels E et al.; ATP-binding cassette (ABC) transporters have often been refractory to over-expression . Using the C41(DE3) E . coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to P-glycoprotein multidrug transporters, were obtained . The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342.

Biosci Biotechnol Biochem, 2002 Jul, 66(7), 1583 - 6
DnaK chaperone machine and trigger factor are only partially required for normal growth of Bacillus subtilis; Reyes DY et al.; While dnaK and tig are the essential components for nascent polypeptide folding in E . coli, deletion did not confer synthetic lethality in B . subtilis, suggesting that under normal growth conditions, another system or mechanism with a specific role prevails . Likewise, survival at high temperature suffered dramatically, resulting from deletion of several sets of heat shock genes, thus during sudden stress various heat shock genes act synergistically to protect the proteins.

Biosci Biotechnol Biochem, 2002 Jul, 66(7), 1555 - 8
Production of long-chain levan by a sacC insertional mutant from Bacillus subtilis 327UH; Shida T et al.; A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20% sucrose, and the yield of levan after 10 hours was more than 60%, when based on the fructose amount of sucrose . After transformation of 327UH with a levanase-deficient 168SC (sacC::Cm(r)) chromosomal DNA, a Cm(r) transformant 327UHSC (sacC::Cm(r) degSU(Hy)) produced 3 times longer levan than that of the wild type.

J Microbiol Methods, 2002 Nov, 51(3), 379 - 85
Two-vector assay as a tool for examining Spo0A gene transcription regulation; Blaskovic D et al.; We have modified an assay using two compatible vectors that coexist in Escherichia coli cells and applied it in the investigation of the transcriptional activity of Spo0A, a key regulator of sporulation in Bacillus subtilis . We have chosen the promoters of the Spo0A dependent genes, spoIIE and spoIIA, involved in sporulation, in order to study the transcription activity solely of the DNA binding domain of Spo0A . We have prepared the two-vector system so that one vector contained the cloned C-Spo0A under the control of an inducible promoter, and the second vector (the promoter probe vector), was composed of the Spo0A dependent spoIIE and spoIIA promoters . Using this two-vector system in E . coli, we proved that C-Spo0A is able to interact with the E . coli transcription apparatus, recognizes both promoters and activates transcription from these promoters .

Cell Mol Life Sci, 2002 Jul, 59(7), 1223 - 32
The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin; Sauvage E et al.; Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics . Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams . We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A . A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity . The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site . In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam . This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis . Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.

Environ Microbiol, 2002 Sep, 4(9), 525 - 31
Is bioconvection enhancing bacterial growth in quiescent environments?
Janosi IM, Czirok A, Silhavy D, Holczinger A.
Bioconvection is an intriguing pattern-forming phenomenon driven by the swimming activity of various aquatic microorganisms . It is generally assumed that bioconvection has a positive effect on the entire microbial population by carrying oxygen into deep layers of non-aerated suspensions . In order to examine the presence of such a biological benefit, we analysed the correlation between bioconvective pattern formation and population growth of several Bacillus subtilis and Bacillus licheniformis strains under non-aerated conditions . Bioconvection is a robust phenomenon, we observed its development in numerous cultures of various strains and growth phases . Nevertheless, evaluation of the data has not revealed detectable positive effects on population growth, questioning the potential biological relevance of bioconvection in natural habitats.

Biochemistry, 2002 Sep 17, 41(37), 11109 - 17
YjeQ, an essential, conserved, uncharacterized protein from Escherichia coli, is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics; Daigle DM et al.; The Escherichia coli protein YjeQ represents a protein family whose members are broadly conserved in bacteria and have been shown to be indispensable to the growth of E . coli and Bacillus subtilis {Arigoni, F., et al . (1998) Nat . Biotechnol . 16, 851} . Proteins of the YjeQ family contain all sequence motifs typical of the vast class of P-loop-containing GTPases, but show a circular permutation, with a G4-G1-G3 pattern of motifs as opposed to the regular G1-G3-G4 pattern seen in most GTPases . All YjeQ family proteins display a unique domain architecture, which includes a predicted N-terminal OB-fold RNA-binding domain, the central permuted GTPase module, and a zinc knuckle-like C-terminal cysteine cluster . This domain architecture suggests a possible role for YjeQ as a regulator of translation . YjeQ was overexpressed, purified to homogeneity, and shown to contain 0.6 equiv of GDP . Steady state kinetic analyses indicated slow GTP hydrolysis, with a k(cat) of 9.4 h(-)(1) and a K(m) for GTP of 120 microM (k(cat)/K(m) = 21.7 M(-)(1) s(-)(1)) . YjeQ also hydrolyzed other nucleoside triphosphates and deoxynucleotide triphosphates such as ATP, ITP, and CTP with specificity constants (k(cat)/K(m)) ranging from 0.2 to 1.0 M(-)(1) s(-)(1) . Pre-steady state kinetic analysis of YjeQ revealed a burst of nucleotide hydrolysis for GTP described by a first-order rate constant of 100 s(-)(1) as compared to a burst rate of 0.2 s(-)(1) for ATP . In addition, a variant in the G1 motif of YjeQ (S221A) was substantially impaired for GTP hydrolysis (0.3 s(-)(1)) with a less significant impact on the steady state rate (1.8 h(-)(1)) . In summary, E . coli YjeQ is an unusual, circularly permuted P-loop-containing GTPase, which catalyzes GTP hydrolysis at a rate 45 000 times greater than that of turnover.

Biochemistry, 2002 Sep 10, 41(36), 10906 - 13
Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions; Brown KR et al.; Heme A, an obligatory cofactor in eukaryotic cytochrome c oxidase, is produced from heme B (protoheme) via two enzymatic reactions catalyzed by heme O synthase and heme A synthase . Heme O synthase is responsible for the addition of a farnesyl moiety, while heme A synthase catalyzes the oxidation of a methyl substituent to an aldehyde . We have cloned the heme O synthase and heme A synthase genes from Bacillus subtilis (ctaB and ctaA) and overexpressed them in Escherichia coli to probe the oxidative mechanism of heme A synthase . Because E . coli does not naturally produce or utilize heme A, this strategy effectively decoupled heme A biosynthesis from the native electron transfer pathway and heme A transport, allowing us to observe two previously unidentified hemes . We utilized HPLC, UV/visible spectroscopy, and tandem mass spectrometry to identify these novel hemes as derivatives of heme O containing an alcohol or a carboxylate moiety at position C8 on pyrrole ring D . We interpret these derivatives to be the putative alcohol intermediate and an overoxidized byproduct of heme A synthase . Because we have shown that all hemes produced by heme A synthase require O(2) for their synthesis, we propose that heme A synthase catalyzes the oxidation of the C8 methyl to an aldehyde group via two discrete monooxygenase reactions.

Life Sci Space Res, 1971, 9, 119 - 24
Survival of bacterial spores under some simulated lunar surface conditions; Horneck G et al.; Spores of Bacillus subtilis were exposed to simulated lunar environmental factors, in order to estimate the chance of living matter to survive on the moon . Vacuum, radiation and extreme temperature were selected and their individual and combined influence was tested . High vacuum up to 2 x 10(-7) torr and ultra-high vacuum up to 5 x 10(-9) torr, ultraviolet rays (254 nm) and a temperature of 80 degrees C were used . The results were compared with those of experiments on vegetative cells.

J Bacteriol, 2002 Oct, 184(19), 5393 - 401
Forespore signaling is necessary for pro-sigmaK processing during Bacillus subtilis sporulation despite the loss of SpoIVFA upon translational arrest; Kroos L et al.; The sigmaK checkpoint coordinates gene expression in the mother cell with signaling from the forespore during Bacillus subtilis sporulation . The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore . Activation of the complex allows proteolytic processing of pro-sigmaK, and the resulting sigmaK RNA polymerase transcribes genes in the mother cell . To investigate activation of the pro-sigmaK processing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis . The SpoIVFA level decreased when pro-sigmaK processing began during sporulation . In extracts of a spoIVB mutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-sigmaK was observed . Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation . Under these conditions, loss of SpoIVFA was independent of spoIVB . The inability to process pro-sigmaK under these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-sigmaK . Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling . This suggests that ongoing translation is normally required for efficient pro-sigmaK processing because synthesis of the SpoIVB signal protein is needed to activate the processing complex . When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA . Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked . The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes.

J Bacteriol, 2002 Oct, 184(19), 5339 - 47
Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization; Azarkina N et al.; At a pH of <or=7, respiration of Bacillus subtilis cells on endogenous substrates shut down almost completely upon addition of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone {CCCP}) and a K+-ionophore (valinomycin) . The same effect was observed with cell spheroplasts lacking the cell wall . The concentration of CCCP required for 50% inhibition of the endogenous respiration in the presence of K+-valinomycin was below 100 nM . Either CCCP or valinomycin alone was much less efficient than the combination of the two . The inhibitory effect was easily reversible and depended specifically on the H+ and K+ concentrations in the medium . Similar inhibition was observed with respect to the reduction of the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine cation (TMPD+), which intercept reducing equivalents at the level of menaquinol . Oxidation of the reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling . The same loss of the electron transfer activities as induced by the uncoupling was observed upon disruption of the cells during isolation of the membranes; the residual activities were not further inhibited by the uncoupler and ionophores . We conclude that the menaquinone-dependent electron transfer in the B . subtilis respiratory chain is facilitated, thermodynamically or kinetically, by membrane energization . A requirement for an energized state of the membrane is not a specific feature of succinate oxidation, as proposed in the literature, since it was also observed in a mutant of B . subtilis lacking succinate:quinone reductase as well as for substrates other than succinate . Possible mechanisms of the energy-dependent regulation of menaquinone-dependent respiration in B . subtilis are discussed.

J Bacteriol, 2002 Oct, 184(19), 5317 - 22
Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo; Lindow JC et al.; Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms . Members of this protein family are involved in chromosome condensation and sister chromatid cohesion . Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning . To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo . We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin . Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant . Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells . In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid . Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.

J Bacteriol, 2002 Oct, 184(19), 5275 - 81
Cold shock response in sporulating Bacillus subtilis and its effect on spore heat resistance; Movahedi S et al.; Cold shock and ethanol and puromycin stress responses in sporulating Bacillus subtilis cells have been investigated . We show that a total of 13 proteins are strongly induced after a short cold shock treatment of sporulating cells . The cold shock pretreatment affected the heat resistance of the spores formed subsequently, with spores heat killed at 85 or 90 degrees C being more heat resistant than the control spores while they were more heat sensitive than controls that were heat treated at 95 or 100 degrees C . However, B . subtilis spores with mutations in the main cold shock proteins, CspB, -C, and -D, did not display decreased heat resistance compared to controls, indicating that these proteins are not directly responsible for the increased heat resistance of the spores . The disappearance of the stress proteins later in sporulation suggests that they cannot be involved in repairing heat damage during spore germination and outgrowth but must alter spore structure in a way which increases or decreases heat resistance . Since heat, ethanol, and puromycin stress produce similar proteins and similar changes in spore heat resistance while cold shock is different in both respects, these alterations appear to be very specific.

J Struct Biol, 2002 Jun, 138(3), 237 - 40
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 TS 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 . HutP and its mutant, 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 dimensions a=b=c=95.6A for HutP and a=b=c=96.8A for the mutant . Complete data sets of 3.0-A resolution for wild-type HutP and of 2.70-A resolution for the mutant HutP were collected.

Trends Microbiol, 2002 Sep, 10(9), 393 - 5
Is there a role for replication fork asymmetry in the distribution of genes in bacterial genomes?
Rocha E.
Replication generates bacterial chromosomes with strands that differ in the number of genes and base composition . It has been suggested that in bacteria such as Bacillus subtilis, PolC is responsible for the synthesis of the leading strand and DnaE for the lagging strand, whereas in many other bacteria DnaE is responsible for the synthesis of both strands . Here, I show that the possession of PolC correlates with leading strands that contain an average of 78% of genes compared with 58% for genomes that do not contain PolC . This suggests that asymmetrical replication forks could have a major role in defining and constraining the structure of the bacterial chromosome . The presence of PolC is not correlated with compositional strand bias, suggesting that the two biases result from different types of structural asymmetry.

Biochim Biophys Acta, 2002 Sep 13, 1577(2), 240 - 50
Transcription attenuation; Gollnick P et al.; In this review, we describe a variety of mechanisms that bacteria use to regulate transcription elongation in order to control gene expression in response to changes in their environment . Together, these mechanisms are known as attenuation and antitermination, and both involve controlling the formation of a transcription terminator structure in the RNA transcript prior to a structural gene or operon . We examine attenuation and antitermination from the point of view of the different biomolecules that are used to influence the RNA structure . Attenuation of many amino acid biosynthetic operons, particularly in enteric bacteria, is controlled by ribosomes translating leader peptides . RNA-binding proteins regulate attenuation, particularly in gram-positive bacteria such as Bacillus subtilis . Transfer RNA is also used to bind to leader RNAs and influence transcription antitermination in a large number of amino acyl tRNA synthetase genes and several biosynthetic genes in gram-positive bacteria . Finally, antisense RNA is involved in mediating transcription attenuation to control copy number of several plasmids.

Biotechnol Bioeng, 2002 Sep 5, 79(5), 532 - 8
Analysis of fermentation processes using flow microfluorometry: Single-parameter observations of batch bacterial growth; Fazel-Madjlessi J et al.; The laser flow microfluorometer (FMF) can determine the amounts of certain components in single cells at sample rates of several thousand cells per second . This technique has been employed to characterize Bacillus subtilis populations in batch fermentations with different inocula . Protein and nucleic acid distributions obtained by FMF analyses at different times during the batch have been decomposed using an optimized fit of summed subpopulation distributions . The results of these decomposition calculations, some of which have been approximately confirmed by independent microscopic observations, indicate that the relative numbers of single rods, cell chains, spores, and swollen rounded cells change dramatically during the entire fermentation including the stationary phase . The dynamics of these subpopulations may be related to secondary metabolite production .

J Photochem Photobiol B, 2002 Aug, 68(1), 23 - 32
Influence of ice and snow covers on the UV exposure of terrestrial microbial communities: dosimetric studies; Cockell CS et al.; Bacillus subtilis spore biological dosimeters and electronic dosimeters were used to investigate the exposure of terrestrial microbial communities in micro-habitats covered by snow and ice in Antarctica . The melting of snow covers of between 5- and 15-cm thickness, depending on age and heterogeneity, could increase B . subtilis spore inactivation by up to an order of magnitude, a relative increase twice that caused by a 50% ozone depletion . Within the snow-pack at depths of less than approximately 3 cm snow algae could receive two to three times the DNA-weighted irradiance they would receive on bare ground . At the edge of the snow-pack, warming of low albedo soils resulted in the formation of overhangs that provided transient UV protection to thawed and growing microbial communities on the soils underneath . In shallow aquatic habitats, thin layers of heterogeneous ice of a few millimetres thickness were found to reduce DNA-weighted irradiances by up to 55% compared to full-sky values with equivalent DNA-weighted diffuse attenuation coefficients (K(DNA)) of >200 m(-1) . A 2-mm snow-encrusted ice cover on a pond was equivalent to 10 cm of ice on a perennially ice covered lake . Ice covers also had the effect of stabilizing the UV exposure, which was often subject to rapid variations of up to 33% of the mean value caused by wind-rippling of the water surface . These data show that changing ice and snow covers cause relative changes in microbial UV exposure at least as great as those caused by changing ozone column abundance .

Mol Microbiol, 2002 Sep, 45(5), 1379 - 88
Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis acyl-lipid desaturase; Cybulski LE et al.; The Des pathway of Bacillus subtilis regulates the expression of the acyl-lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids (UFAs) from saturated phospholipid precursors . Previously, we showed that the master switch for the Des pathway is a two-component regulatory system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene . Activation of this pathway takes place when cells are shifted to low growth temperature . Here, we report on the mechanism by which isoleucine regulates the Des pathway . We found that exogenous isoleucine sources, as well as its alpha-keto acid derivative, which is a branched-chain fatty acid precursor, negatively regulate the expression of the des gene at 37 degrees C . The DesK-DesR two-component system mediates this response, as both partners are required to sense and transduce the isoleucine signal at 37 degrees C . Fatty acid profiles strongly indicate that isoleucine affects the signalling state of the DesK sensor protein by dramatically increasing the incorporation of the lower-melting-point anteiso-branched-chain fatty acids into membrane phospholipids . We propose that both a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism . Thus, the Des pathway would provide a novel mechanism to optimize membrane lipid fluidity at a constant temperature.

Mol Microbiol, 2002 Sep, 45(5), 1267 - 76
Antibiotics that inhibit cell wall biosynthesis induce expression of the Bacillus subtilis sigma(W) and sigma(M) regulons; Cao M et al.; Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors . The sigma(W) regulon includes functions involved in detoxification and protection against antimicrobials, whereas sigma(M) is essential for growth at high salt concentrations . We now report that antibiotics that inhibit cell wall biosynthesis induce both sigma(W) and sigma(M) regulons as monitored using DNA microarrays . Induction of selected sigma(W)-dependent genes was confirmed using lacZ reporter fusions and Northern blot analysis . The ability of vancomycin to induce the sigma(W) regulon is dependent on both sigma(W) and the cognate anti-sigma, RsiW, but is independent of the transition state regulator AbrB . These results suggest that the membrane-localized RsiW anti-sigma(W) factor mediates the transcriptional response to cell wall stress . Our findings are consistent with the idea that one function of ECF sigma factors is to coordinate antibiosis stress responses and cell envelope homeostasis.

J Antimicrob Chemother, 2002 Sep, 50(3), 313 - 21
Genetic characterization of the fusidic acid and cadmium resistance determinants of Staphylococcus aureus plasmid pUB101; O'Brien FG et al.; We report the cloning of the fusidic acid and cadmium resistance determinants from Staphylococcus aureus plasmid pUB101 . The pUB101 fusidic acid resistance determinant was located on a 2.9 kb HindIII fragment . Sequencing of this fragment revealed three putative open reading frames (ORFs) of 213 (far1), 152 (orf152) and 170 amino acids (orf170), which are flanked by the right-hand end of insertion sequence IS431/257 (IS431/257RH) and a partial ORF . Far1 and Orf152 demonstrated homology with a chromosomally encoded fibronectin-binding protein of Listeria monocytogenes and the putative protein YosT, found on the SP beta c2 prophage of Bacillus subtilis, respectively . Transformation of S . aureus with a construct containing a 949 bp far1-specific amplicon led to the isolation of a fusidic acid-resistant transformant, thereby identifying the pUB101 fusidic acid resistance structural gene . Between orf152 and far1 we identified a unique 113 bp symmetrical element and other repeat elements that may be involved with the control of orf152 and/or far1 expression . Hybridization of Southern blots revealed that far1 was not located on the chromosome or plasmid content of a limited number of Australian, UK and Hong Kong fusidic acid-resistant isolates . The pUB101 cadmium resistance determinant was located on a 3.6 kb HindIII fragment that carried a cadDX operon, remnants of two putative plasmid replication protein genes and IS431/257RH . Sequence analysis also demonstrated the presence of a single-stranded origin of replication, normally found on rolling circle replicating plasmids, within the putative promoter region of the cadDX operon.

Acta Crystallogr D Biol Crystallogr, 2002 Sep, 58(Pt 9), 1490 - 3 Epub 2002 Aug 23.
Spore-coat laccase CotA from Bacillus subtilis: crystallization and preliminary X-ray characterization by the MAD method; Enguita FJ et al.; Bacterial endospores are highly resistant structures that allow survival for long periods of time in adverse environments . The spore-forming Gram-positive bacterium Bacillus subtilis synthesizes a coat around the endospore during development composed of several assembled polypeptides . The role of these components of the spore coat remains unclear; however, some of them appear to be enzymes possibly involved in the assembly process or in the final properties of the spore . The outer spore-coat protein CotA is a 65 kDa polypeptide showing a high degree of sequence similarity with copper-dependent oxidases, including fungal and plant laccases, ascorbate oxidase and CueO from Esherichia coli . CotA has been recently characterized as a copper-dependent laccase . Unlike previously reported laccases, CotA shows increased thermostability . Here, the crystallization of a recombinant CotA protein produced in E . coli and the preliminary characterization of the crystals is reported . Structure solution by the MAD method at the copper K edge is also reported.

Biochem Soc Trans, 2002 Aug, 30(4), 590 - 5
Terminal steps of haem biosynthesis; Dailey HA; The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem . Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (coproporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms . The terminal step is catalysed by the enzyme ferrochelatase . This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a {2Fe-2S} cluster . Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics . Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.

J Bacteriol, 2002 Sep, 184(18), 5179 - 86
Global expression profile of Bacillus subtilis grown in the presence of sulfate or methionine; Auger S et al.; DNA arrays were used to investigate the global transcriptional profile of Bacillus subtilis grown in the presence of sulfate or methionine as the sole sulfur source . The expression of at least 56 genes differed significantly under the two growth conditions . The expression of several genes belonging to the S-box regulon was repressed in the presence of methionine probably in response to S-adenosylmethionine availability . The expression of genes encoding transporters (yhcL, ytmJKLMN, and yxeMO) was high when the sulfur source was methionine or taurine and reduced when it was sulfate.

J Bacteriol, 2002 Sep, 184(18), 5174 - 8
Insufficient expression of the ilv-leu operon encoding enzymes of branched-chain amino acid biosynthesis limits growth of a Bacillus subtilis ccpA mutant; Ludwig H et al.; Bacillus subtilis ccpA mutant strains exhibit two distinct phenotypes: they are defective in catabolite repression, and their growth on minimal media is strongly impaired . This growth defect is largely due to a lack of expression of the gltAB operon . However, growth is impaired even in the presence of glutamate . Here, we demonstrate that the ccpA mutant strain needs methionine and the branched-chain amino acids for optimal growth . The control of expression of the ilv-leu operon by CcpA provides a novel regulatory link between carbon and amino acid metabolism.

Protein Expr Purif, 2002 Aug, 25(3), 409 - 15
Design, production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins; Qureshi MH et al.; To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host . By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion . It remained in the monomeric state in the presence or absence of biotin . Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor . Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower . The dissociation constant was determined to be 1.3 x 10(-8)M . These properties make it an attractive agent for affinity purification of biotinylated proteins . An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract . Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity . Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.

Mol Microbiol, 2002 Aug, 45(4), 1119 - 30
The cell differentiation protein SpoIIE contains a regulatory site that controls its phosphatase activity in response to asymmetric septation; Feucht A et al.; Starvation induces Bacillus subtilis to initiate a -simple, two-cell developmental process that begins with an asymmetric cell division . Activation of the first compartment-specific transcription factor, sigmaF, is coupled to this morphological event . SpoIIE, a bifunctional protein, is essential for the compartment-specific activation of sigmaF and also has a morphogenic activity required for asymmetric cell division . SpoIIE consists of three domains: a hydrophobic N-terminal domain, which targets the protein to the membrane; a central domain, involved in oligomerization of SpoIIE and interaction with the cell division protein FtsZ; and a C-terminal domain comprising a PP2C protein phosphatase . Here, we report the isolation of mutations at the very beginning of the central domain of spoIIE, which are capable of activating sigmaF inde-pendently of septum formation . Purified mutant proteins showed the same phosphatase activity as the wild-type protein in vitro . The mutant proteins were fully functional in respect of their localization to sites of asymmetric septation and support of asymmetric division . The data provide strong evidence that the phosphatase domain of SpoIIE is tightly regulated in a way that makes it respond to the formation of the asymmetric septum.

Mol Microbiol, 2002 Aug, 45(4), 997 - 1005
A peroxide-induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis; Gaballa A et al.; In Bacillus subtilis, hydrogen peroxide (H2O2) induces expression of the PerR regulon including catalase (KatA), alkyl hydroperoxide reductase and the DNA-binding protein MrgA . We have identified the P-type metal-transporting ATPase ZosA (formerly YkvW) as an additional member of the perR regulon . Expression of zosA is induced by H2O2 and repressed by the PerR metalloregulatory protein, which binds to two Per boxes in the promoter region . Physiological studies implicate ZosA in Zn(II) uptake . ZosA functions together with two Zur-regulated uptake systems and one known efflux system to maintain Zn(II) homeostasis . ZosA is the major pathway for zinc uptake in cells growing with micromolar levels of Zn(II) that are known to repress the two Zur-regulated transporters . A perR mutant is sensitive to high levels of zinc, and this sensitivity is partially suppressed by a zosA mutation . ZosA is important for resistance to both H2O2 and the thiol-oxidizing agent diamide . This suggests that increased intracellular Zn(II) may protect thiols from oxidation . In contrast, catalase is critical for H2O2 resistance but does not contribute significantly to diamide resistance . Growth of cells with elevated zinc significantly increases resistance to high concentrations of H2O2, and this effect requires ZosA . Our results indicate that peroxide stress leads to the upregulation of a dedicated Zn(II) uptake system that plays an important role in H2O2 and disulphide stress resistance.

Microbiology, 2002 Aug, 148(Pt 8), 2591 - 8
Identification and characterization of novel small RNAs in the aspS-yrvM intergenic region of the Bacillus subtilis genome; Suzuma S et al.; A novel RNA species was isolated from Bacillus subtilis, and its sequence was determined and mapped to its genetic position . This RNA was termed BS190 RNA from the length of its mature form (190 nt), and the gene encoding it is located within the aspS-yrvM intergenic region of the B . subtilis genome . Northern blotting revealed that the novel RNA species is transcribed in vegetative cells as a larger precursor (BS201 RNA, 201 nt) . After transcription, the 5' end of the precursor is processed to generate the mature form, BS190 RNA . A computer-aided prediction of the secondary structure of BS190 RNA showed that it can be folded into a single hairpin structure with some bulge structures . The authors found that the growth rate of a DeltaBS190 mutant strain of B . subtilis was reduced when compared to the wild-type . A phylogenetic comparison of the sequence of the BS190 RNA gene with sequences from the databases suggests that RNA related to BS190 RNA appears to be encoded in the genomes of Bacillus halodurans and Listeria monocytogenes.

Microbiology, 2002 Aug, 148(Pt 8), 2383 - 92
Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination; Chirakkal H et al.; The location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested . The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B . subtilis spore germination . Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument . The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction . Neither protein changes location or size as the spore germinates . The ypeB gene is the second gene in a bicistronic operon with sleB . The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization . In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB - in both the inner membrane and the integument . As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme's hydrolytic specificity could not be defined . The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD(600) fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores . The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore's ability to complete germination . Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B . subtilis . Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally . The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination: the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

Philos Trans R Soc Lond B Biol Sci, 2002 Jul 29, 357(1423), 895 - 907
Coping with the cold: the cold shock response in the Gram-positive soil bacterium Bacillus subtilis; Weber MH et al.; All organisms examined to date, respond to a sudden change in environmental temperature with a specific cascade of adaptation reactions that, in some cases, have been identified and monitored at the molecular level . According to the type of temperature change, this response has been termed heat shock response (HSR) or cold shock response (CSR) . During the HSR, a specialized sigma factor has been shown to play a central regulatory role in controlling expression of genes predominantly required to cope with heat-induced alteration of protein conformation . In contrast, after cold shock, nucleic acid structure and proteins interacting with the biological information molecules DNA and RNA appear to play a major cellular role . Currently, no cold-specific sigma factor has been identified . Therefore, unlike the HSR, the CSR appears to be organized as a complex stimulon rather than resembling a regulon . This review has been designed to draw a refined picture of our current understanding of the CSR in Bacillus subtilis . Important processes such as temperature sensing, membrane adaptation, modification of the translation apparatus, as well as nucleoid reorganization and some metabolic aspects, are discussed in brief . Special emphasis is placed on recent findings concerning the nucleic acid binding cold shock proteins, which play a fundamental role, not only during cold shock adaptation but also under optimal growth conditions.

J Bacteriol, 2002 Sep, 184(17), 4936 - 40
The spectrum of spontaneous rifampin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from that of vegetative cells and resembles that of Mycobacterium tuberculosis; Nicholson WL et al.; Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the beta subunit of RNA polymerase . In this study, we demonstrated that spores of B . subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells . In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB, H482R, from vegetative cells . Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L . The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis . The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.

J Bacteriol, 2002 Sep, 184(17), 4881 - 90
Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis; Britton RA et al.; Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase in Bacillus subtilis . Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase . To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotated B . subtilis open reading frames . In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H . This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes . In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H . Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.

J Bacteriol, 2002 Sep, 184(17), 4819 - 28
Bacillus subtilis mutant LicT antiterminators exhibiting enzyme I- and HPr-independent antitermination affect catabolite repression of the bglPH operon; Lindner C et al.; The Bacillus subtilis antiterminator LicT regulates the expression of bglPH and bglS, which encode the enzymes for the metabolism of aryl-beta-glucosides and the beta-glucanase BglS . The N-terminal domain of LicT (first 55 amino acids) prevents the formation of rho-independent terminators on the respective transcripts by binding to target sites overlapping these terminators . Proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulate the antitermination activity of LicT by phosphorylating histidines in its two PTS regulation domains (PRDs) . Phosphorylation at His-100 in PRD-1 requires the PTS proteins enzyme I and HPr and the phosphorylated permease BglP and inactivates LicT . During transport and phosphorylation of aryl-beta-glucosides, BglP is dephosphorylated, which renders LicT active and thus leads to bglPH and bglS induction . In contrast, phosphorylation at His-207 and/or His-269 in PRD-2, which requires only enzyme I and HPr, is absolutely necessary for LicT activity and bglPH and bglS expression . We isolated spontaneous licT mutants expressing bglPH even when enzyme I and HPr were absent (as indicated by the designation "Pia" {PTS-independent antitermination}) . Introduced in a ptsHI(+) strain, two classes of licT(Pia) mutations could be distinguished . Mutants synthesizing LicT(Pia) antiterminators altered in PRD-2 still required induction by aryl-beta-glucosides, whereas mutations affecting PRD-1 caused constitutive bglPH expression . One of the two carbon catabolite repression (CCR) mechanisms operative for bglPH requires the rho-independent terminator and is probably prevented when LicT is activated by P approximately His-HPr-dependent phosphorylation in PRD-2 (where the prefix "P approximately " stands for "phospho") . During CCR, the small amount of P approximately His-HPr present in cells growing on repressing PTS sugars probably leads to insufficient phosphorylation at PRD-2 of LicT and therefore to reduced bglPH expression . In agreement with this concept, mutants synthesizing a P approximately His-HPr-independent LicT(Pia) had lost LicT-modulated CCR.

J Bacteriol, 2002 Sep, 184(17), 4722 - 32
Tet(L) and tet(K) tetracycline-divalent metal/H+ antiporters: characterization of multiple catalytic modes and a mutagenesis approach to differences in their efflux substrate and coupling ion preferences; Jin J et al.; The Tet(L) protein encoded in the Bacillus subtilis chromosome and the closely related Tet(K) protein from Staphylococcus aureus plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me(2+)) complex that bears a net single positive charge, Na+, and K+ . Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H+ as the coupling ion . In this study, competitive cross-inhibition between K+ and other cytoplasmic efflux substrates was demonstrated . Tet(L) and Tet(K) had also been shown to use K+ as an alternate coupling ion in support of Na+ or K+ efflux . Here they were shown to couple TC-Me(2+) efflux to K+ uptake as well, exhibiting greater use of K+ as a coupling ion as the external pH increased . The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K+, both as a cytoplasmic efflux substrate and as an external coupling ion . Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative "antiporter motif," motif C, of Tet proteins would be involved in these characteristic preferences . Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K+ preference both as coupling ion and efflux substrate . A reciprocal S157A mutant of Tet(K) exhibited reduced K+ preference . Competitive inhibition among substrates and the parallel effects of the single mutation upon K+ preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion . However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates.

J Bacteriol, 2002 Sep, 184(17), 4681 - 9
Identification of Bacillus subtilis CysL, a regulator of the cysJI operon, which encodes sulfite reductase; Guillouard I et al.; The way in which the genes involved in cysteine biosynthesis are regulated is poorly characterized in Bacillus subtilis . We showed that CysL (formerly YwfK), a LysR-type transcriptional regulator, activates the transcription of the cysJI operon, which encodes sulfite reductase . We demonstrated that a cysL mutant and a cysJI mutant have similar phenotypes . Both are unable to grow using sulfate or sulfite as the sulfur source . The level of expression of the cysJI operon is higher in the presence of sulfate, sulfite, or thiosulfate than in the presence of cysteine . Conversely, the transcription of the cysH and cysK genes is not regulated by these sulfur sources . In the presence of thiosulfate, the expression of the cysJI operon was reduced 11-fold, whereas the expression of the cysH and cysK genes was increased, in a cysL mutant . A cis-acting DNA sequence located upstream of the transcriptional start site of the cysJI operon (positions -76 to -70) was shown to be necessary for sulfur source- and CysL-dependent regulation . CysL also negatively regulates its own transcription, a common characteristic of the LysR-type regulators . Gel mobility shift assays and DNase I footprint experiments showed that the CysL protein specifically binds to cysJ and cysL promoter regions . This is the first report of a regulator of some of the genes involved in cysteine biosynthesis in B . subtilis.

RNA, 2002 Jul, 8(7), 933 - 47
Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P; Crary SM et al.; Ribonuclease P (RNase P) is a ribonucleoprotein that requires magnesium ions to catalyze the 5' maturation of transfer RNA . To identify interactions essential for catalysis, the properties of RNase P containing single sulfur substitutions for nonbridging phosphodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic experiments . Sulfur substitution at the nonbridging oxygens of the phosphodiester bond of nucleotide U51 only modestly affects catalysis . However, phosphorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300-4,000-fold for P RNA and 500-15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of pre-tRNA(Asp), highlighting the importance of this region for catalysis . Furthermore, addition of manganese enhances pre-tRNA cleavage catalyzed by B . subtilis RNase P RNA containing an Sp phosphorothioate modification at A49, as observed for Escherichia coli P RNA {Christian et al., RNA, 2000, 6:511-519}, suggesting that an essential metal ion may be coordinated at this site . In contrast, no manganese rescue is observed for the A49 Sp phosphorothioate modification in RNase P holoenzyme . These differential manganese rescue effects, along with affinity cleavage, suggest that the protein component may interact with a metal ion bound near A49 in helix P4 of P RNA.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11121 - 6 Epub 2002 Aug 06.
tRNA-mediated transcription antitermination in vitro: codon-anticodon pairing independent of the ribosome; Grundy FJ et al.; Uncharged tRNA acts as the effector for transcription antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria . Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA . The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA . It was not known whether the leader RNA-tRNA interaction requires additional factors . We show here that tRNA-dependent antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA-RNA interaction is sufficient to control gene expression by antitermination . The tRNA response exhibits similar specificity in vivo and in vitro, and the antitermination reaction in vitro is independent of NusA and functions with either B . subtilis or Escherichia coli RNA polymerase.

Fitoterapia, 2002 Aug, 73(5), 431 - 3
Isolation of antibacterial fatty acids from Schotia brachypetala; McGaw LJ et al.; In southern Africa, the roots of Schotia brachypetala are used by traditional healers to treat dysentery and diarrhoea . Activity-directed fractionation of the ethanol extract of the dried leaves yielded 9,12,15-octadecatrienoic (linolenic) acid and methyl-5,11,14,17-eicosatetraenoate . These fatty acids displayed antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and to a lesser extent, against the Gram-negative Escherichia coli and Klebsiella pneumoniae.

DNA Cell Biol, 2002 Jul, 21(7), 527 - 34
Complete nucleotide sequence of the S10-spc operon of phytoplasma: gene organization and genetic code resemble those of Bacillus subtilis; Miyata S et al.; An 11.4-kbp region of genomic DNA containing the complete S10-spc operon was constructed by an integrative mapping technique with eight plasmid vectors carrying ribosomal protein sequences from onion yellows phytoplasma . Southern hybridization analysis indicated that phytoplasmal S10-spc is a single-copy operon . This is the first complete S10-spc operon of a phytoplasma to be reported, although only a part of six serial genes of the S10 operon is reported previously . The operon has a context of 5'-rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, rps17, rpl14, rpl24, rpl5, rps14, rps8, rpl6, rpl18, rps5, rpl30, rpl15, SecY-3', and is composed of 21 ribosomal protein subunit genes and a SecY protein translocase subunit gene . Resembling Bacillus, this operon contains an rpl30 gene that other mollicutes (Mycoplasma genitalium, M . pneumoniae, and M . pulmonis) lack . A phylogenetic tree based on the rps3 sequence showed that phytoplasmas are phylogenetically closer to acholeplasmas and bacillus than to mycoplasmas . In the S10-spc operon, translation may start from either a GTG codon or an ATG codon, and stop at a TGA codon, as has been reported for acholeplasmas and bacillus . However, in mycoplasmas, GTG was found as a start codon, and TGA was found not as a stop codon, but instead as a tryptophan codon . These data derived from the gene organization, and the genetic code deviation support the hypothesis that phytoplasmal genes resemble those of acholeplasmas and Bacillus more than those of other mollicutes.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11067 - 72 Epub 2002 Aug 02.
NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism invitro; Yakhnin AV et al.; The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms . Both mechanisms require the binding of tryptophan-activated TRAP to the 11 (G/U)AG-repeat segment in the trp leader transcript . To promote termination, TRAP must bind to the nascent RNA before the antiterminator structure forms . Because only 20 nucleotides separate the TRAP-binding site from the 3' end of the antiterminator, TRAP has a short time frame to control this regulatory decision . Synchronization of factor binding and/or RNA folding with the RNA polymerase position is a major challenge in all attenuation mechanisms . Because RNA polymerase pausing allows this synchronization in many attenuation mechanisms, we performed experiments in vitro to determine whether pausing participates in the B . subtilis trp attenuation mechanism . We identified two NusA-stimulated pause sites in the trp leader region . Formation of pause hairpins participates in pausing at both positions . The first pause occurred at the nucleotide just preceding the critical overlap between the alternative antiterminator and terminator structures . TRAP binding to transcripts containing preexisting pause complexes releases RNA polymerase, suggesting that pausing provides additional time for TRAP to bind and promote termination . The second pause is downstream from the trp leader termination point, raising the possibility that this pause event participates in the trpE translation control mechanism . NusA also increases the efficiency of termination in the trp leader region and shifts termination one nucleotide upstream . Finally, NusA-stimulated termination is cooperative, suggesting that binding of multiple NusA molecules influences termination.

Cell Calcium . 2002 Aug;32(2):93.
Expression of a P-type Ca(2+)-transport ATPase in Bacillus subtilis during sporulation; Raeymaekers L et al.; The open reading frame designated yloB in the genomic sequence of Bacillus subtilis encodes a putative protein that is most similar to the typically eukaryotic type IIA family of P-type ion-motive ATPases, including the endo(sarco)plasmic reticulum (SERCA) and PMR1 Ca(2+)-transporters, located respectively in the SERCA and the Golgi apparatus . The overall amino acid sequence is more similar to that of the Pmr1s than to the SERCAs, whereas the inverse is seen for the 10 amino acids that form the two Ca(2+)-binding sites in SERCA . Sporulating but not vegetative B . subtilis cells express the predicted protein, as shown by Western blotting and by the formation of a Ca(2+)-dependent phosphorylated intermediate . Half-maximal activation of phosphointermediate formation occurred at 2.5 microM Ca(2+) . Insertion mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect 45Ca accumulation during sporulation . However, spores from knockouts were less resistant to heat and showed a slower rate of germination . It is concluded that the P-type Ca(2+)-transport ATPase from B . subtilis is not essential for survival, but assists in the formation of resistant spores . The evolutionary relationship of the transporter to the eukaryotic P-type Ca(2+)-transport ATPases is discussed.

Eur J Med Chem, 2002 Aug, 37(8), 659 - 69
Preparations of heterospirostanols and their pharmacological activities; Quan HJ et al.; (3beta,20S,22S,25R)-22-Thiospirosol-5-en-3-ol (9) and (3beta,20S,22S,25R)-22-seleno-spirosol-5-en-3-ol (11) were prepared from diosgenin (3) via 26-iodopseudodiosgenin (6) as a key intermediate . Diosgenone (15), solasodinone (16), (20S,22S,25R)-22-thio-spirosol-4-en-3-one (17), (20S,22S,25R)-22-selenospirosol-4-en-3-one (18) and (20R,22S,25R)-spirosol-4-en-3-one (19) were prepared by Oppenauer oxidation of 3, solasodine 4, 9, 11 and (3beta,20R,22R,25R)-spirosol-5-en-3-ol 14, respectively . Oxidations of 15 and 16 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) provided corresponding dienone products, (20S,22S,25R)-spirosol-1,4-dien-3-one (20) and (20S,22S,25R)-22-thiospirosol-1,4-dien-3-one (21), respectively, while oxidation of 19 (C-20 diastereoisomer of 15) gave no dienone product but 21-exo vinyl product 22 . 26-Thioacetylpseudodiosgenone (24) and 26-cyanoselenopseudodiosgenone (25) were prepared by treatment of 26-iodopseudodiosgenose (23), which was obtained by Oppenauer oxidation of 6, with potassium thioacetate and potassium selenocyanate, respectively . Compounds 15 and 19 exhibited more than 80% inhibitions in INF-gamma productions at 10.0 microM . Compounds 4 and 25 showed cytotoxic activities (IC(50) = 6 and 5 microM, respectively) against cancerous HCT 116 cell lines . Compounds 12 and 25 had antiurease activities (IC(50) = 12.4 and 11.4 microM, respectively), in which only the latter showed an inhibition zone (mean zone diameter = 12.2 mm) formed by Bacillus subtilis 168 trp.

Comp Biochem Physiol A Mol Integr Physiol, 2002 Sep, 133(1), 95 - 104
Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var . natto; Samanya M et al.; Two experiments were conducted . In experiment 1, chickens were fed dried Bacillus subtilis var . natto for 3 or 28 days . Growth performance and internal organs were not different from controls, but feed efficiency tended to be improved in the 28-day feeding . In these birds, blood ammonia concentration was decreased (P<0.05) . Blood glucose concentration, and amylase and lipase activity in the intestinal content were not significantly different among dietary groups . These results suggest that the B . subtilis natto depressed ammonia concentration . In experiment 2, chickens were fed dietary B . subtilis natto for 28 days . These birds had a tendency to display greater growth performance and intestinal histologies, such as villus height, cell area and cell mitosis, than the controls . Flat cell outline on the duodenal villus surface in controls developed large, protruded cell clusters and cell protuberances after feeding of dietary B . subtilis natto . These results indicate that intestinal function was activated by the depressed blood ammonia concentration in the body of the chicken . The present results may suggest that the B . subtilis natto has the potential to be a beneficial microorganism in chickens.

Res Microbiol, 2002 Jun, 153(5), 269 - 76
Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats; Pinchuk IV et al.; One of the most interesting groups of phenolic compounds is comprised of the low molecular weight phenylpropanol derivative substances named isocoumarins, which possess important biological activities . In this study, the isocoumarin production and genetic diversity of 51 Bacillus strains isolated from different geographical and ecological niches were studied . Using molecular identification techniques, 47 strains were identified as B . subtilis, three as B . licheniformis and one as B . pumilus . When these strains were screened for isocumarin production, 11 belonging to the species B . subtilis produced amicoumacins, antibiotics of the isocoumarin group . RAPD analysis demonstrated that these strains fell into two groups which contained only these amicoumacin producers . No association was detected between RAPD profiles and the geographic origin or habitat of the strains tested . In conclusion, production of amicoumacin antibiotics by B . subtilis is a common characteristic of individual strains that presented genetic and physiological homogeneity.

Environ Microbiol, 2002 Aug, 4(8), 482 - 6
Factors affecting PCR-mediated recombination; Shafikhani S; In the past decade, polymerase chain reaction (PCR) has become an important tool for the identification of previously unknown microorganisms and the analysis of environmental microbial diversity . Several studies published during recent years, however, have demonstrated that products obtained after PCR using Taq or Vent DNA polymerases will contain hybrid molecules when several homologous target sequences such as multigene families, alleles, or RNA viruses are co-amplified . In this report, we examined the recombination frequency and the extent of template switching during PCR using Taq, Pfu and RTth/Vent DNA polymerases . As a test system we constructed a series of plasmids carrying between one and three frame shift mutations in the gene coding for the protease subtilisin or deletions of approximately 100 bp in the lacZ alpha . Highest recombination frequencies were observed when these mutants were co-amplified with Taq followed by RTth/Vent DNA polymerases . Pfu DNA polymerase displayed no discernable recombination activity under normal PCR conditions . Data also suggest that in vivo repair of heteroduplex DNA molecules in Escherichia coli by a RecA-independent mechanism, perhaps the mismatch repair, results in the formation of chimeric molecules . Using Bacillus subtilis as the host, however, can significantly diminish non-PCR RecA-independent in vivo recombination, owing to the fact that transforming DNA molecules enter B . subtilis as single strands . Combined, these results suggest that using Pfu DNA polymerase for amplification and B . subtilis as the host for transformation may significantly reduce chimera formation.

J Org Chem, 2002 Aug 9, 67(16), 5807 - 16
Design, synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase; Cushman M et al.; A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine synthase . The compounds were designed to bind to both the ribitylpurine binding site and the phosphate binding site of lumazine synthase . In the carboxyalkyl series, maximum activity against both enzymes was observed with the 3'-carboxypropyl compound 22 . Lengthening or shortening the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity . In the phosphonoxyalkyl series, the 3'-phosphonoxypropyl compound 33 was more potent than the 4'-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against riboflavin synthase . Molecular modeling suggested that the terminal carboxyl group of 6-(3'-carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of the enzyme . A hypothetical molecular model was also constructed for the binding of 6-(2'-carboxyethyl)-7-oxolumazine (15) in the active site of E . coli riboflavin synthase, which demonstrated that the active site could readily accommodate two molecules of the inhibitor.

J Appl Microbiol, 2002, 93(2), 316 - 25
Studies on the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide; Melly E et al.; AIMS: To determine the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide . METHODS AND RESULTS: Killing of spores of B . subtilis with hydrogen peroxide caused no release of dipicolinic acid (DPA) and hydrogen peroxide-killed spores were not appreciably sensitized for DPA release upon a subsequent heat treatment . Hydrogen peroxide-killed spores appeared to initiate germination normally, released DPA and hydrolysed significant amounts of their cortex . However, the germinated killed spores did not swell, did not accumulate ATP or reduced flavin mononucleotide and the cores of these germinated spores were not accessible to nucleic acid stains . CONCLUSIONS: These data indicate that treatment with hydrogen peroxide results in spores in which the core cannot swell properly during spore germination . SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide further information on the mechanism of killing of spores of Bacillus species by hydrogen peroxide.

EMBO J, 2002 Aug 1, 21(15), 4001 - 11
A large dispersed chromosomal region required for chromosome segregation in sporulating cells of Bacillus subtilis; Wu LJ et al.; The cis-acting sequences required for chromosome segregation are poorly understood in most organisms, including bacteria . Sporulating cells of Bacillus subtilis undergo an unusual asymmetric cell division during which the origin of DNA replication (oriC) region of the chromosome migrates to an extreme polar position . We have now characterized the sequences required for this migration . We show that the previously characterized soj-spo0J chromosome segregation system is not essential for chromosome movement to the cell pole, so this must be driven by an additional segregation mechanism . Observations on a large set of precisely engineered chromosomal inversions and translocations have identified a polar localization region (PLR), which lies approximately 150-300 kbp to the left of oriC . Surprisingly, oriC itself has no involvement in this chromosome segregation system . Dissection of the PLR showed that it has internal functional redundancy, reminiscent of the large diffuse centromeres of most eukaryotic cells.

J Mol Biol, 2002 Aug 9, 321(2), 355 - 62
A partially buried site in homologous HPr proteins is not optimized for stability; Nicholson EM et al.; The energetic consequences of site-specific replacement of a residue at a partially buried site in the two homologous HPr proteins from Escherichia coli and Bacillus subtilis is described . We determined previously that the replacement of a partially buried Lys residue with Glu at position 49 in E.coli HPr increased the conformational stability of the protein substantially because the side-chain of the latter residue could act as a hydrogen-bond acceptor . Here, we extend this analysis to other side-chains with different chemical properties and abilities to form hydrogen bonds to compare the properties of this position in the backgrounds of two different homologous HPr proteins . We find that the variants with polar residues that can form a tertiary hydrogen bond with a nearby site in the protein are more stable than either hydrophobic residues or polar residues that become buried yet are incapable of forming a new hydrogen bond . Furthermore, the protein with the wild-type residue in each HPr variant is not among the most stable of the proteins studied . These results suggest a general strategy for designing variants in which the overall stability of a protein can be modulated in a defined fashion.

J Mol Biol, 2002 Aug 9, 321(2), 341 - 53
Formation of metal nanoclusters on specific surface sites of protein molecules; Braun N et al.; During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration) . Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development . Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters . On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography . In all three cases, decoration of the same protein results in different cluster distributions for gold and silver . Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions . A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues . The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods . Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters . This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities.

Planta Med, 2002 Jul, 68(7), 615 - 20
Antimicrobial flavonoids from Bolusanthus speciosus; Bojase G et al.; A new isoflavanone namely 3,5,7,2',4'-pentahydroxy-8,3'-di(gamma,gamma-dimethylallyl)isoflavanone (bolusanthin II) and four new pterocarpans identified as 3-hydroxy-6',6'-dimethylpyrano{2',3':1,2} {6a R,11a R}-8,9-methylenedioxypterocarpan (bolucarpan A), 3-hydroxy-6',6'-dimethyl-4',5'-dihydropyrano{2',3':1,2}{6a R,11a R}- 8,9-methylenedioxypterocarpan (bolucarpan B), 3-hydroxy-9-methoxy-6',6'-dimethylpyrano-{2',3':1,2}{6a R,11a R}-pterocarpan (bolucarpan C) and 3-hydroxy-9-methoxy-6',6'-dimethyl-4',5'-dihydropyrano{2',3':1,2}{6a R,11a R}-pterocarpan (bolucarpan D) and three known isoflavonoids were isolated from the methanolic extracts of the root bark, while eight known isoflavonoids were isolated from the stem bark of Bolusanthus speciosus . These compounds showed antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Saccharomyces cerevisiae and Candida mycoderma using the agar overlay technique.

Annu Rev Microbiol, 2002, 56, 599 - 624 Epub 2002 Jan 30.
Impact of genomic technologies on studies of bacterial gene expression; Rhodius V et al.; The ability to simultaneously monitor expression of all genes in any bacterium whose genome has been sequenced has only recently become available . This requires not only careful experimentation but also that voluminous data be organized and interpreted . Here we review the emerging technologies that are impacting the study of bacterial global regulatory mechanisms with a view toward discussing both perceived best practices and the current state of the art . To do this, we concentrate upon examples using Escherichia coli and Bacillus subtilis because prior work in these organisms provides a sound basis for comparison.

J Bacteriol, 2002 Aug, 184(16), 4636 - 9
Mutations in the Bacillus subtilis glnRA operon that cause nitrogen source-dependent defects in regulation of TnrA activity; Fisher SH et al.; The Bacillus subtilis nitrogen transcriptional factor TnrA is inactive in cells grown with excess nitrogen, e.g., glutamine or glutamate plus ammonium, because feedback-inhibited glutamine synthetase (product of glnA) binds to TnrA and blocks its DNA-binding activity . Two conditional mutations that allow TnrA-dependent gene expression in cells grown with glutamate plus ammonium, but not in glutamine-grown cells, were characterized . One mutant contained a mutation in the glnA ribosome binding site, while the other mutant synthesized a truncated GlnR protein that constitutively repressed glnRA expression . The levels of glutamine synthetase were reduced in both mutants . As a result, when these mutants are grown with excess nitrogen in the absence of glutamine, there is insufficient production of the feedback inhibitors necessary to convert glutamine synthetase into its feedback-inhibited form and TnrA-activated genes are expressed at high levels.

J Biotechnol, 2002 Sep 25, 98(2-3), 243 - 54
Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion; van Dijl JM et al.; Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year . However, standard export technologies, based on the well-characterised general secretory (Sec) pathway, are frequently inapplicable for the production of proteins . The recently discovered twin-arginine translocation (Tat) pathway offers additional potential to transport proteins . Here we review the use of functional genomic and proteomic approaches to explore the Tat pathway of Bacillus subtilis . The properties of Tat pathway components and the twin-arginine signal peptides that direct proteins into this pathway are discussed . Where appropriate, a comparison is made with Tat systems from other organism, such as Escherichia coli . Recent findings with the latter organism in particular provide proof-of-principle that the Tat pathway can be exploited for the production of Sec-incompatible proteins.

Biochemistry (Mosc), 2002 Jul, 67(7), 802 - 6
Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme; Skvortsova MA et al.; The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene . The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel . The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg . The molecular weight of binase II is 30 kD . The enzyme is activated by Mg2+ and virtually completely inhibited by EDTA . The pH optimum for the reaction of RNA hydrolysis is 8.5 . The properties of the enzyme are close to those of RNase Bsn from B . subtilis . The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.

Infect Control Hosp Epidemiol, 2002 Jul, 23(7), 393 - 6
Sterilization of heat-resistant instruments with infrared radiation; Mata-Portuguez VH et al.; OBJECTIVE: To evaluate the lethality profile of an infrared radiation (IR) prototype sterilizer . METHOD: Simulated use and D value tests were conducted with Bacillus subtilis spores American Type Culture Collection-9372 . A spore suspension (1.06 +/- 0.03 x 10(6)) in 5% bovine serum albumin was air dried on stainless steel instruments . IR cycles were completed and the instruments were immersed in tryptic soy broth for 120 hours at 37 degrees C . Direct enumeration of survivors was performed to evaluate IR death . Instrument loads contained carriers laden with spores (1.06 +/- 0.3 x 10(6)) . The spores were seeded on tryptic soy agar and survivors were counted after 120 hours of incubation at 37 degrees C . RESULTS: All instruments exposed to IR (n = 50) were culture negative . In contrast, all unprocessed instruments (n = 30) showed B . subtilis growth . The prototype's D value was 0.56, and the death rate's slope was -1.76 (r = -0.99741; P < .0001) . The 10(6) sterility assurance level was reached after 8 minutes and 40 seconds of exposure, from cold start . CONCLUSIONS: IR destroys B . subtilis spores . Instrument sterilization with IR may provide another technology for infection control.

Nucleic Acids Res, 2002 Jul 15, 30(14), 3026 - 33
Transfer RNA-mediated antitermination in vitro; Putzer H et al.; The threonyl-tRNA synthetase gene (thrS) is a member of the T-box family of approximately 250 genes, found essentially in Gram-positive bacteria, regulated by a tRNA-dependent antitermination mechanism in response to starvation for the cognate amino acid . While interaction between uncharged tRNA and the untranslated leader region of these genes has been firmly established by genetic means, attempts to show this interaction or to reconstitute the antitermination mechanism in vitro using purified tRNAs have so far failed . In addition, a number of conserved sequences have been identified in the T-box leaders, for which no function has yet been assigned . This suggests that factors other than the tRNA are important for this type of control . Here we demonstrate tRNA-mediated antitermination for the first time in vitro, using the regulatory tRNA(Thr) isoacceptor isolated from Bacillus subtilis and a partially purified protein fraction . As predicted by the model, aminoacylation of tRNA(Thr(GGU)) with threonine completely abolishes its ability to act as an effector . The role of the partially purified protein fraction can be functionally substituted by high concentrations of spermidine . However, this polyamine does not play a significant role in the induction of thrS expression in vivo, suggesting that it is specific protein co-factors that promote T-box gene regulation in conjunction with uncharged tRNA.

Biochemistry, 2002 Jul 30, 41(30), 9718 - 26
Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics; Eppelmann K et al.; Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs) . By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs . Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket . In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo . First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket . Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency . Second, according to the selectivity-conferring code, the binding pockets of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively . Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of L-Asn . Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch . Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster . The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide {Asn(5)}surfactin . This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.

Biochemistry, 2002 Jul 30, 41(30), 9545 - 58
The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit; Kurz JC et al.; The RNA subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of tRNA 5'-maturation in vitro . The protein component of RNase P, required for cleavage of precursor tRNA in vivo, enhances pre-tRNA binding by directly contacting the 5'-leader sequence . Using a combination of transient kinetics and equilibrium binding measurements, we now demonstrate that the protein component of RNase P also facilitates catalysis by specifically increasing the affinities of magnesium ions bound to the RNase P x pre-tRNA(Asp) complex . The protein component does not alter the number or apparent affinity of magnesium ions that are either diffusely associated with the RNase P RNA polyanion or required for binding mature tRNA(Asp) . Nor does the protein component alter the pH dependence of pre-tRNA(Asp) cleavage catalyzed by RNase P, providing further evidence that the protein component does not directly stabilize the catalytic transition state . However, the protein subunit does increase the affinities of at least four magnesium sites that stabilize pre-tRNA binding and, possibly, catalysis . Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme x pre-tRNA complex . These results suggest that the protein component enhances the magnesium affinity of the RNase P x pre-tRNA complex indirectly by binding and positioning pre-tRNA . Furthermore, RNase P is inhibited by cobalt hexammine (K(I) = 0.11 +/- 0.01 mM) while magnesium, manganese, cobalt, and zinc compete with cobalt hexammine to activate RNase P . These data are consistent with the hypothesis that catalysis by RNase P requires at least one metal-water ligand or one inner-sphere metal contact.

J Biol Chem, 2002 Sep 20, 277(38), 35567 - 73 Epub 2002 Jul 19.
Using hetero-11-mers composed of wild type and mutant subunits to study tryptophan binding to TRAP and its role in activating RNA binding; Li PT et al.; Expression of genes involved in tryptophan metabolism in Bacillus subtilis is regulated by the TRAP protein in response to changes in l-tryptophan levels . TRAP binding to several RNA targets that contain between 9 and 11 (G/U)AG repeats regulates transcription and/or translation of these genes . TRAP consists of 11 identical subunits and is activated to bind RNA by binding up to 11 molecules of tryptophan . To investigate the mechanism by which tryptophan binding activates TRAP, we generated hetero-11-mers containing different proportions of subunits from wild type (WT) TRAP that bind tryptophan and from a mutant TRAP (Thr(25) to Ala) defective in tryptophan binding . Studies of these hetero-11-mers show that tryptophan-binding sites created from active subunits bind tryptophan with similar affinity to those in WT homo-11-mers, whereas sites containing the T25A substitution do not bind tryptophan . Hetero-11-mers with very few (one or two) bound tryptophans show only 10-fold lower affinity than WT TRAP for an RNA with 11 GAG repeats, whereas TRAP with no bound tryptophan shows no detectable binding to this RNA . We also demonstrate that tryptophan binding induces a conformational change in TRAP in the vicinity of the RNA-binding site, suggesting a possible mechanism for activation of RNA binding.

Z Naturforsch {C}, 2002 May-Jun, 57(5-6), 465 - 70
Trichosetin, a novel tetramic acid antibiotic produced in dual culture of Trichoderma harzianum and Catharanthus roseus Callus; Marfori EC et al.; The dual culture of Trichoderma harzianum and Catharanthus roseus callus produced an antimicrobial compound with a remarkable activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis . Structural elucidation revealed that this compound, which we have named trichosetin, is a novel tetramic acid (2,4-pyrrolidinedione) antibiotic and a homolog of the fungal metabolite equisetin . This compound however, was not produced in the individual culture of T . harzianum or C . roseus callus.

Eur J Med Chem, 2002 Jul, 37(7), 553 - 64
Hydrazones of 1,2-benzisothiazole hydrazides: synthesis, antimicrobial activity and QSAR investigations; Vicini P et al.; A series of hydrazones of 1,2-benzisothiazole hydrazides 1a-m, 2a-m, 3a-m, 4a-m, 5a-m as well as their cyclic (1 and 4) and acyclic (2, 3 and 5) 1,2-benzisothiazole parent hydrazides, were synthesised and evaluated as antibacterial and antifungal agents . All of the 2-amino-1,2-benzisothiazol-3(2H)-one derivatives, belonging to series I and IV, showed a good antibacterial activity against Gram positive bacteria . Most of them were active against yeasts too . Compounds 1 and 4, together with 1l, proved to be the most effective compounds . Quantitative structure-activity relationship (QSAR) investigation with a 2D-QSAR analysis was applied to find a correlation between different experimental or calculated physicochemical parameters of the compounds studied . A 3D-QSAR study was performed, applying Comparative Molecular Similarity Indices Analysis (CoMSIA) method, to derive quantitative models relating the structural features of 1,2-benzisothiazole derivatives 1, 1a-m and 4, 4a-m and their antimicrobial activity against Bacillus subtilis, resulted the most sensitive micro-organism.

J Mol Microbiol Biotechnol, 2002 Jul, 4(4), 427 - 52
A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins; Mittenhuber G; Regulation of expression of the general stress regulon of Bacillus subtilis is mediated by the activation of the alternative sigma factor sigmaB . Activation of sigmaB is accomplished by a complex regulatory network involving protein-protein interactions and reversible protein phosphorylation . PSI-BLAST searches were performed and phylogenetic trees for sigmaB and its regulatory proteins were constructed . Occurrence of sigmaB is restricted to a small group of gram-positive bacteria (Bacillus, Staphylococcus, Listeria) . Related sigma factors also involved in stress responses are present in Mycobacterium tuberculosis, Streptomyces species and even in cyanobacteria (Synechocystis species) . Putative regulatory proteins found in several other bacterial species can be broadly catagorized into three categories: Anti sigma factors, anti-anti sigma factors and phosphatases . Anti sigma factors are able to bind to sigma factors and are also kinases of anti sigma factor antagonists . Only in their nonphosphorylated state, these antagonists are able to bind to the anti sigma factor . Phosphorylated antagonists can be dephosphorylated by PP2C phosphatases . These phosphatases are of pivotal importance for activation of the sigma factor . Different phosphatases identified in this search contain a wide variety of domains found in signal transducing proteins (PAS/PAC, GAF, REC, HATase_c, HAMP) . The HATPase_c domain found in several phosphatases most probably constitutes a serine/threonine kinase domain of anti sigma factors . Such proteins are most probably bifunctional anti-anti sigma factor kinases and phosphatases . The regulatory network of anti-anti sigma factors anti sigma factors and phosphatases is probably ancient and most likely evolved from a structurally similar network found in the Deinococcus radiodurans genome . In completely sequenced genomes of several bacterial species, some elements of the network are missing . The N-terminus of RsbU, a phosphatase activated in response to environmental stress exhibits similarities to a region in the beta chain of phenylalanyl-tRNA synthetases.

J Biol Chem, 2002 Sep 27, 277(39), 35969 - 79 Epub 2002 Jul 17.
Homologous-pairing activity of the Bacillus subtilis bacteriophage SPP1 replication protein G35P; Ayora S et al.; Genetic evidence suggests that the SPP1-encoded gene 35 product (G35P) is essential for phage DNA replication . Purified G35P binds single-strand DNA (ssDNA) and double-strand (dsDNA) and specifically interacts with SPP1-encoded replicative DNA helicase G40P and SSB protein G36P . G35P promotes joint molecule formation between a circular ssDNA and a homologous linear dsDNA with an ssDNA tail . Joint molecule formation requires a metal ion but is independent of a nucleotide cofactor . Joint molecules formed during these reactions contain a displaced linear ssDNA strand . Electron microscopic analysis shows that G35P forms a multimeric ring structure in ssDNA tails of dsDNA molecules and left-handed filaments on ssDNA . G35P promotes strand annealing at the AT-rich region of SPP1 oriL on a supercoiled template . These results altogether are consistent with the hypothesis that the homologous pairing catalyzed by G35P is an integral part of SPP1 DNA replication . The loading of G40P at a d-loop (ori DNA or at any stalled replication fork) by G35P could lead to replication fork reactivation.

Anal Biochem, 2002 Jul 15, 306(2), 222 - 7
Macromolecular assembly of the transition state regulator AbrB in its unbound and complexed states probed by microelectrospray ionization mass spectrometry; Benson LM et al.; The Bacillus subtilis global transition-state regulator AbrB specifically recognizes over 60 different DNA regulatory regions of genes expressed during cellular response to suboptimal environments . Most interestingly the DNA regions recognized by AbrB share no obvious consensus base sequence . To more clearly understand the functional aspects of AbrB activity, microelectrospray ionization mass spectrometry has been employed to resolve the macromolecular assembly of unbound and DNA-bound AbrB . Analysis of the N-terminal DNA binding domain of AbrB (AbrBN53, residues 1-53) demonstrates that AbrBN53 is a stable dimer, showing no apparent exchange with a monomeric form as a function of pH, ionic strength, solvent, or protein concentration . AbrBN53 demonstrates a capacity for DNA binding, underscoring the role of the N-terminal domain in both DNA recognition and dimerization . Full-length AbrB is shown to exist as a homotetramer . An investigation of the binding of AbrBN53 and AbrB to the natural DNA target element sinIR shows that AbrBN53 binds as a dimer and AbrB binds as a tetramer . This study represents the first detailed characterization of the stoichiometry of a transition-state regulator binding to one of its target promoters.

Mol Microbiol, 2002 Jul, 45(2), 569 - 83
GabR, a member of a novel protein family, regulates the utilization of gamma-aminobutyrate in Bacillus subtilis; Belitsky BR et al.; The Bacillus subtilis ycnG (gabT) and ycnH (gabD) genes were shown to encode gamma-aminobutyrate (GABA) aminotransferase and succinic semi-aldehyde dehydrogenase, respectively, and to form a GABA-inducible operon . Null mutations in gabT, gabD or the divergently transcribed ycnF (gabR) gene blocked the utilization of GABA as sole nitrogen source . GabR proved to be a transcriptional activator of the gabTD operon and a negative autoregulator . The target of GabR action was localized to an 87 bp region that includes both gabR and gabT promoters . GabR is a member of a novel but widespread family of chimeric bacterial proteins that have apparent DNA-binding and aminotransferase domains . Mutations in conserved residues of the putative aminotransferase domain abolished GabR function as a transcriptional activator, but did not affect its activity as a negative autoregulator.

Mol Microbiol, 2002 Jul, 45(2), 555 - 68
The role of heterologous receptors in McpB-mediated signalling in Bacillus subtilis chemotaxis; Zimmer MA et al.; Asparagine chemotaxis in Bacillus subtilis appears to involve two partially redundant adaptation mechanisms: a receptor methylation-independent process that operates at low attractant concentrations and a receptor methylation-dependent process that is required for optimal responses to high concentrations . In order to elucidate these processes, chemotactic responses were assessed for strains expressing methylation-defective mutations in the asparagine receptor, McpB, in which all 10 putative receptors (10del), five receptors (5del) or only the native copy of mcpB were deleted . This was done in both the presence and the absence of the methylesterase CheB . We found that: (i) only responses to high concentrations of asparagine were impaired; (ii) the presence of all heterologous receptors fully compensated for this defect, whereas responses progressively worsened as more receptors were taken away; (iii) methyl-group turnover occurred on heterologous receptors after the addition of asparagine, and these methylation changes were required for the restoration of normal swimming behaviour; (iv) in the absence of the methyleste-rase, the presence of heterologous receptors in some cases caused impaired chemotaxis; and (v) either a certain threshold number of receptors must be present to promote basal CheA activity, or one or more of the receptors missing in the 10del background (but present in the 5del background) is required for establishing basal CheA activity . Taken together, these findings suggest that many or all chemoreceptors work as an ensemble that constitutes a robust chemotaxis system . We propose that the ability of non-McpB receptors to compensate for the methylation-defective McpB mutations involves lateral transmission of the adapted conformational change across the ensemble.

Mol Microbiol, 2002 Jul, 45(2), 543 - 53
Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation; Ludwig H et al.; Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis . Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars . Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA) . CcpA is required for induction of the gapA operon by glucose . Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region . Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS) . A functional CcpA is needed for efficient transport of these sugars . This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS . In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer . A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon . We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.

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