Nat Struct Biol, 2000 Apr, 7(4), 303 - 8 Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase; Eriksen TA et al.; Here we report the first three-dimensional structure of a phosphoribosylpyrophosphate (PRPP) synthetase . PRPP is an essential intermediate in several biosynthetic pathways . Structures of the Bacillus subtilis PRPP synthetase in complex with analogs of the activator phosphate and the allosteric inhibitor ADP show that the functional form of the enzyme is a hexamer . The individual subunits fold into two domains, both of which resemble the type I phosphoribosyltransfereases . The active site is located between the two domains and includes residues from two subunits . Phosphate and ADP bind to the same regulatory site consisting of residues from three subunits of the hexamer . In addition to identifying residues important for binding substrates and effectors, the structures suggest a novel mode of allosteric regulation. Biopolymers, 1999, 52(1), 57 - 63 Specificity of hydroxylmethyluracil-containing DNA for transcription factor 1: structural insights; Vu HM et al.; The genomic materials from some Bacillus subtilis bacteriophages are found to contain 5-(hydroxymethyl)-2'-deoxyuridine in place of thymine . Phage-encoded proteins such as transcription factor 1 specifically and preferentially bind to the minor grooves of these hmU-containing DNA but not to thymine-containing DNA . Data from electrophoretic mobility shift assays suggest that the inherent, localized flexibility of hmU-DNA, which is sequence-specific, is responsible for its discriminative binding . We discuss here, from the NMR-derived structural point of view, how differential DNA flexibility can contribute to specific binding of TF1 to hmU-DNA . Biosci Biotechnol Biochem, 2000 Feb, 64(2), 386 - 92 An efficient method for production of uridine 5'-diphospho-N-acetylglucosamine; Okuyama K et al.; Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5'-UMP and glucosamine, in which yeast cells catalyze the conversion of 5'-UMP to 5'-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5'-UTP and glucosamine as substrates . However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product . We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N-acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP-GlcNAc biosynthesis . The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine . The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc . Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5'-UMP and 100 mM GlcNAc. Biosci Biotechnol Biochem, 2000 Feb, 64(2), 275 - 9 Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens; Akamatsu T et al.; Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B . subtilis or B . amyloliquefaciens . The interspecific transformation frequency of B . subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA . Increased interspecific transformation frequencies of B . subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA) . An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B . subtilis chromosome . Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation . The involvement of mutS in the interspecific transformation was not significant. Dev Comp Immunol, 2000 Jun, 24(4), 367 - 79 Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria; da Silva C et al.; The hemocytes to which bacteria adhere were defined and the contribution of the prophenoloxidase system of fifth instar nymphs of Acheta domesticus to adhesion were examined . The physicochemical parameters affecting hemocyte and phenoloxidase activity were determined . Both plasmatocytes and granular cells responded to bacteria, the latter cells entrapping the microorganisms on filopodial extensions . The optimum pH for hemocyte adhesion to glass slides was 6.5, the granular cells being the most sensitive hemocyte type . Although hydrophobic resin beads and positively-charged beads favoured hemocyte attachment, these parameters did not contribute to differential bacterial adhesion to hemocytes . Activation of phenoloxidase was neither enhanced nor inhibited by 0.1 and 1 mg/ml of laminarin or zymosan nor by dead Bacillus subtilis . However, live B . subtilis activated the enzyme and dead Xenorhabdus nematophilus inhibited enzyme activation . Serine protease components of the prophenoloxidase system had opsonic properties for B . subtilis but not for X . nematophilus . Phenoloxidase activity was enhanced by Ca(2+) and Mg(2+) and inhibited by SO(2-)(4). J Bacteriol, 2000 Apr, 182(8), 2329 - 31 A Bacillus subtilis gene of previously unknown function, yhaG, is translationally regulated by tryptophan-activated TRAP and appears to be involved in tryptophan transport; Sarsero JP et al.; Computer analysis of the Bacillus subtilis genome sequence revealed a gene with no previously attributed function, yhaG, specifying a transcript containing a presumptive binding site for the tryptophan-activated regulatory protein, TRAP . The presumptive TRAP binding site overlaps the yhaG Shine-Dalgarno sequence and translation initiation region . TRAP was shown to regulate expression of yhaG translationally . Production of the yhaG transcript in vivo was found to compete for the binding of TRAP to other known TRAP binding sites . YhaG is likely to be a transmembrane protein involved in tryptophan transport. J Bacteriol, 2000 Apr, 182(8), 2311 - 3 A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins; Jack DL et al.; The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps . Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon, ykkCD, but not expression of either ykkC or ykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs. J Bacteriol, 2000 Apr, 182(8), 2307 - 10 A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis; Masaoka Y et al.; Genes (ebrAB) responsible for ethidium resistance were cloned from chromosomal DNA of Bacillus subtilis ATCC 9372 . The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in Escherichia coli but also in B . subtilis . It also caused an elevated energy-dependent efflux of ethidium in E . coli . EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps . Neither ebrA nor ebrB was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance . Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump . In known members of the SMR family, only one gene produces drug efflux . Thus, EbrAB is a novel SMR family multidrug efflux pump with two components. J Bacteriol, 2000 Apr, 182(8), 2119 - 24 Probable identification of a membrane-associated repressor of Bacillus subtilis DNA replication as the E2 subunit of the pyruvate dehydrogenase complex; Stein A et al.; Two Bacillus subtilis lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of B . subtilis DNA replication (J . Laffan and W . Firshein, Proc . Natl . Acad . Sci . USA 85:7452-7456, 1988) . Three clones that reacted strongly with the antibody contained an entire open reading frame . Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase . An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound . This sequence was almost identical to one detected in Bacillus thuringiensis that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T . Walter and A . Aronson, J . Biol . Chem., 274:7901-7906, 1999) . However, the exact sequence was not as important in B . subtilis as the AT-rich core region . Binding would occur as long as most of the AT character of the core remained . Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered . The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay. J Bacteriol, 2000 Apr, 182(8), 2088 - 95 Two types of Bacillus subtilis tetA(L) deletion strains reveal the physiological importance of TetA(L) in K(+) acquisition as well as in Na(+), alkali, and tetracycline resistance; Wang W et al.; The chromosomally encoded TetA(L) protein of Bacillus subtilis is a multifunctional tetracycline-metal/H(+) antiporter that also exhibits monovalent cation/H(+) antiport activity and a net K(+) uptake mode . In this study, B . subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types of tetA(L) deletion strains that are generated in the same selection . Both strains exhibited increased sensitivity to low tetracycline concentrations as expected . The mutants also had significantly reduced ability to grow in media containing low concentrations of K(+), indicating that the net K(+) uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C . JC112 also exhibited (i) greater impairment of Na(+)- or K(+)-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co(+2) as well as Na(+) sensitivity . Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants . Increased expression of two loci has thus far been shown . Increased expression of czcD-trkA, a locus with a proposed involvement in K(+) uptake, occurred in both mutants . The increase was highest in the presence of Co(2+) and was higher in JC112 than in JC112C . Deletion of czcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low {K(+)}, supporting a significant role for this locus in K(+) uptake . Expression of yheL, which is a homologue of the Na(+)/H(+) antiporter-encoding nhaC gene from Bacillus firmus OF4, was also increased in both tetA(L) deletion strains, again with higher up-regulation in JC112 . The phenotypes resulting from deletion of yheL were consistent with a modest role for YheL in Na(+)-dependent pH homeostasis in the wild type . No major role for YheL was indicated in the mutants in spite of the overexpression . The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na(+), and alkali resistance and K(+) acquisition . The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions. J Appl Microbiol, 2000 Jan, 88(1), 132 - 41 Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes; Coffey A et al.; Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread . This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L . monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease . High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate . The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values . It can also degrade actin, the most abundant protein in many eukaryotic cells . The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity . A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics. Int J Food Microbiol, 1999 Nov 15, 52(3), 189 - 96 Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese; Fernandez-Garcia E et al.; The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp . cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese . Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media . Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6 . Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses . Tyramine and histamine were detected in all batches of cheese from day 30 . Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively) . Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses . After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture. J Med Entomol, 2000 Mar, 37(2), 265 - 70 Response of the tick Dermacentor variabilis (Acari: Ixodidae) to hemocoelic inoculation of Borrelia burgdorferi (Spirochetales); Johns R et al.; When Borrelia burgdorferi B31 low passage strain spirochetes were directly injected into the hemocoel of Dermacentor variabilis (Say) females, the bacteria were cleared from the hemocoel within < 24 h . Viable spirochetes were not found in hemolymph, salivary gland, or ovary tissues by subculturing or by IFA . The hemocyte population increased approximately 6 times within the first 6 h after inoculation compared with the uninoculated controls . In contrast, the soluble total hemolymph protein content decreased inversely with the increase in hemocytes . Borreliacidal activity was demonstrated with cell-free hemolymph from D . variabilis . In vitro antimicrobial assays using hemolymph from borrelia-challenged and nonchallenged ticks resulted in 72% spirochete reductions compared with only 11.5%, respectively, within 1 h . Additional evidence of induced antimicrobial hemolymph protein activity was demonstrated by SDS-PAGE, which revealed upregulation of a lysozyme-like peptide (approximately 15 kDa) (22% increase) and the induction of a approximately 5.8 kDa peptide in the B . burgdorferi-challenged ticks . In contrast with the nonvector borne Bacillus subtilis, D . variabilis presented a rapid and robust response to challenge with cultured B . burgdorferi spirochetes and lead to their early elimination . The role of the tick immune system, including possible differences between vector and nonvector ticks, in determining the success of invasive bacteria is discussed. Spectrochim Acta A Mol Biomol Spectrosc, 2000 Feb 1, 56A(2), 341 - 9 Cu(II) complexes in bacterial growth medium: electron spin resonance study; Jung K et al.; In this study we report a spectroscopic investigation on the structure and stability of Cu(II)-complexes that are formed in a minimum growth medium (MM), normally used for Bacillus subtilis cultures . As other transition metals, Cu(II) compounds are toxic to this bacterium and the toxicity depends on the Cu(II) concentration . MM contained NH4+ ions and asparagine (asn) as the source of inorganic and organic nitrogen . Both ESR and electronic spectra demonstrated the very important role played by the amino acid asparagine in the coordinative behaviour of Cu(II) . In particular, three different complexes were evidenced: Cu(H2O)6(2+); Cu(asn)+ and Cu(asn)2 . The relative amount of these three species strongly depended on pH, on Cu:asn ratio and on the presence of the phosphate ions . They were identified and evaluated quantitatively by extensive simulation of the electron spin resonance (ESR) spectra recorded in different experimental conditions . The bis-complex was found to be more stable in MM than in an asparagine-containing water solution with the same Cu:asn ratio . A comparison of the spectroscopic results with microbiological investigations is also made. Biochimie, 2000 Jan, 82(1), 85 - 91 The L17 ribosomal protein of Bacillus subtilis binds preferentially to curved DNA; Zouine M et al.; We searched in Bacillus subtilis for proteins that bind preferentially to curved DNA . Two proteins of 9 and 15 kDa displaying this property were purified from exponentially growing cells of B . subtilis strain 168 . Sequencing of N-terminal amino acids identified them as the proteins HBsu and L17 respectively . The overproduction of L17 from B . subtilis in Escherichia coli was shown to have a strong effect on nucleoid morphology and segregation. J Mol Biol, 2000 Mar 24, 297(2), 335 - 53 Inferring regulatory elements from a whole genome . An analysis of Helicobacter pylori sigma(80) family of promoter signals; Vanet A et al.; Helicobacter pylori is adapted to life in a unique niche, the gastric epithelium of primates . Its promoters may therefore be different from those of other bacteria . Here, we determine motifs possibly involved in the recognition of such promoter sequences by the RNA polymerase using a new motif identification method . An important feature of this method is that the motifs are sought with the least possible assumptions about what they may look like . The method starts by considering the whole genome of H . pylori and attempts to infer directly from it a description for a family of promoters . Thus, this approach differs from searching for such promoters with a previously established description . The two algorithms are based on the idea of inferring motifs by flexibly comparing words in the sequences with an external object, instead of between themselves . The first algorithm infers single motifs, the second a combination of two motifs separated from one another by strictly defined, sterically constrained distances . Besides independently finding motifs known to be present in other bacteria, such as the Shine-Dalgarno sequence and the TATA-box, this approach suggests the existence in H . pylori of a new, combined motif, TTAAGC, followed optimally 21 bp downstream by TATAAT . Between these two motifs, there is in some cases another, TTTTAA or, less frequently, a repetition of TTAAGC separated optimally from the TATA-box by 12 bp . The combined motif TTAAGCx(21+/-2)TATAAT is present with no errors immediately upstream from the only two copies of the ribosomal 23 S-5 S RNA genes in H . pylori, and with one error upstream from the only two copies of the ribosomal 16 S RNA genes . The operons of both ribosomal RNA molecules are strongly expressed, representing an encouraging sign of the pertinence of the motifs found by the algorithms . In 25 cases out of a possible 30, the combined motif is found with no more than three substitutions immediately upstream from ribosomal proteins, or operons containing a ribosomal protein . This is roughly the same frequency of occurrence as for TTGACAx(15-19)TATAAT (with the same maximum number of substitutions allowed) described as being the sigma(70 )promoter sequence consensus in Bacillus subtilis and Escherichia coli . The frequency of occurrence of the new motif obtained, TTAAGCx(19-23)TATAAT, remains high when all protein genes in H . pylori are considered, as is the case for the TTGACAx(15-19)TATAAT motif in B . subtilis but not in E . coli . J Bacteriol, 2000 Apr, 182(7), 1987 - 94 Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores; Behravan J et al.; The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine . The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold . The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant . Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete . The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced . Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence . A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B . cereus, though somewhat less extreme and equally repairable by spore permeabilization . Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme . The B . subtilis operon is expressed solely during sporulation, and is sigma K-inducible . We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore. J Bacteriol, 2000 Apr, 182(7), 1942 - 8 The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA; Ross MA et al.; HBsu, the Bacillus subtilis homolog of the Escherichia coli HU proteins and the major chromosomal protein in vegetative cells of B . subtilis, is present at similar levels in vegetative cells and spores ( approximately 5 x 10(4) monomers/genome) . The level of HBsu in spores was unaffected by the presence or absence of the alpha/beta-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores . In developing forespores, HBsu colocalized with alpha/beta-type SASP on the nucleoid, suggesting that HBsu could modulate alpha/beta-type SASP-mediated properties of spore DNA . Indeed, in vitro studies showed that HBsu altered alpha/beta-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing alpha/beta-type SASP-mediated positive supercoiling, and greatly ameliorated the alpha/beta-type SASP-mediated increase in DNA persistence length . However, HBsu did not significantly interfere with the alpha/beta-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation . These data strongly support a role for HBsu in modulating the effects of alpha/beta-type SASP on the properties of DNA in the developing and dormant spore. J Bacteriol, 2000 Apr, 182(7), 1916 - 22 Purification and characterization of the DeoR repressor of Bacillus subtilis; Zeng X et al.; Transcription of the Bacillus subtilis dra-nupC-pdp operon is repressed by the DeoR repressor protein . The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step . Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer . Binding of DeoR to the operator DNA of the dra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay . An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative . In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM . The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate . DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them. J Bacteriol, 2000 Apr, 182(7), 1883 - 8 The Bacillus subtilis yabG gene is transcribed by SigK RNA polymerase during sporulation, and yabG mutant spores have altered coat protein composition; Takamatsu H et al.; The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes . Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T(4)) in wild-type cells and in a gerE36 (GerE(-)) mutant but not in spoIIAC (SigF(-)), spoIIGAB (SigE(-)), spoIIIG (SigG(-)), and spoIVCB (SigK(-)) mutants . The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase . Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme . The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores . On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions . We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively . The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy . These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B . subtilis. J Bacteriol, 2000 Apr, 182(7), 1828 - 33 Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat; Ozin AJ et al.; During endospore formation in Bacillus subtilis, over two dozen polypeptides are assembled into a multilayered structure known as the spore coat, which protects the cortex peptidoglycan (PG) and permits efficient germination . In the initial stages of coat assembly a protein known as CotE forms a ring around the forespore . A second morphogenetic protein, SpoVID, is required for maintenance of the CotE ring during the later stages, when most of proteins are assembled into the coat . Here, we report on a protein that appears to associate with SpoVID during the early stage of coat assembly . This protein, which we call SafA for SpoVID-associated factor A, is encoded by a locus previously known as yrbA . We confirmed the results of a previous study that showed safA mutant spores have defective coats which are missing several proteins . We have extended these studies with the finding that SafA and SpoVID were coimmunoprecipitated by anti-SafA or anti-SpoVID antiserum from whole-cell extracts 3 and 4 h after the onset of sporulation . Therefore, SafA may associate with SpoVID during the early stage of coat assembly . We used immunogold electron microscopy to localize SafA and found it in the cortex, near the interface with the coat in mature spores . SafA appears to have a modular design . The C-terminal region of SafA is similar to those of several inner spore coat proteins . The N-terminal region contains a sequence that is conserved among proteins that associate with the cell wall . This motif in the N-terminal region may target SafA to the PG-containing regions of the developing spore. J Bacteriol, 2000 Apr, 182(7), 1819 - 27 trp RNA-binding attenuation protein-5' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon; Du H et al.; The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mechanism . Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure . TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator . A third stem-loop structure that forms at the extreme 5' end of the trp leader transcript also plays a role in the transcription attenuation mechanism . The 5' stem-loop increases the affinity of TRAP for trp leader RNA . Results from RNA structure mapping experiments demonstrate that the 5' stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure . Footprinting results indicate that TRAP interacts with the 5' stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript . Expression studies with trpE'-'lacZ translational fusions demonstrate that TRAP-5' stem-loop interaction is required for proper regulation of the trp operon . 3' RNA boundary experiments indicate that the 5' structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association . Thus, TRAP-5' stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation. FEMS Microbiol Lett, 2000 Mar 15, 184(2), 285 - 9 Genetic analysis of SecA-SecY interaction required for spore development in Bacillus subtilis; Kobayashi H et al.; All spontaneous suppressor mutations obtained from a secA12 sporulation-defective mutant in Bacillus subtilis were localized in highly conserved membrane-spanning regions of SecY . The expression of early sporulation genes, kinA and spo0A encoding a histidine kinase and a transcription regulator for several sporulation genes, respectively, was restored in these suppressor mutants . These results indicate that the secretion function of translocase combined with Sec proteins is required for sporulation in B . subtilis. Enzyme Microb Technol, 2000 Mar 1, 26(5-6), 406 - 413 Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes* Yang J, Shih I I, Tzeng Y, Wang S. A protease-producing microorganism was isolated in northern Taiwan and identified as a strain of Bacillus subtilis . B . subtilis Y-108 thus isolated can be used for deproteinization of crustacean wastes in the preparation of chitin . For deproteinization tests, liquid phase fermentation of untreated shrimp shell, crab shell, and lobster shell wastes with this microbe showed protein removal of 88, 67, and 83%, respectively . In contrast, the protein removal of the acid treated wastes was 76, 62, 56%, respectively . The optimized conditions for protease production was found when the culture was shaken at 30 degrees C for 3 days in 100 ml of medium (phosphate buffer adjusted to pH 6.0) containing 7% shrimp and crab shell powder (SCSP), 0.1% K(2)HPO(4), 0.05% MgSO(4), 1.0% arabinose, 1.5% NaNO(3), and 1.5% CaCl(2) . Under such conditions, the protease of B . subtilis Y-108 attained the highest activity . It was as high as 20.2 U/ml . The protease was purified in a three-step procedure involving ammonium sulfate precipitation, DEAE-Sepharose CL-6B ionic exchange chromatography, and Sephacryl S-200 gel permeation chromatography . The enzyme was shown to have a relative molecular weight of 44 kDa by SDS polyacrylamide gel electrophoresis . The protease was most active at pH 8.0 and 50 degrees C with casein as substrate . The protease was activated by Mn(+2), Fe(+2), Zn(+2), Mg(+2), Co(+2), but inhibited completely by Hg(+2) . The protease was also inhibited by metal-chelating agent such as EDTA, sulfhydryl reagents as beta-mercaptoethanol, and by cysteine hydrochloride, histidine, glycerol . The EDTA was the most effective inhibitor that caused complete inhibition of protease . It was concluded that this enzyme is a metal-chelator-sensitive neutral protease. Mol Microbiol, 2000 Mar, 35(5), 1244 - 54 cis-acting regulatory sequences for antitermination in the transcript of the Bacillus subtilis hut operon and histidine-dependent binding of HutP to the transcript containing the regulatory sequences; Oda M et al.; The location of the cis-acting regulatory region for histidine-dependent antitermination of the Bacillus subtilis hut operon was determined . A secondary structure, whose sequences partially overlap with the downstream terminator, was found in the regulatory region of the hut transcript . Mutational analysis of the regulatory region showed that the secondary structure was required for histidine-dependent antitermination . An electrophoretic mobility-shift assay demonstrated that, in response to the presence of histidine and Mg2+, purified HutP bound hut RNA bearing putative secondary structure but not RNA lacking the potential to form putative secondary structure . Native gel electrophoresis showed that HutP existed as a hexamer . A filter-binding assay revealed that the concentration of histidine required for half-maximal binding of HutP to RNA was 3.1 mM and that the Kd for binding of HutP to RNA was approximately 0.56 microM in the presence of histidine . These results suggested that putative secondary structure in the regulatory region of hut mRNA could function as an antiterminator to inhibit the formation of the terminator structure and that HutP causes expression of the hut structural genes by binding to the putative antiterminator structure in response to the presence of histidine. Mol Microbiol, 2000 Mar, 35(5), 1110 - 9 Characterization of ylbF, a new gene involved in competence development and sporulation in Bacillus subtilis; Tortosa P et al.; We used mini Tn10 transposition to generate a library of Bacillus subtilis insertion mutants, with the goal of identifying and characterizing new competence genes . Two new regulatory genes were identified in our screen: ypuN (also known as rsiX, the anti-sigmaX factor) and ylbF . The disruption of ylbF leads to a dramatic decrease in the expression of comK, encoding the competence transcription factor . Our data show that ylbF positively controls ComK at a post-transcriptional level . It has been reported previously that ComK is degraded in vivo and in vitro by a multimeric protein complex composed of ClpP, ClpC and MecA . This proteolysis is inhibited by the ComS peptide . We show that both the overexpression of comS and the inactivation of mecA individually suffice to bypass the competence phenotype of the ylbF mutation . This mutation does not seem to alter the cellular concentrations of MecA or ClpP, and we propose a role for YlbF in modulating the translation, stability or activity of ComS . In addition to its role in competence, ylbF also appears to regulate sporulation by acting before stage II. Mutat Res, 2000 Feb 16, 465(1-2), 27 - 38 Rec effect of certain textile dyes in Bacillus subtilis; Sharma MK et al.; A large number of compounds are toxic, genotoxic, mutagenic, teratogenic and/or carcinogenic . The genotoxicity of four textile dyes commonly used in India namely Sulphur Red Brown 360 (SRB), Jade Green 2G (JG), Reactofix Turquoise Blue 5GFL (RTB) and Direct Scarlet 4BS (DS) was determined by Bacillus subtilis spore Rec assay, both in the presence and absence of metabolizing activation mixture (S9 mix) . Each dye was toxic at higher dose levels . A dose-dependent increase in the depth of growth inhibition zones was observed for all dyes . Zones of inhibition were usually clearer at higher doses of the dyes and with Rec- bacteria, but were translucent with Rec+ bacteria . SRB and DS were toxic to Rec+ and Rec- bacteria . JG was less genotoxic in the absence of S9 mix, however, its genotoxic potential increased in the presence of S9 mix . Reactofix T blue was more genotoxic in the absence of S9 mixture. Microbiology, 2000 Feb, 146 ( Pt 2), 263 - 71 Regulation of the transport system for C4-dicarboxylic acids in Bacillus subtilis; Asai K et al.; Transport systems for C4-dicarboxylates, such as malate, fumarate and succinate, are poorly understood in Gram-positive bacteria . The whole genome sequence of Bacillus subtilis revealed two genes, ydbE and ydbH, whose deduced products are highly homologous to binding proteins and transporters for C4-dicarboxylates in Gram-negative bacteria . Between ydbE and ydbH, genes ydbF and ydbG encoding a sensor-regulator pair, were located . Inactivation of each one of the ydbEFGH genes caused a deficiency in utilization of fumarate or succinate but not of malate . Expression of ydbH, encoding a putative transporter, was stimulated in a minimal salt medium containing 0-05% yeast extract but repressed by the addition of malate to the medium . Inactivation of the putative sensor-regulator pair or solute-binding protein, ydbFG or ydbE, caused complete loss of ydbH expression . The utilization of fumarate and stimulation of ydbH expression resumed in a ydbE null mutant in which ydbFGH were overproduced . Based on these observations, together with analysis of the sequence similarities of the deduced product, we conclude that YdbH is a C4-dicarboxylate-transport protein and its expression is regulated by a C4-dicarboxylate sensor kinase-regulator pair, YdbF and YdbG . Furthermore, it is suggested that YdbE does not directly participate in transport of C4-dicarboxylates, but plays a sensory role in the ydbF-ydbG two-component system, giving rise to specificity or increased efficiency to the system . Deletion analysis of the promoter region of ydbH revealed that a direct repeat sequence was required for the activation of ydbH expression . A catabolite-responsive element (CRE) was also found in the -10 region of the promoter, suggesting negative regulation by a CRE-binding protein. Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2656 - 61 A Bacillus subtilis operon containing genes of unknown function senses tRNATrp charging and regulates expression of the genes of tryptophan biosynthesis; Sarsero JP et al.; Strains of Bacillus subtilis containing a temperature-sensitive tryptophanyl-tRNA synthetase produce elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess tryptophan . This increase is because of reduced availability of the tryptophan-activated trp RNA-binding attenuation protein (TRAP) . To test the hypothesis that this elevated trp gene expression was caused by the overproduction of a transcript capable of binding and sequestering TRAP, a computer program was designed to search the B . subtilis genome sequence for additional potential TRAP binding sites . A region containing a stretch of (G/A)AG trinucleotide repeats, characteristic of a TRAP binding site, was identified in the yczA-ycbK operon . We show that transcriptional regulation of the yczA-ycbK operon is controlled by the T-box antitermination mechanism in response to the level of uncharged tRNA(Trp), and that the presence of a trpS1 mutant allele increases production of the yczA-ycbK transcript . Elevated yczA-ycbK expression was shown to activate transcription of the trp operon . Deletion of the yczA-ycbK operon abolishes the trpS1 effect on trp gene expression . The purpose of increasing expression of the genes of tryptophan biosynthesis in the trpS mutant would be to provide additional tryptophan to overcome the charged tRNA(Trp) deficiency . Therefore, in B . subtilis, as in Escherichia coli, transcription of the tryptophan biosynthetic genes is regulated in response to changes in the extent of charging of tRNA(Trp) as well as the availability of tryptophan. Biosci Biotechnol Biochem, 2000 Jan, 64(1), 181 - 3 Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2; Nagano H et al.; A collagenase in the culture supernatant of B . subtilis FS-2, isolated from traditional fish sauce, was purified . The enzyme had a molecular mass of about 125 kDa . It degraded gelatin with maximum activity at pH 9 and a temperature of 50 degrees C . The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60 degrees C and 65 degrees C for 30 min, respectively . Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-beta-mercaptoethanol and DFP . The protease from B . subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile. Biochim Biophys Acta, 2000 Mar 15, 1464(1), 18 - 26 Cold shock in Bacillus subtilis: different effects of benzyl alcohol and ethanol on the membrane organisation and cell adaptation; Konopasek I et al.; A temperature shift-down of Bacillus subtilis from 40 to 20 degrees C induces an 80 min growth lag . Benzyl alcohol reduced this period to 51 min, whereas ethanol prolonged it up to 102 min . The effect of the two alcohols on the membrane state was investigated by measuring the steady-state fluorescence anisotropy and analysing the lifetime distribution of diphenylhexatriene (DPH) and its polar derivative, TMA-DPH . As followed from the fluorescence anisotropy, the two alcohols exerted similar (fluidizing) effects on the cytoplasmic membranes of B . subtilis . However, benzyl alcohol significantly shortened the main DPH lifetime component and widened its distribution, while ethanol had no effect . The benzyl alcohol activity was interpreted in terms of an increased membrane hydration due to disordering of the membrane structure . Such an effect imitates the cold shock induced synthesis of unsaturated fatty acids in B . subtilis . The fatty acid analysis revealed that ethanol hindered this adaptive synthesis of fatty acids . At the same time, its effect on the membrane state (membrane order) was very low and could not substitute the physiological response as was the case with benzyl alcohol . It can thus be concluded that the adaptation of the membrane physical state contributes significantly to the cold shock response of B . subtilis. Biochemistry, 2000 Mar 14, 39(10), 2517 - 29 Porphyrin interactions with wild-type and mutant mouse ferrochelatase; Franco R et al.; Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe(2+) chelation into protoporphyrin IX . Resonance Raman and UV-vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin . The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively . Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure . On the basis of changes in the UV-vis absorption spectrum, addition of free-base or metalated porphyrins to wild-type ferrochelatase and H207N variant yields a 1:1 complex, most likely a monomeric protein-bound species at the active site . In contrast, the addition of porphyrin (either free base or metalated) to E287Q is substoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification . The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive lines and the vinyl vibrational mode in the resonance Raman spectra . Shifts in the resonance Raman lines of free-base and metalated porphyrins bound to the wild-type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle . However, the magnitude of the distortion cannot be determined without first defining the specific type of deformation . Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins . In fact, resonance Raman spectral decompositions indicate a homogeneous ruffled deformation for the nickel protoporphyrin bound to the wild-type ferrochelatase, whereas both planar and ruffled conformations are present for the H207N-bound porphyrin . Perhaps more revealing is the unusual resonance Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free-base protoporphyrin in solution . This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle . Taken together, these findings suggest that distortion occurs in murine ferrochelatase for some porphyrins, even without metal binding, which is apparently required for the yeast ferrochelatase. J Biotechnol, 2000 Feb 28, 78(1), 1 - 9 Isolation of a Bacillus sp . capable of transforming isoeugenol to vanillin; Shimoni E et al.; Natural aroma compounds are of major interest to the flavor and fragrance industry . Due to the limited sources for natural aromas, there is a growing interest in developing alternative sources for natural aroma compounds, and in particular aromatic aldehydes . In several microbial species aromatic aldehydes are detected as intermediates in the degradation pathway of phenylpropanoids . Thus, bioconversion of phenylpropanoids is one possible route for the production of these aroma compounds . The present work describes the isolation of microbial strains, capable of producing vanillin from isoeugenol . Bacterial strains isolated from soil, were screened for their ability to transform isoeugenol to vanillin . One of these strains, strain B2, was found to produce high amounts of vanillin when grown in the presence of isoeugenol, and was also capable of growing on isoeugenol as the sole carbon source . Based on its fatty acids profile, strain B2 was identified as a Bacillus subtilis sp . The bioconversion capabilities of strain B2 were tested in growing cultures and cell free extracts . In the presence of isoeugenol, a growing cultures of B . subtilis B2 produced 0.61 g l-1 vanillin (molar yield of 12.4%), whereas cell free extracts resulted in 0.9 g l-1 vanillin (molar yield of 14%). J Pharm Biomed Anal, 1999 Jun, 20(1-2), 217 - 24 Interlaboratory study comparing the microbiological potency of spiramycins I, II and III; Liu L et al.; An interlaboratory study has been performed to determine the relative potencies of spiramycins (SPMs) I, II and III by diffusion or/and turbidimetric assays with Bacillus subtilis or Staphylococcus aureus as the test organisms . Six laboratories from three countries participated . Experimental procedures were according to the European Pharmacopoeia, 3rd ed . The activity of SPM I is markedly higher than that of SPM II and III . By diffusion, the activities of SPM II and III relative to SPM I were found to be 57 and 72%, respectively . The interlaboratory relative standard deviations (RSD) varied from 3.6 to 16.3% . By turbidimetry, the activities of SPM II and III relative to SPM I were found to be 45 and 52%, respectively . The interlaboratory RSD values varied from 2.6 to 7.7% . The results of the study were analyzed according to the ISO 5725-2 guidelines to determine the repeatability, the between-laboratory and the reproducibility variances of both methods. Appl Environ Microbiol, 2000 Mar, 66(3), 1220 - 2 Transient growth requirement in Bacillus subtilis following the cessation of exponential growth; Sung HM et al.; During an investigation of the parameters controlling mutations in Bacillus subtilis we observed that this bacterium exhibits a transient growth requirement for two nonessential amino acids (glutamic acid and isoleucine) during a type of postexponential growth on a minimal medium. J Endod, 1975 Aug, 1(8), 273 - 5 The sporicidal activity of glass bead sterilizers; Windeler AS Jr et al.; Endodontic glass bead sterilizers were evaluated for sporicidal effectiveness . Small metal endodontic instruments contaminated with Bacillus subtilis spores were effectively sterilized when the instruments were exposed for 15 seconds at a temperature of 218 C . Nonmetal objects such as cotton pellets and paper points could not be sterilized because they charred and decomposed under the required temperature conditions. J Biol Chem, 2000 Mar 10, 275(10), 6712 - 6 NMR studies of Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli . Assignment of imino proton signals and determination of thermal stability; Yan X et al.; 15N-Labeled Bacillus subtilis tRNA(Trp) wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques . These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNA(Trp) molecules . Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs whose peaks were not clearly observed . Several base triplets found in the crystal structure of tRNA were observed in the present study as well . These studies also revealed two components of tRNA(Trp), which could not be separated by high pressure liquid chromatography, corresponding to s(4)U and U at position 8 of the tRNA(Trp), as indicated by two different sets of peaks for the TpsiC and D arms . The modification at position 8 altered the local conformation of the core region of the tRNA . Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to the s(4)U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA. J AOAC Int, 2000 Jan-Feb, 83(1), 145 - 55 Recovery and sporicidal resistance of various B . subtilis spore preparations on porcelain penicylinders compared with results from AOAC test methods; Danielson JW et al.; Sporicidal test results obtained from carriers inoculated with 4 types of defined Bacillus subtilis spore preparations were compared with the standard AOAC sporicidal test using soil extract nutrient broth (SENB) B . subtilis 19659 spores . Recoveries of spores inoculated on penicylinders from B . subtilis clean spores (washed and suspended in water) and B . subtilis 19659 spores inoculated from culture filtrates according to the AOAC method were compared . Spores were exposed to 6 concentrations (0.5-3.0% w/v) of glutaraldehyde in phosphate buffer (pH 7.5) for 10 h . Concentrations were established by titrimetry and liquid chromatography . Recoveries of surviving spores were determined for 3 types of clean B . subtilis var . niger preparations, one clean B . subtilis 19659 preparation, and the SENB B . subtilis 19659 filtrates . Spore carriers, inoculated by the standard AOAC protocol, resulted in as much as a 2-log number difference in runs 1-12, but not more than 0.5 log number for each clean spore preparation . The SENB spores varied most in resistance to glutaraldehyde, with no growth in recovery media from 3 different batches of 1, 1.5, and 2% glutaraldehyde . Separate batches of SENB preparations of B . subtilis 19659 were resistant and destroyed by 1.0% glutaraldehyde, with 3.98 and 6.0 log numbers of spores on penicylinders, respectively . Clean spore preparations of B . subtilis 19659 on porcelain penicylinders were more resistant to glutaraldehyde than were SENB spores . Nutrient agar/Mg/Ca and nutrient agar/Mg spore preparations of B . subtilis var . niger showed the most uniform resistance to glutaraldehyde . Spores with calcium added showed increased resistance to glutaraldehyde . B . subtilis 19659 spores from the Columbia broth spore preparation were the most resistant and were recovered after exposure to 3.0% glutaraldehyde. J Bacteriol, 2000 Mar, 182(6), 1764 - 7 Nitrate assimilation genes of the marine diazotrophic, filamentous cyanobacterium Trichodesmium sp . strain WH9601; Wang Q et al.; A 4.0-kb DNA fragment of Trichodesmium sp . strain WH9601 contained gene sequences encoding the nitrate reduction enzymes, nirA and narB . A third gene positioned between nirA and narB encodes a putative membrane protein with similarity to the nitrate permeases of Bacillus subtilis (NasA) and Emericella nidulans (CrnA) . The gene was shown to functionally complement a DeltanasA mutant of B . subtilis and was assigned the name napA (nitrate permease) . NapA was involved in both nitrate and nitrite uptake by the complemented B . subtilis cells . napA is distinct from the nrt genes that encode the nitrate transporter of freshwater cyanobacteria. J Bacteriol, 2000 Mar, 182(6), 1650 - 8 Penicillin-binding protein-related factor A is required for proper chromosome segregation in Bacillus subtilis; Pedersen LB et al.; Previous work has shown that the ponA gene, encoding penicillin-binding protein 1 (PBP1), is in a two-gene operon with prfA (PBP-related factor A) (also called recU), which encodes a putative 206-residue basic protein (pI = 10.1) with no significant sequence homology to proteins with known functions . Inactivation of prfA results in cells that grow slower and vary significantly in length relative to wild-type cells . We now show that prfA mutant cells have a defect in chromosome segregation resulting in the production of approximately 0.9 to 3% anucleate cells in prfA cultures grown at 30 or 37 degrees C in rich medium and that the lack of PrfA exacerbates the chromosome segregation defect in smc and spoOJ mutant cells . In addition, overexpression of prfA was found to be toxic for and cause nucleoid condensation in Escherichia coli. J Bacteriol, 2000 Mar, 182(6), 1592 - 9 Expression of ykdA, encoding a Bacillus subtilis homologue of HtrA, is heat shock inducible and negatively autoregulated; Noone D et al.; There are three members of the HtrA family of serine proteases, YkdA, YvtA, and YyxA, encoded in the chromosome of Bacillus subtilis . In this study, we report on the promoter structure and regulation of ykdA expression . The ykdA gene is heat inducible, exhibiting a biphasic pattern of expression during a 60-min interval after heat shock . Increased expression after heat shock occurs at the transcriptional level . The heat-shock-inducible promoter has a single mismatch with a SigA-type -10 motif, but does not exhibit similarity to a SigA -35 region . There are six octamer repeats with a consensus TTTTCACA positioned at, and upstream of, the normal position of a -35 region . While repeats V and VI appear dispensable, repeat IV is essential for normal thermoinducible expression . This promoter structure is also found in the control region of yvtA, encoding a second member of this family of proteases . Expression of ykdA is negatively autoregulated both during the growth cycle and during heat shock . Our evidence suggests that YkdA protease activity is not required for this form of regulation . Null mutants of ykdA display increased tolerance to heat and are 80-fold more resistant to 10 mM hydrogen peroxide than wild-type cells . However, ykdA expression is not induced by hydrogen peroxide . These results indicate that the regulon to which YkdA belongs is linked to the oxidative stress response in B . subtilis. J Bacteriol, 2000 Mar, 182(6), 1499 - 506 A novel spore peptidoglycan hydrolase of Bacillus cereus: biochemical characterization and nucleotide sequence of the corresponding gene, sleL; Chen Y et al.; The exudate of germinated spores of B . cereus IFO 13597 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme {CFLE}), in addition to a previously characterized germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme {SCLE}) (R . Moriyama, S . Kudoh, S . Miyata, S . Nonobe, A . Hattori, and S . Makino, J . Bacteriol . 178:5330-5332, 1996) . CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam . This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination . CFLE was purified in an active form and identified as a 48-kDa protein which functions as an N-acetylglucosaminidase . Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer . A gene encoding the enzyme, sleL, was cloned in Escherichia coli, and the nucleotide sequence was determined . The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136 . The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins . Inspection of the data banks showed no similarity of CFLE with N-acetylglucosaminidases found so far, suggesting that CFLE is a novel type of N-acetylglucosaminidase . The B . subtilis genome sequence contains genes, yaaH and ydhD, which encode putative proteins showing similarity to SleL. Mol Microbiol, 2000 Feb, 35(4), 800 - 11 CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes; Nair S et al.; Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens . In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth . The first gene of the clpC operon of L . monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes . An L . monocytogenes ctsR-deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42 degrees C), but its level of virulence in the mouse was not affected . The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response . Regulation of the L . monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B . subtilis as a host . The L . monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B . subtilis . The purified CtsR protein of L . monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting . Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B . subtilis . This is the first description of a stress response regulatory gene in a pathogen. J Endod, 1999 Jul, 25(7), 498 - 501 Rapid decontamination of gutta-percha cones with sodium hypochlorite; Cardoso CL et al.; Gutta-percha cones are now widely used to fill root canals . Because they cannot be sterilized by conventional autoclaving or in a hot-air oven, gutta-percha cones require rapid chairside decontamination before use to maintain the aseptic chain, an essential factor in successful endodontic therapy . The purpose of this study was to compare the effectiveness of different concentrations of sodium hypochlorite (0.25% to 4%) in sterilizing gutta-percha cones artificially contaminated with Staphylococcus aureus and Escherichia coli strains, and Bacillus subtilis spores . After 1 min of treatment, the solutions tested showed bactericidal and sporicidal effects at concentrations of 0.25% and 1%, respectively . At a concentration of 0.25%, the solutions tested were effective in destroying spores after 5 min of exposure . Based on this study, treatment of the cones for 1 min with 1% sodium hypochlorite (Milton's solution) or for 5 min with Dakin's liquid (0.5% sodium hypochlorite) is recommended. Protein Expr Purif, 2000 Mar, 18(2), 242 - 8 Staphylococcal protein A as a fusion partner directs secretion of the e1alpha and e1beta subunits of pea mitochondrial pyruvate dehydrogenase by Bacillus subtilis; Moreno JI et al.; Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1alpha and E1beta subunits of Pisum sativum mitochondrial pyruvate dehydrogenase from Bacillus subtilis . These proteins were not exported when the signal peptide from levansucrase (SacBSP) was fused to their N-termini . Both SacBSP-E1alpha and SacBSP-E1beta fusion proteins were insoluble in the cytoplasm . However, when the SPA open-reading frame was inserted between SacBSP and E1alpha or E1beta, corresponding fusion proteins were secreted from the cells . The first (E) IgG-binding domain of SPA was sufficient to direct low level secretion of both fusion proteins (SacBSP-E-E1alpha and SacBSP-E-E1beta) . Adding the second (D) IgG-binding domain improved extracellular protein yields 3- to 4-fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins . All constructs were based on the pUB110-derived multicopy plasmid pWB705 . Separate B . subtilis strains transformed with SacBSP-E-E1alpha-His(6) or SacBSP-E1beta were cocultivated in the presence of Ni-NTA agarose . The native pyruvate dehydrogenase alpha2beta2 structure was bound to the affinity matrix, demonstrating assembly after secretion . The use of SPA as a fusion partner during expression of heterologous proteins by B . subtilis provides the basis of a versatile system that can be used to study both secretion and protein:protein interactions . Dermatology, 2000, 200(1), 1 - 5 Personal UV dosimetry by Bacillus subtilis spore films; Moehrle M et al.; BACKGROUND: Ultraviolet radiation (UVR) is known to be the most important risk factor for melanoma and non-melanoma skin cancers . Until today it has been impossible to measure reliably UVR in the frame of epidemiological studies . The recent development of a spore film containing spores of Bacillus subtilis resulted in a new method of UV measurement by personal dosimetry . METHODS: The practical application of dosimeters was tested in 18 study persons under different circumstances of UV exposure and in 4 different geographical regions . RESULTS: Eleven children carried dosimeters on their shoulders for 1 day, playing in- and outdoors on a sunny day in summertime . Their whole-day values ranged from 0.1 to 1.5 minimal erythema doses (MED) per day with a mean of 0.71 MED (+/-0.44) . Four lifeguards in a public swimming-pool carried dosimeters on their shoulders for 11 days and received UV exposures ranging from 3.6 to 9.5 MED (mean 5.9 +/- 1.88) . Three mountain guides with dosimeters attached to the lateral head in different mountain regions at 23 mountaineering activities received daily exposures of 4.44-17.07 MED (mean 11.9 +/- 3.8) . CONCLUSION: B . subtilis spore film dosimeters can be applied to different study persons including children and mountain guides under different climatic conditions . A broad range of UV exposures can reliably be measured with this method . Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2005 - 10 The crystal structure and mechanism of orotidine 5'-monophosphate decarboxylase; Appleby TC et al.; The crystal structure of Bacillus subtilis orotidine 5'-monophosphate (OMP) decarboxylase with bound uridine 5'-monophosphate has been determined by multiple wavelength anomalous diffraction phasing techniques and refined to an R-factor of 19.3% at 2.4 A resolution . OMP decarboxylase is a dimer of two identical subunits . Each monomer consists of a triosephosphate isomerase barrel and contains an active site that is located across one end of the barrel and near the dimer interface . For each active site, most of the residues are contributed by one monomer with a few residues contributed from the adjacent monomer . The most highly conserved residues are located in the active site and suggest a novel catalytic mechanism for decarboxylation that is different from any previously proposed OMP decarboxylase mechanism . The uridine 5'-monophosphate molecule is bound to the active site such that the phosphate group is most exposed and the C5-C6 edge of the pyrimidine base is most buried . In the proposed catalytic mechanism, the ground state of the substrate is destabilized by electrostatic repulsion between the carboxylate of the substrate and the carboxylate of Asp60 . This repulsion is reduced in the transition state by shifting negative charge from the carboxylate to C6 of the pyrimidine, which is close to the protonated amine of Lys62 . We propose that the decarboxylation of OMP proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys62 occur in a concerted reaction. Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 365 - 9 The metal dependence of Bacillus subtilis phytase; Kerovuo J et al.; The metal ion requirement of a Bacillus subtilis phytase has been studied . Removal of metal ions from the enzyme by EDTA resulted in complete inactivation . Circular dichroism spectroscopy was used to study the effect of metal ion removal on the protein conformation . The loss of enzymatic activity is most likely due to a conformational change, as the circular dichroism spectra of holoenzyme and metal-depleted enzyme were different . Metal-depleted enzyme was partially able to restore the active conformation when incubated in the presence of calcium . Only minor reactivation was detected with other divalent metal ions and their combinations . Based on the data we conclude that B . subtilis phytase requires calcium for active conformation . Calcium has also a strong stabilizing effect on the enzyme against thermal denaturation . However, the conformational change resulted by calcium depletion does not affect the protease susceptibility . J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 277 - 84 New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography; Lauer I et al.; Proteases are commonly used in the biscuit and cracker industry as processing aids . They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness . Commercial bacterial proteases are derived from Bacillus fermentation broth . As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities . The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties . Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step . Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields . The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2 . This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities . Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease . This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing . Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities . The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38 . pH and temperature optima were also determined on the fraction. J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 267 - 75 Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213; Steller S et al.; The purification of the multienzyme system producing the lipodecapeptide fengycin in Bacillus subtilis b213 was investigated . By gel filtration of a cell free extract of this organism three enzyme fractions were obtained from which five multifunctional components of fengycin synthetase were separated by high resolution anion-exchange FPLC procedures . These proteins were characterized by their thioester formation activities with 14C-labeled substrate amino acids and by N-terminal sequencing . Correlation of these data with the DNA sequences of the pps (fen) operons in three B . subtilis strains provided detailed knowledge on the structural and functional organization of fengycin synthetase. Nature, 2000 Feb 3, 403(6769), 540 - 4 Myoglobin-like aerotaxis transducers in Archaea and Bacteria; Hou S et al.; Haem-containing proteins such as haemoglobin and myoglobin play an essential role in oxygen transport and storage . Comparison of the amino-acid sequences of globins from Bacteria and Eukarya suggests that they share an early common ancestor, even though the proteins perform different functions in these two kingdoms . Until now, no members of the globin family have been found in the third kingdom, Archaea . Recent studies of biological signalling in the Bacteria and Eukarya have revealed a new class of haem-containing proteins that serve as sensors . Until now, no haem-based sensor has been described in the Archaea . Here we report the first myoglobin-like, haem-containing protein in the Archaea, and the first haem-based aerotactic transducer in the Bacteria (termed HemAT-Hs for the archaeon Halobacterium salinarum, and HemAT-Bs for Bacillus subtilis) . These proteins exhibit spectral properties similar to those of myoglobin and trigger aerotactic responses. Biochim Biophys Acta, 2000 Feb 15, 1463(2), 209 - 18 Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells; Shin SY et al.; A 20-residue hybrid peptide (CA(1-8)-MA(1-12): KWKLFKKIGIGKFLHSAKKF-NH(2)) incorporating 1-8 residues of cecropin A (CA) and 1-12 residues of magainin 2 (MA) has potent antibiotic activity without hemolytic activity . In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly of CA(1-8)-MA(1-12) (CA-MA) on antibiotic activity, CA-MA and its three analogues, CA-MA1, CA-MA2 and CA-MA3 were synthesized . The Gly-Ile-Gly sequence of CA-MA was deleted in CA-MA1 and replaced with Pro and Gly-Pro-Gly in CA-MA2 and CA-MA3, respectively . CA-MA1 and CA-MA3 caused a significant decrease in the bactericidal rate against Escherichia coli and Bacillus subtilis and the tumoricidal activity against four different tumor cells, and the PC/PS (4:1, w/w) vesicle-aggregating and disrupting activities . However, CA-MA2 showed a similar bactericidal rate and antitumor, vesicle-aggregating and disrupting activities, as compared with CA-MA . These results suggested that the flexibility or beta-turn induced by Gly-Ile-Gly or Pro in the central part of CA-MA may be important in the electrostatic interaction of the cationic short alpha-helical region in the N-terminus with the cell membrane surface and the hydrophobic interaction of amphipathic alpha-helical region in the C-terminus with the hydrophobic acyl chains in the cell membrane . CA-MA3 exhibited lower activity in antibacterial, antitumor, and vesicle-aggregating and disrupting activities than CA-MA and CA-MA2 . This result suggested that the excessive beta-turn structure by Gly-Pro-Gly in CA-MA3 seems to interrupt the ion channel/pore formation on the lipid bilayer . It was concluded that the appropriate flexibility or beta-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure. EMBO J, 2000 Feb 15, 19(4), 710 - 8 Compartmentalization of transcription and translation in Bacillus subtilis; Lewis PJ et al.; Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis . Unexpectedly, we found that RNAP resides principally within the nucleoid . Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division . This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid . Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes . We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains . At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis . These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus. FEMS Microbiol Lett, 2000 Feb 15, 183(2), 247 - 51 The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis deficiency of a CtaB-deficient mutant; Throne-Holst M et al.; Heme A is a prosthetic group in many respiratory oxidases . It is synthesised from heme B (protoheme IX) with heme O as an intermediate . In Bacillus subtilis two genes required for heme A synthesis, ctaA and ctaB, have been identified . CtaB is the heme O synthase and CtaA is involved in the conversion of heme O to heme A . A ctaB paralogue, yjdK, has been identified through the B . subtilis genome sequencing project . In this study we show that when carried on a low copy number plasmid, the yjdK gene can complement a ctaB deletion mutant with respect to heme A synthesis . Our results indicate that YjdK has heme O synthase activity . We therefore suggest that yjdK be renamed as ctaO. Mol Microbiol, 2000 Feb, 35(3), 686 - 96 A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes; Simon J et al.; Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor . Two forms of nitrite reductase were isolated from the membrane fraction of W . succinogenes . One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase . The other form consisted of NrfA and a 22 kDa polypeptide (NrfH) . Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol . Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane . It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W . succinogenes nitrite reductase is mediated by NrfH . The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes . The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues) . NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli . NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family . The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis . The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts . The residual N-terminal part of NrfI resembles Ccs1 proteins . The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA . The properties of three deletion mutants of W . succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied . Mutants DeltanrfAIJ and DeltanrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant DeltanrfJ showed wild-type properties . The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation. Mol Microbiol, 2000 Feb, 35(3), 612 - 22 The putative DNA translocase SpoIIIE is required for sporulation of the symmetrically dividing coccal species Sporosarcina ureae; Chary VK et al.; The spoIIIE gene of Sporosarcina ureae encodes a 780-residue protein, showing 58% identity to the SpoIIIE protein of Bacillus subtilis, which is thought to be a DNA translocase . Expression of the S . ureae spoIIIE gene is able to restore sporulation in a B . subtilis spoIIIE mutant . Inactivation of the S . ureae spoIIIE gene blocks sporulation of S . ureae at stage III . Within the limits of detection, the sporulation division in S . ureae shows the same symmetry, or near symmetry, as the vegetative division (in contrast to the highly asymmetric location of the sporulation division for B . subtilis), and so it is inferred that SpoIIIE facilitates chromosome partitioning during sporulation, even when the division is not grossly asymmetric . It is suggested that chromosome partitioning lags behind division during sporulation but not during vegetative growth. Genes Cells, 2000 Feb, 5(2), 79 - 88 Temporal and selective association of multiple sigma factors with RNA polymerase during sporulation in Bacillus subtilis; Fujita M; BACKGROUND: During sporulation in Bacillus subtilis, an asymmetric division produces two cells, a forespore and mother cell, with which follow different developmental paths . The highly ordered programme of temporal and spatial gene activation during sporulation is governed by the principal RNA polymerase holoenzyme (EsigmaA) and alternative holoenzyme forms containing the developmental sigma factors sigmaH, sigmaF, sigmaE, sigmaG and sigmaK, which appear successively during development . The control mechanism(s) of temporal and selective association of multiple sigma factors with core RNA polymerase is unclear . As a first step to addressing these issues, this report quantifies the amount of each subunit of RNA polymerase that is present in the sporangium during sporulation, and analyses in vitro the relative affinities of each sigma subunit for core RNA polymerase . RESULTS: Using quantitative immunoblot analysis, the amounts of EsigmaA, EsigmaH, EsigmaE and EsigmaK in relation to the total amount of RNA polymerase at appropriate time-points were found to be 15%, 1%, 6% and 2%, respectively . Therefore, the core RNA polymerase is predicted to be in excess . The level of core RNA polymerase and sigmaA remained constant during the transition from vegetative growth to sporulation, whereas the sporulation-specific sigma factors appeared successively, in the order sigmaH, sigmaE and sigmaK . Competition experiments between sigma factors in an in vitro transcription system revealed the dominance of sigmaA over sigmaH and sigmaE for open promoter complex formation . These results are inconsistent with the idea that late appearing sigma factors can displace earlier appearing sigmas from the core enzyme . CONCLUSIONS: As the core RNA polymerase is in excess, the results suggest that successive sigma factors can bind to core RNA polymerase without having to displace earlier appearing sigma factors . Thus, the programme of gene expression during sporulation might not require mechanisms for the substitution of one sigma factor by another on the core RNA polymerase. Eur J Biochem, 2000 Feb, 267(4), 1230 - 8 Alkaline phosphatase from the Antarctic strain TAB5 . Properties and psychrophilic adaptations; Rina M et al.; The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined . A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified . The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources . Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology-based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations . The AP gene was expressed in E . coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined . The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity . The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols. J Bacteriol, 2000 Mar, 182(5), 1452 - 6 Stress triggers a process that limits activation of the Bacillus subtilis stress transcription factor sigma(B); Scott JM et al.; Stress-induced activation of the Bacillus subtilis transcription factor sigma(B) is transitory . To determine whether the process that limits sigma(B) activation is itself triggered by stress, B . subtilis strains in which the stress pathway was artificially activated by the induced expression of a positive regulatory protein (RsbT) were exposed to ethanol stress and were monitored for the persistence of sigma(B) activity . Without ethanol treatment, the induced cultures displayed continuously high sigma(B) activity . Ethanol treatment restricted ongoing sigma(B) activity, but only in strains with intact rsbX and -S genes . The loss of other gene products (RsbR and Obg) known to participate in the stress activation pathway had little influence in blocking the ethanol effect . The data argue that stress upregulates the activity of the RsbX-S regulatory pair to restrict sigma(B) induction following stress. J Bacteriol, 2000 Mar, 182(5), 1448 - 51 Utilization of subsidiary chromosomal replication terminators in Bacillus subtilis; Griffiths AA et al.; The Bacillus subtilis merodiploid strain GSY1127 contains a large nontandem duplication of a portion of its chromosome within its left (anticlockwise) replication segment . This causes displacement of the replication terminus region to a noticeably asymmetric location relative to oriC . The utilization of the subsidiary replication terminators, TerIII and TerV, in the merodiploid strain has been compared with that in B . subtilis 168 . It is shown that TerIII is utilized to a significant extent in GSY1127 and that TerV is used only marginally at the most . Neither of these terminators is used to a measurable extent in the 168 strain . It is concluded that TerIII and TerV do indeed function as backups to the major terminator TerI, as has been generally thought . It is further concluded that, in the 168 strain, the vast majority of clockwise forks are arrested at the highly efficient TerI terminator, with fork fusion between the approaching forks occurring frequently while the clockwise fork is stationary at TerI. J Bacteriol, 2000 Mar, 182(5), 1313 - 20 Partitioning of the linear chromosome during sporulation of Streptomyces coelicolor A3(2) involves an oriC-linked parAB locus; Kim HJ et al.; Candidate partitioning genes (parA and parB) for the linear chromosome of Streptomyces coelicolor were identified by DNA sequencing in a series of seven genes located between rnpA and trxA near the chromosomal replication origin . The most likely translation start point of parB overlapped the parA stop codon, suggestive of coregulation, and transcription analysis suggested that the two genes formed an operon . Deletion of part of parB had no effect on the growth or appearance of colonies but caused a deficiency in DNA partitioning during the multiple septation events involved in converting aerial hyphae into long chains of spores . At least 13% of spore compartments failed to inherit the normal DNA allocation . The same phenotype was obtained with a deletion removing a segment of DNA from both parA and parB . Reinforcing the idea of a special role for the par locus during sporulation, the stronger of two parAB promoters was greatly upregulated at about the time when sporulation septation was maximal in colonies . Three copies of a 14-bp inverted repeat (GTTTCACGTGAAAC) were found in or near the parAB genes, and at least 12 more identical copies were identified within 100 kb of oriC from the growing genome sequence database . Only one perfect copy of the 14-bp sequence was present in approximately 5 Mb of sequence available from the rest of the genome . The 14-bp sequence was similar to sequences identified as binding sites for Spo0J, a ParB homologue from Bacillus subtilis believed to be important for DNA partitioning (D . C.-H . Lin and A . D . Grossman, Cell 92:675-685, 1998) . One of these sites encompassed the transcription start point of the stronger parA promoter. J Bacteriol, 2000 Mar, 182(5), 1226 - 31 Expression of a new operon from Bacillus subtilis, ykzB-ykoL, under the control of the TnrA and PhoP-phoR global regulators; Robichon D et al.; The ykzB and ykoL genes encode two peptides, of 51 and 60 amino acids, the functions of which are unknown . The ykzB and tnrA genes are contiguous and transcribed divergently . Expression of ykzB and ykoL is induced by glutamate and is under the control of the TnrA global regulator of nitrogen utilization . TnrA regulated its own synthesis in glutamate minimal medium . Two DNA sequences (TnrAB1 and TnrAB2) homologous to the TnrA binding site are present in the region between tnrA and ykzB . Deletion mapping indicated that the TnrAB2 binding site was involved in activation of the ykzB promoter . In addition, transcription of tnrA depends on the presence of the TnrAB1 binding site . The ykzB and ykoL genes are probably in the same transcriptional unit . A single promoter involved in transcription in the presence of glutamate was mapped by primer extension . ykoL expression was induced by phosphate limitation and depended on the PhoP-PhoR two-component regulatory system . Its promoter was mapped to the region between ykoL and ykzB . Four boxes similar to the PhoP binding site are present upstream from the ykoL promoter . These boxes are probably recognized by PhoP approximately P during the activation of transcription in phosphate limitation conditions. Chin J Biotechnol, 1999, 15(1), 15 - 21 Purification and properties of genetic expressing product of thermostable protease from Bacillus stearothermophilus HY-69; Sun C et al.; The thermostable metal protease gene from Bacillus stearothermophilus HY-69 had been cloned and expressed in Bacillus subtilis MI113 . The genetic expressing product of the enzyme was purified by CM-cellulose chromatography . The product shows homogeneity on PAGE . Its molecular weight is 27,000 +/- 1000 by SDS-PAGE and Sephadex G100 filtration, respectively . The alpha-helix content of the protease is estimated to be about 66%, the beta-turn about 28%, the random coil about 6%, but not beta-sheet, calculated from the circular dichroism data . The optimal temperature of the enzyme was 70 degrees C . When the enzyme was denatured in 3 mol/L of Gdn--HCl in phosphate buffer pH6.0 for 20 min, it remained about 40% of original activity . It shows that it is rather resistant to heat and Gdn-HCl denaturation . Its conformational variety coursed by Gdn-HCl was investigated by the far UV circular dichroism and fluorescence spectra . The results show that the enzyme has more compact conformation and internal hydrophobility. Nucleic Acids Res, 2000 Mar 1, 28(5), 1206 - 10 Evaluation and characterization of catabolite-responsive elements (cre) of Bacillus subtilis; Miwa Y et al.; A global mechanism of catabolite repression of the genus Bacillus comprises negative regulation exerted through the binding of the CcpA protein to the catabolite-responsive elements (cres) of the target genes . We searched for cre sequences in the Bacillus subtilis genome using a query sequence, WTGNAANCGNWNNCW (N and W stand for any base and A or T, respectively), picking out 126 putative and known cre sequences . To examine their cre function, we integrated spac promoter (P spac )-cre-lacZ fusions into the amyE locus . Examination of catabolite repression of beta-galactosidase synthesis in the integrants led us to the following conclusions: (i) lower mismatching of cre sequences to the query sequence is required for their function; (ii) although cre sequences are partially palindromic, low mismatching in the same direction as that of transcription of the target genes is more critical for their function than that in the inverse direction; and (iii) yet, a more palindromic nature of cre sequences is desirable for a better function . Furthermore, the alignment of 22 cre s that function in vivo implicated a consensus sequence, WWTGNAARCGNWWWCAWW (R stands for G or A) . Interestingly, in the case where cre sequences are located in the protein-coding regions of the target genes, their conserved bases are preferentially the third bases of codons where base degeneracy is allowed. Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2236 - 9 Antibacterial activities of cryptotanshinone and dihydrotanshinone I from a medicinal herb, Salvia miltiorrhiza Bunge; Lee DS et al.; Cryptotanshinone and dihydrotanshinone I, constituents of a medicinal plant, Salvia miltiorrhiza Bunge, had antibacterial activity against a broad range of Gram positive bacteria . These compounds generated superoxide radicals in Bacillus subtilis lysates . A recombination-deficient mutant strain of B . subtilis was 2- to 8-fold more sensitive than a wild strain, and this hypersensitivity was reduced in the presence of dithiothreitol as an antioxidant . DNA, RNA, and protein syntheses in B . subtilis were non-selectively inhibited by these compounds . These results suggest that superoxide radicals are important in the antibacterial actions of the agents. Biotechnol Prog, 2000 Jan-Feb, 16(1), 92 - 101 Modeling conductive heat transfer and process uniformity during batch high-pressure processing of foods; Denys S et al.; A numerical model for predicting conductive heat transfer during batch high hydrostatic pressure (HHP) processing of foods was developed and tested for a food simulator (agar gel) . For a comprehensive evaluation of the proposed method, both "conventional" HHP processes, HHP processes with gradual, step-by-step pressure buildup and pressure release, and pressure cycling HHP processes were included . In all cases, good agreement between experimental and predicted temperature profiles was observed . The model provides a very useful tool to evaluate batch HHP processes in terms of uniformity of any heat- and/or pressure-related effect . This is illustrated for inactivation of Bacillus subtilis alpha-amylase, an enzymatic model system with known pressure-temperature degradation kinetics. J Biol Chem, 2000 Feb 11, 275(6), 4519 - 24 Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis; Yakhnin AV et al.; The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP) . Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA . The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP . We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation . We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions . Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity . Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations . However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP . One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function . Another mutant protein exhibited tryptophan-independent RNA binding activity . We also found that trp leader RNA increases the affinity of TRAP for tryptophan. Curr Biol, 2000 Jan 13, 10(1), R19 - 21 RNA-binding proteins: TRAPping RNA bases; Muto Y et al.; In Bacillus subtilis, tryptophan biosynthesis is regulated by a mechanism called attenuation . The new crystal structure of the 'trp RNA binding attenuation protein', TRAP, in complex with RNA has provided new structural insights into how proteins can bind RNA to regulate transcription and translation. Microbiology, 2000 Jan, 146 ( Pt 1), 97 - 105 Changes in protein synthesis during the adaptation of Bacillus subtilis to anaerobic growth conditions; Marino M et al.; After a shift of Bacillus subtilis from aerobic to anaerobic growth conditions, nitrate ammonification and various fermentative processes replace oxygen-dependent respiration . Cell-free extracts prepared from wild-type B . subtilis and from mutants of the regulatory loci fnr and resDE grown under aerobic and various anaerobic conditions were compared by two-dimensional gel electrophoresis . Proteins involved in the adaptation process were identified by their N-terminal sequence . Induction of cytoplasmic lactate dehydrogenase (LctE) synthesis under anaerobic fermentative conditions was dependent on fnr and resDE . Anaerobic nitrate repression of LctE formation required fnr-mediated expression of narGHJI, encoding respiratory nitrate reductase . Anaerobic induction of the flavohaemoglobin Hmp required resDE and nitrite . The general anaerobic induction of ywfl, encoding a protein of unknown function, was modulated by resDE and fnr . The ywfl gene shares its upstream region with the pta gene, encoding the fermentative enzyme acetyl-CoA:orthophosphate acetyltransferase . Anaerobic repression of the synthesis of a potential membrane-associated NADH dehydrogenase (YjlD, Ndh), and anaerobic induction of fructose-1,6-bisphosphate aldolase (FbaA) and dehydrolipoamide dehydrogenase (PhdD, Lpd) formation, did not require fnr or resDE participation . Synthesis of glycerol kinase (GlpK) was decreased under anaerobic conditions . Finally, the effect of anaerobic stress induced by the immediate shift from aerobic to strictly anaerobic conditions was analysed . The induction of various systems for the utilization of alternative carbon sources such as inositol (IoIA, IoIG, IoIH, IoII), melibiose (MeIA) and 6-phospho-alpha-glucosides (GIvA) indicated a catabolite-response-like stress reaction. Microbiology, 2000 Jan, 146 ( Pt 1), 77 - 88 Chaperone-like activities of the CsaA protein of Bacillus subtilis; Muller JP et al.; The growth and protein export defects of Escherichia coli secA51(Ts) strains can be suppressed by the CsaA protein of Bacillus subtilis . The present studies indicate that this effect can be attributed to chaperone-like activities of CsaA . First, CsaA stimulated protein export in secB, groES and dnaJ mutant strains of E . coli . Second, CsaA suppressed the growth defects of dnaK, dnaJ and grpE mutants of E . coli . Third, and most importantly, CsaA exhibited chaperone-like properties by stimulating the reactivation of heat-denatured firefly luciferase in groEL, groES, dnaK and grpE mutant strains of E . coli, and by preventing the aggregation of heat-denatured luciferase in vitro . Thus, it seems that CsaA suppresses the growth and secretion defects of E . coli secA(Ts) strains either by improving the translocation competence of exported pre-proteins, thereby making them better substrates for mutant SecA proteins, or by stimulating the translocation activity of mutant SecA proteins. Microbiology, 2000 Jan, 146 ( Pt 1), 65 - 75 Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study; Hirose I et al.; To analyse the proteome of Bacillus subtilis extracellular proteins, extracellular protein samples were prepared from culture media (minimal medium containing 0.4% glucose) of parental B . subtilis 168, a secA-temperature sensitive mutant and an ffh conditional mutant, and examined by two-dimensional gel electrophoresis . Approximately 100 to 110 spots were visualized in a gel of B . subtilis 168 extracellular proteins . Over 90% and 80% of these disappeared in the absence of SecA and Ffh, respectively . Thirty-eight obvious spots on the gel of the B . subtilis 168 preparation were selected and compared with spots obtained under SecA- or Ffh-deficient conditions . The appearance of 36 of these 38 spots depended on SecA and Ffh . Nineteen additional extracellular proteins were detected in cultures maintained in cellobiose, maltose and soluble starch . Among 23 proteins of which the N-terminal amino acid sequences were determined, 17 were extracellular proteins having signal peptides in their precursor form . Two membrane proteins, Yfnl and YflE, were cleaved behind 226Ala-Tyr-Ala228 and 213Ala-Leu-Ala215, respectively, and of which products seemed to be liberated into the culture medium . The production of Yfnl and YflE were also dependent on SecA and Ffh . These results indicate that most extracellular proteins target to and translocate across the cytoplasmic membrane by co-operation between the signal-recognition particle and Sec protein-secretion pathways . In contrast, a spot for Hag appeared independent from SecA and Ffh . Intracellular proteins Gap, SodA and KatA were identified in the extracellular protein samples . On the basis of these results and computer searches, it was predicted that B . subtilis produces 150 to 180 proteins extracellularly. Microbiology, 2000 Jan, 146 ( Pt 1), 57 - 64 Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB; Boland FM et al.; The role of the sleB gene of Bacillus subtilis, which encodes a putative spore-cortex-lytic enzyme, and the downstream ypeB gene were investigated . Both SleB and YpeB were required for normal germination to occur . The corresponding mutants formed phase-bright, heat-resistant spores with no apparent defects in dormancy . However, mutant spore suspensions lost optical density slower than the wild-type and spores were phase-grey even 12 h after the triggering of germination . Since the loss of heat resistance and release of dipicolinic acid was similar to the wild-type, these mutants were blocked in the later stages of germination . The mutants were nevertheless capable of outgrowth on rich agar to form colonies, indicating that other spore components can compensate for their function sufficiently to allow outgrowth . The expression and regulation of the operon was examined using a lacZ transcriptional fusion . Expression of the operon began 2 h after the onset of sporulation and was under the control of RNA polymerase containing the forespore-specific sigma factor, sigmaG . The application of reverse phase HPLC revealed that the mutants do not have any structural defect in the dormant spore cortex and therefore these genes are not required for normal spore-cortex synthesis . The analysis of peptidoglycan dynamics during germination showed, however, that the cortex was only partially hydrolysed in both mutants . This analysis also revealed that the likely hydrolytic bond specificity of SleB is likely to be that of a lytic transglycosylase. Microbiology, 2000 Jan, 146 ( Pt 1), 49 - 55 Effects of hydration on molecular mobility in phase-bright Bacillus subtilis spores; Leuschner RG et al.; The molecular mobility of 31P and 13C in dormant Bacillus subtilis spore samples with different water concentrations was investigated by high-resolution solid-state NMR . Lowest molecular mobility was observed in freeze-dried preparations . Rehydration to a 10% weight increase resulted in increases in molecular motions and addition of excess water furthered this effect . A spore slurry which had been freeze-dried displayed after addition of excess water similar NMR spectra to native wet preparations . Dipicolinic acid (DPA), which is mainly located in the core, was detected at all hydration levels in 13C cross-polarization magic angle spinning (CPMAS) but not in single-pulse magic angle spinning (SPMAS) spectra, indicating that hydration had no effect on its mobility . The molecular mobility of 31P, present mainly in core-specific components, was strongly dependent on hydration . This result suggests reversible water migration between inner spore compartments and the environment, whereas 13C spectra of DPA indicate that it is immobilized in a water-insoluble network in the core . Scanning transmission electron microscopy revealed that freeze-dried spores were significantly longer and narrower than fully hydrated spores and had a 3% smaller volume. J Mol Biol, 2000 Feb 11, 296(1), 117 - 32 Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly; Droge A et al.; The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7 . The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid . Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures . Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro . Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold . Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein . The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis . Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids . Predominant formation of T=7 procapsids requires presence of the portal protein . This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process . Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection . A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced . Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins . Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure . J Mol Biol, 2000 Feb 11, 296(1), 103 - 15 In vitro packaging of DNA of the Bacillus subtilis bacteriophage SPP1; Droge A et al.; In vitro packaging of bacteriophage SPP1 DNA into procapsids is described and the requirements of this process were determined . Combination of proheads with an extract supplying terminase, DNA and phage tails yielded up to 10(7 )viable phages per milliliter of in vitro reaction under optimized conditions . The presence of neutral polymers and polyamines had a concentration and type dependent effect in the packaging reaction . The terminase donor extract lost rapidly activity at 30 degrees C in contrast to the stability of the prohead donor extract . Maturation to infective virions was observed using both procapsids assembled in SPP1 infected cells and procapsid-like structures assembled in Escherichia coli that overexpressed the SPP1 prohead gene clusters . Neither a majority of aberrant capsid-related structures present in the latter material nor procapsids lacking the portal protein inhibited DNA packaging . Addition of purified portal protein reduced DNA packaging activity in vitro only at concentrations 20-fold higher than those found in the SPP1 infected cell . The SPP1 DNA packaged in vitro originated exclusively from the terminase donor extract . This packaging selectivity was not observed in vivo during mixed infections . The data are compatible with a model for processive headful DNA packaging in which terminase and DNA co-produced in the same cell are tightly associated and can effectively discriminate the portal vertex of DNA packaging-proficient proheads from aberrant structures, from portal-less procapsids, and from isolated portal protein . J Mol Biol, 2000 Jan 28, 295(4), 865 - 78 CcpC, a novel regulator of the LysR family required for glucose repression of the citB gene in Bacillus subtilis; Jourlin-Castelli C et al.; Synergistic carbon catabolite repression of the Bacillus subtilis aconitase (citB) gene by glucose and a source of 2-ketoglutarate is dependent on DNA sequences located upstream of the gene . Mutations in a dyad symmetry element centered at position -66 and in a repeat of the downstream arm of the dyad symmetry at position -27 cause derepressed citB expression . In this work, a protein able to bind to a DNA fragment containing these elements was purified and identified . This protein, named CcpC (Catabolite control protein C), shares sequence similarity with members of the LysR family of transcriptional regulators . In addition to binding to the citB promoter, CcpC bound to the promoter of the citZ gene, which encodes the cell's major citrate synthase and is subject to carbon catabolite repression . In a ccpC null mutant, expression of both citB and citZ was derepressed in glucose-glutamine minimal medium, indicating that CcpC is a negative regulator of citB and citZ gene expression . DNase I footprinting experiments showed that CcpC binds to two sites within the citB promoter region, corresponding to the dyad symmetry and -27 elements . In the presence of citrate, a putative inducer, only the dyad symmetry element was fully protected by CcpC . When the dyad symmetry element was mutated, CcpC was no longer able to bind to either the dyad symmetry or -27 elements . Repression of citB and citZ gene expression during anaerobiosis also proved to be mediated by CcpC . Z Naturforsch {C}, 1999 Nov, 54(11), 859 - 65 The structure of two fengycins from Bacillus subtilis S499; Schneider J et al.; The structures of the two fengycins, lipopeptides from Bacillus subtilis, were elucidated by spectroscopic methods and chemical degradation . They show a close structural relationship to the plipastatins from Bacillus cereus differing only in the stereochemistry of the Tyr residues. J Antibiot (Tokyo), 1999 Nov, 52(11), 960 - 70 Stresgenin B, an inhibitor of heat-induced heat shock protein gene expression, produced by Streptomyces sp . AS-9; Akagawa H et al.; Stresgenin B was isolated as an inhibitor of heat-induced heat shock protein (HSP) gene expression from a culture broth of Streptomyces sp . AS-9 by silica gel chromatography and HPLC . The molecular formula of the novel compound was determined as C11H13NO5 by high resolution FAB-MS analysis, and the structure was determined by UV, 1H NMR, 13C NMR, HMQC, HMBC, and NOESY spectra . Stresgenin B inhibited heat-induced luciferase reporter-gene expression directed by the human hsp70B promoter in Chinese hamster ovary (CHO) cells at concentrations lower than the concentrations for inhibition of dexamethasone-induced luciferase reporter-gene expression directed by the mouse mammary tumor virus (MMTV)-LTR promoter . The inhibition of heat-induced reporter gene expression was evident even when cells were exposed to stresgenin B only during heat stress treatment . Moreover, the compound inhibited heat-induced syntheses of hsp72/73, hsp90, and hsp110 and thereby suppressed the induction of thermotolerance . Stresgenin B showed moderate cytotoxic activities against several neoplastic cell lines and also showed antibacterial activities against Micrococcus luteus, Bacillus subtilis and Staphylococcus aureus strains. Anal Chem, 2000 Jan 1, 72(1), 119 - 27 Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and Fourier transform infrared spectroscopy; Goodacre R et al.; Thirty-six strains of aerobic endospore-forming bacteria confirmed by polyphasic taxonomic methods to belong