Bacillus subtilis

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 to Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis (including Bacillus niger and Bacillus globigii), Bacillus sphaericus, and Brevi laterosporus were grown axenically on nutrient agar, and vegetative and sporulated biomasses were analyzed by Curie-point pyrolysis mass spectrometry (PyMS) and diffuse reflectance-absorbance Fourier-transform infrared spectroscopy (FT-IR) . Chemometric methods based on rule induction and genetic programming were used to determine the physiological state (vegetative cells or spores) correctly, and these methods produced mathematical rules which could be simply interpreted in biochemical terms . For PyMS it was found that m/z 105 was characteristic and is a pyridine ketonium ion (C6H3ON+) obtained from the pyrolysis of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a substance found in spores but not in vegetative cells; this was confirmed using pyrolysis-gas chromatography/mass spectrometry . In addition, a pyridine ring vibration at 1447-1439 cm-1 from DPA was found to be highly characteristic of spores in FT-IR analysis . Thus, although the original data sets recorded hundreds of spectral variables from whole cells simultaneously, a simple biomarker can be used for the rapid and unequivocal detection of spores of these organisms.

J Nat Prod, 1999 Dec, 62(12), 1595 - 9
Antibacterial and antiandrogen flavonoids from Sophora flavescens; Kuroyanagi M et al.; Sixteen flavanones, three flavanonols, and four pterocarpans were isolated from the MeOH extract of the roots of Sophora flavescens . Twelve of these were new compounds, including eight prenylflavanones (1-8), one prenylflavanonol (9) and three novel pterocarpane derivatives (10-12) . Their structures were elucidated using NMR and mass spectral methods . Some of these compounds have irregular C10 prenyl moieties at C-8 of the flavanone skeleton . These compounds exhibited significant antibacterial activities against the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, S . epidermidis, and Propionibacterium acnes . They also exhibited antiandrogen activities.

Appl Environ Microbiol, 2000 Feb, 66(2), 620 - 6
Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation; Riesenman PJ et al.; Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer . The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances . In this study, spore coat-defective mutants of B . subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments . Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain . Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were . Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were . In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were . Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores . Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.

Appl Environ Microbiol, 2000 Feb, 66(2), 476 - 80
Enhancement of secretion and extracellular stability of staphylokinase in Bacillus subtilis by wprA gene disruption; Lee SJ et al.; Staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases . A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame . However, the amount of SAK secretion was marginal (45 mg/liter) . In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter) . In addition, the extracellular stability of mature SAK was dramatically enhanced . These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.

Radiat Environ Biophys, 1999 Dec, 38(4), 249 - 59
The track structures of ionizing particles and their application to radiation biophysics . II . Results for various organisms irradiated with protons, deuterons and alpha-particles; Briden PE et al.; Using knowledge of the track structure generated by ionizing particles together with details of the organisms being irradiated, the application of a new analytical method to two biophysical models to explain the inactivation of cells by radiation has been developed . It is shown that both models are equally successful in predicting experimental results and that good agreement is found with the data for single-strand phage, Bacillus subtilis spores, various strains of Escherichia coli, haploid and diploid yeast, and human diploid fibroblasts . The only significant discrepancy arose with T1-phage, for which a tentative explanation is offered . The differences in inherent radiosensitivity between organisms, after allowance is made for differences in target size, are attributed to differences in enzymatic repair systems and in the packing of the DNA.

Mol Microbiol, 2000 Jan, 35(2), 324 - 40
Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution; Loessner MJ et al.; A118 is a temperate phage isolated from Listeria monocytogenes . In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA . Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules . No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism . Partial denaturation mapping of DNA cross-linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development . Seventy-two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle-specific manner into at least three major transcriptional units . N-terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control-associated functions and proteins necessary for DNA recombination, modification and replication . Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules . We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site-specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

Mol Microbiol, 2000 Jan, 35(2), 299 - 311
Role of penicillin-binding protein PBP 2B in assembly and functioning of the division machinery of Bacillus subtilis; Daniel RA et al.; We have characterized the role of the penicillin-binding protein PBP 2B in cell division of Bacillus subtilis . We have shown that depletion of the protein results in an arrest in division, but that this arrest is slow, probably because the protein is relatively stable . PBP 2B-depleted filaments contained, at about their mid-points, structures resembling partially formed septa, into which most, if not all, of the division proteins had assembled . Although clearly deficient in wall material, membrane invagination seemed to continue, indicating that membrane and wall ingrowth can be uncoupled . At other potential division sites along the filaments, no visible ingrowths were observed, although FtsZ rings assembled at regular intervals . Thus, PBP 2B is apparently required for both the initiation of division and continued septal ingrowth . Immunofluorescence microscopy showed that the protein is recruited to the division site . The pattern of localization suggested that this recruitment occurs continually during septal ingrowth . During sporulation, PBP 2B was present transiently in the asymmetrical septum of sporulating cells, and its availability may play a role in the regulation of sporulation septation.

Biochemistry, 2000 Feb 1, 39(4), 727 - 35
Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis; Morandi P et al.; To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit . The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered . On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme . The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH . By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+) . During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed . Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E . coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum . Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 143 - 6
Effect of host bacteria genotype on spontaneous reversions of Bacillus subtilis bacteriophage phi29 sus17 nonsense codon; Fucik V et al.; Gene 17 of Bacillus subtilis bacteriophage Phi29 is an early gene playing a role in DNA replication . Its mutant sus17(112) carries the TAA nonsense triplet at the fifth codon of the gene . We isolated and sequenced 73 spontaneous revertants producing normal-size plaques on bacteria without an informational suppressor gene . In all revertants, the TAA triplet was changed by a one-base substitution and the sequences CAA, AAA, TTA, TAC and TAT were recovered at its place . The spectrum of these mutations was markedly influenced by the genotype of the bacteria in which the revertants arose . In agreement with the results described in Escherichia coli, the ratio of transversions to transitions (CAA being the only transition acceptable) was higher in strains harboring the functional allele recA(+) than in those with recA4 . Our results support the idea that also in the Gram-positive B . subtilis, the spectra of spontaneous mutations are specifically modified by an SOS function . It is assumed that the single-stranded DNA chains generated in the course of phage DNA replication might act as an inducing factor.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 9 - 14
Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis; Jan J et al.; The aprE gene of Bacillus subtilis encodes the major serine alkaline protease known as subtilisin . It is expressed during the transition state and transcribed by the sigma(A) form of the RNA polymerase (RNAP) . In this work, we characterized the regulatory region of the aprE gene (rraprE) from B . subtilis . By computer analysis and site-directed mutagenesis, we localized the aprE promoter sequence 7 bp upstream from its transcription initiation site (TIS) . We also characterized the static curvature properties of the rraprE DNA and found two different areas of DNA bending, within the first 400 bp upstream of its TIS . We postulate that these particular curved DNA regions could play a role in the interaction with some regulatory proteins and discuss possible implications related to aprE transcription regulation.

J Nat Prod, 2000 Jan, 63(1), 37 - 40
Isoprene biosynthesis in Bacillus subtilis via the methylerythritol phosphate pathway; Wagner WP et al.; Isoprene (2-methyl-1,3-butadiene), an abundant natural product of unknown function in plants, has recently been found to be one of the major volatiles formed by Bacillus subtilis . To understand the metabolic origins of isoprene in B . subtilis, we used (13)C- and (2)H-labeling methods with GC-MS analysis of released isoprene . The results indicate that, in this bacterium, isoprene is not formed by the mevalonate pathway or from catabolism of leucine, but, as in plant systems, it is a product of the methylerythritol phosphate pathway of isoprenoid synthesis . This work supports the idea that B . subtilis could be used as a microbial model for studying the biochemistry of isoprene formation.

J Bacteriol, 2000 Feb, 182(4), 1096 - 108
SpoIIB localizes to active sites of septal biogenesis and spatially regulates septal thinning during engulfment in bacillus subtilis; Perez AR et al.; A key step in the Bacillus subtilis spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum . We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy . We have found that spoIIB mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in spoIID, spoIIM, and spoIIP mutants . However, unlike the sporangia of those three mutants, spoIIB mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment . Electron micrographs showed that in spoIIB mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan . These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan . In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.

J Bacteriol, 2000 Feb, 182(4), 1046 - 52
Molecular analysis of the tagF gene, encoding CDP-Glycerol:Poly(glycerophosphate) glycerophosphotransferase of Staphylococcus epidermidis ATCC 14990; Fitzgerald SN et al.; Staphylococcus epidermidis ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of Bacillus subtilis 168 . The S . epidermidis tagF gene was cloned from genomic DNA and sequenced . When introduced on a plasmid vector into B . subtilis 1A486 carrying the conditionally lethal temperature-sensitive mutation tagF1 (rodC1), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature . This showed that the cloned S . epidermidis gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase . An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation . Unlike B . subtilis, where the tagF gene is part of the tagDEF operon, the tagF gene of S . epidermidis is not linked to any other tag genes . We attempted to disrupt the chromosomal tagF gene in S . epidermidis TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5' end of tagF on a similarly sized DNA fragment was able to integrate . This suggests that the tagF gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.

J Bacteriol, 2000 Feb, 182(4), 919 - 27
Amino acid transport and metabolism in mycobacteria: cloning, interruption, and characterization of an L-Arginine/gamma-aminobutyric acid permease in Mycobacterium bovis BCG; Seth A et al.; Genes encoding L-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host . In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter from Mycobacterium bovis BCG and the effects of its deletion on L-arginine transport and metabolism . The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the rocE gene of Bacillus subtilis, a putative arginine transporter . A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker . The mutant strain was complemented with the wild-type gene in single copy . Transport analysis of these strains was conducted using (14)C-labeled substrates . Greatly reduced uptake of L-arginine and gamma-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium . However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow), L-{(14)C}arginine uptake by the mutant but not the wild-type strain increased strongly . Exogenous L-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium . L-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM . Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.

J Bacteriol, 2000 Feb, 182(4), 898 - 904
Function of a principal Na(+)/H(+) antiporter, ShaA, is required for initiation of sporulation in Bacillus subtilis; Kosono S et al.; ShaA (sodium/hydrogen antiporter, previously termed YufT {or NtrA}), which is responsible for Na(+)/H(+) antiporter activity, is considered to be the major Na(+) excretion system in Bacillus subtilis . We found that a shaA-disrupted mutant of B . subtilis shows impaired sporulation but normal vegetative growth when the external Na(+) concentration was increased in a low range . In the shaA mutant, sigma(H)-dependent expression of spo0A (P(S)) and spoVG at an early stage of sporulation was sensitive to external NaCl . The level of sigma(H) protein was reduced by the addition of NaCl, while the expression of spo0H, which encodes sigma(H), was little affected, indicating that posttranscriptional control of sigma(H) rather than spo0H transcription is affected by the addition of NaCl in the shaA mutant . Since this mutant is considered to have a diminished ability to maintain a low internal Na(+) concentration, an increased level of internal Na(+) may affect posttranscriptional control of sigma(H) . Bypassing the phosphorelay by introducing the sof-1 mutation into this mutant did not restore spo0A (P(S)) expression, suggesting that disruption of shaA affects sigma(H) accumulation, but does not interfere with the phosphorylation and phosphotransfer reactions of the phosphorelay . These results suggest that ShaA plays a significant role at an early stage of sporulation and not only during vegetative growth . Our findings raise the possibility that fine control of cytoplasmic ion levels, including control of the internal Na(+) concentration, may be important for the progression of the sporulation process.

J Biol Chem, 2000 Jan 28, 275(4), 2472 - 8
Non-bilayer lipids stimulate the activity of the reconstituted bacterial protein translocase; van der Does C et al.; To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside . SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity . The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory . The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation . Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation . In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered . Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex . Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.

Tuber Lung Dis, 1998, 79(2), 107 - 9
Antibacterial spectra of drugs used for chemotherapy of mycobacterial infections; Oliva B et al.; The mechanism of action of many antimycobacterial agents is poorly understood . To obtain preliminary information on whether the targets for some of these drugs might also occur in other bacteria, the in vitro activities of selected agents against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were determined . Dapsone, p-aminosalicylic acid and thiacetazone failed to inhibit the above organisms (MIC values > 100 micrograms/ml) that may therefore lack targets for these drugs . Capreomycin, viomycin and clofazimine demonstrated activity against some of the organisms (MIC values < 100 micrograms/ml) suggesting that the targets of these drugs may not be restricted to mycobacterial species . The agents were all potent inhibitors of Mycobacterium bovis bacille Calmette-Guerin (MIC values 0.08-0.5 microgram/ml).

J Food Prot, 2000 Jan, 63(1), 63 - 70
Modeling UV-induced inactivation of microorganisms on surfaces; Gardner DW et al.; A model is presented to account for inactivation by UV light of microorganisms on the surfaces of solid materials . In the model, the surface is divided into a discrete number of zones, each having a characteristic exposure factor (alpha) . This is the ratio of UV intensity actually "seen" by the microorganism to that incident on the surface . Application of the model requires inactivation data obtained under conditions where the surface microorganisms are fully exposed to incident UV (alpha = 1) as well as kinetic inactivation data for the same microorganisms actually present on the surface of interest during UV irradiation . The kinetics in question may apply either to a single species or to the characteristic microflora associated with a particular material . Standard nonlinear programming techniques were used to determine the number of zones among which the microorganisms are distributed, the alpha for each zone, and the fraction of the microbial population present in each zone . The model was applied to data previously published by Gardner and Shama for UV inactivation of Bacillus subtilis spores on the surfaces of filter papers and also to the data of Stermer et al . for UV irradiation of beef . Good representation of the kinetics was obtained, and a maximum of three zones was required to adequately represent the experimental data . One direct application of the model is that it yields quantitative information about the UV fluences necessary to achieve specified reductions in microbial viability.

Mikrobiol Z, 1999 Sep-Oct, 61(5), 56 - 63
{The effect of the nutritional sources on the synthesis of exopolysaccharides and amino acids by Bacillus subtilis strains}; Osadchaia AI et al.; Dynamics of cell biomass accumulation and secretion to the medium extracellular polysaccharides and amino acids has been studied in Bacillus subtilis cultures No No 39 and 51 used to produce healing biopreparations--probiotics . The investigation data indicate to certain relation between these processes . EPS secretion in the studied cultures proceeded parallel with their growth and started in the logarithmic phase . Maximum EPS yield was observed by the beginning of the stationary phase after 10-12 h of growth . A successible change in the amino acid content in the medium was observed in the growth process of the studied bacteria: the bacteria first consumed amino acids of the initial medium and then excreted amino acids synthesized into the medium . Under the active production of EPS the content of extracellular amino acids in the medium was inconsiderable . The content of EPS was lower during accumulations of high concentrations of extracellular amino acids . Role of the medium components in regulation of the studied processes has been shown . The ratio C:N in the medium was of essential significance . The C:N ratio 2.0-3.0:1.0 was optimal both for the growth and secretion of EPS by the studied cultures while that of 1.0-1.5:1.0 was optimal for production of the extracellular amino acids . The increase of C:N ratio resulted in the decrease of metabolites secretion by the cultures.

Mikrobiol Z, 1999 Sep-Oct, 61(5), 19 - 27
{The effect of the cultivation conditions on the properties of bacilli comprising the basis of probiotics}; Furzikova TM et al.; Such biological factors as bile, gastric juice, blood serum, amino acids and pH of the medium have been studied for their effect on the growth intensity and antagonistic activity of bacilli being the basis of biosporin and subalin . It has been established that pH of the medium within 7.0-9.0 as well as certain concentrations of the gastric juice, bile, blood serum and most of amino acids did not affect the growth of Bacillus subtilis 3 and Bacillus subtilis 2335/105 . A regular decrease of growth intensity in bacilli under the increase of the gastric juice concentration or decrease of pH of the medium to 5.0-2.0 is registered . The mentioned biological factors affect differently the antagonistic properties of the studied cultures.

Comput Chem, 2000 Jan, 24(1), 57 - 70
Detecting localized repeats in genomic sequences: a new strategy and its application to Bacillus subtilis and Arabidopsis thaliana sequences; Klaerr-Blanchard M et al.; A new method for the search of local repeats in long DNA sequences, such as complete genomes, is presented . It detects a large variety of repeats varying in length from one to several hundred bases, which may contain many mutations . By mutations we mean substitutions, insertions or deletions of one or more bases . The method is based on counting occurrences of short words (3-12 bases) in sequence fragments called windows . A score is computed for each window, based on calculating exact word occurrence probabilities for all the words of a given length in the window . The probabilities are defined using a Bernoulli model (independent letters) for the sequence, using the actual letter frequencies from each window . A plot of the probabilities along the sequence for high-scoring windows facilitates the identification of the repeated patterns . We applied the method to the 1.87 Mb sequence of chromosome 4 of Arabidopsis thaliana and to the complete genome of Bacillus subtilis (4.2 Mb) . The repeats that we found were classified according to their size, number of occurrences, distance between occurrences, and location with respect to genes . The method proves particularly useful in detecting long, inexact repeats that are local, but not necessarily tandem . The method is implemented as a C program called EXCEP, which is available on request from the authors.

Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 728 - 33
Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator; de la Hoz AB et al.; Transcription initiation of the copy-number control and better-than-random segregation genes of the broad-host-range and low-copy-number plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded omega product in Bacillus subtilis cells . The promoters of the copS (Pcop1 and Pcop2), delta (Pdelta), and omega (Pomega) genes have been mapped . These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times . The cooperative binding of omega protein to the repeats on the Pcop1, Pcop2, Pdelta, and Pomega promoters represses transcription initiation by a mechanism that does not exclude sigma(A)RNAP from the promoters . These results indicate that omega protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.

Nucleic Acids Res, 2000 Feb 1, 28(3), 720 - 7
Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage; Warnecke JM et al.; Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3'-OH and 5'-phosphate termini, and at least two metal ions are thought to be essential for catalysis . To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3'-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3'- S -phosphorothiolate linkages at the RNase P cleavage site were synthesized . With a 3'- S -phosphorothiolate-modified ptRNA carrying a 7 nt 5'-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5'-direction was observed . Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn(2+)or Cd(2+) . To suppress aberrant cleavage, we also constructed a 3'- S -phosphorothiolate-modified ptRNA with a 1 nt 5'-flank . No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions . Ground state binding of modified ptRNAs was not impaired, suggesting that the 3'- S -phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.

J Biol Chem, 2000 Jan 21, 275(3), 1773 - 80
The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding; Jault JM et al.; Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources . In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46 . We report here that B . subtilis HprK forms homo-oligomers constituted most likely of eight subunits . Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235 . It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations . A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments . Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations . This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.

J Bacteriol, 2000 Feb, 182(3), 689 - 95
Different processing of an mRNA species in Bacillus subtilis and Escherichia coli; Persson M et al.; Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription . The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator . In the presence of G3P, the antiterminator protein GlpP binds to glpD leader mRNA and promotes readthrough of the terminator . Certain mutations in the inverted repeat of the glpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD . The TS phenotype is due to temperature-dependent degradation of the glpD mRNA . In the presence of GlpP, the glpD mRNA is stabilized . glpD leader-lacZ fusions were integrated into the chromosomes of B . subtilis and Escherichia coli . Determination of steady-state levels of fusion mRNA in B . subtilis showed that the stability of the fusion mRNA is determined by the glpD leader part . Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA in B . subtilis and E . coli revealed significant differences . A glpD leader-lacZ fusion transcript that was unstable in B . subtilis was considerably more stable in E . coli . GlpP, which stabilizes the transcript in B . subtilis, did not affect its stability in E . coli . Primer extension analysis showed that the glpD leader-lacZ fusion transcript is processed differently in B . subtilis and in E . coli . The dominating cleavage site in E . coli was barely detectable in B . subtilis . This site was shown to be a target of E . coli RNase III.

Mol Microbiol, 2000 Jan, 35(1), 180 - 8
A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the sigmaB transcription factor of Bacillus subtilis; Vijay K et al.; The sigmaB transcription factor of the bacterium Bacillus subtilis is activated by growth-limiting energy or environmental challenge to direct the synthesis of more than 100 general stress proteins . Although the signal transduction pathway that conveys these stress signals to sigmaB is becoming increasingly well understood, how environmental or energy stress signals enter this pathway remains unknown . We show here that two PP2C serine phosphatases - RsbP, which is required for response to energy stress, and RsbU, which is required for response to environmental stress - each converge on the RsbV regulator of sigmaB . According to the current understanding of sigmaB regulation, in unstressed cells the phosphorylated RsbV anti-anti-sigma is unable to complex the RsbW anti-sigma, which is then free to bind and inactivate sigmaB . We can now advance the model that either PP2C phosphatase, when triggered by its particular class of stress, can remove the phosphate from RsbV and thereby activate sigmaB . The action of the previously described RsbU is known to be controlled by dedicated upstream signalling components that are activated by environmental stress . The action of the RsbP phosphatase described here requires an energy stress, which we suggest is sensed, at least in part, by the PAS domain in the amino-terminal region of the RsbP phosphatase . In other bacterial signalling proteins, similar PAS domains and their associated chromophores directly sense changes in intracellular redox potential to control the activity of a linked output domain.

Mol Microbiol, 2000 Jan, 35(1), 150 - 60
A developmentally regulated catalase required for proper differentiation and osmoprotection of Streptomyces coelicolor; Cho YH et al.; Streptomyces coelicolor produces at least three catalases, the expression of which varies under different conditions . We characterized a gene (catB) for developmentally controlled catalase of 779 amino acids (83408 Da), homologous to KatE of Escherichia coli and Bacillus subtilis . Expression of the catB gene increased at the stationary phase in liquid culture and after the onset of differentiation on solid culture . It was also increased by osmotic treatments . Transcription was initiated from a promoter (catBp), whose sequence (ATGCCTCG-N13-GGGTAC) resembled promoters recognized by sigmaB of B . subtilis . CatB protein underwent proteolytic cleavage of its N-terminal 95 amino acids and was secreted to the medium when cells sporulated . Disruption of the catB gene caused impairment in the formation of aerial mycelium and reduction in the synthesis of undecylprodigiosin . On the contrary, hyperproduction of actinorhodin was observed in accordance with the increase in actII-ORF4 transcription . In addition, catB mutant became hypersensitive to osmotic stresses . These results suggest that regulated synthesis of CatB protein is necessary to ensure proper differentiation as well as to protect S . coelicolor cells against osmotic stresses.

Mol Microbiol, 2000 Jan, 35(1), 123 - 38
Novel Rhodobacter capsulatus genes required for the biogenesis of various c-type cytochromes; Deshmukh M et al.; Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c-type cytochrome (cyt)-deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated . Both mutants were completely deficient in all known c-type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R . capsulatus . Complementation of 771 and K2 with a wild-type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively . The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram-negative species . Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R . capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis . Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function . All R . capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species . In contrast, the R . capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the gram-positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli . Membrane topology of CcdA was established in R . capsulatus using ccdA:phoA and ccdA :lacZ gene fusions . The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded . Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis . This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some gram-negative bacteria such as R . capsulatus.

Mol Microbiol, 2000 Jan, 35(1), 44 - 57
CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis; Kirby JR et al.; The methyl-accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis . In this study, we show that wild-type B . subtilis cells contain approximately 2,000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response . In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine-2-carboxylate . Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half-maximal demethylation of McpB occurring when only 3% of the receptors are titrated . Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine . Together, the data support the hypothesis that methylation changes occur on asparagine-bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.

J Bacteriol, 2000 Jan, 182(2), 555 - 60
The TRAP-like SplA protein is a trans-acting negative regulator of spore photoproduct lyase synthesis during Bacillus subtilis sporulation; Fajardo-Cavazos P et al.; UV resistance of bacterial endospores derives from a unique DNA photochemistry in which the major UV photoproduct is the thymine dimer 5-thyminyl-5,6-dihydrothymine (spore photoproduct {SP}) instead of cyclobutane pyrimidine dimers . Repair of SP during spore germination is due in large part to the activity of the enzyme SP lyase encoded by splB, the second cistron of the splAB operon . Expression of the splAB operon in Bacillus subtilis is transcriptionally activated by the Esigma(G) form of RNA polymerase during morphological stage III in the developing forespore compartment, and SP lyase is packaged into the dormant spore . In addition to temporal and compartmental control of splAB expression, a second regulatory circuit which modulates the level of expression of splB-lacZ fusions without altering their developmental timing or compartmentalization is reported here . This second regulatory circuit involves the negative action of the splA gene product, a 79-amino-acid protein with approximately 50% similarity and 17% identity to TRAP, the tryptophan RNA-binding attenuation protein from B . subtilis and Bacillus pumilus.

J Bacteriol, 2000 Jan, 182(2), 418 - 24
Identification and characterization of a new prespore-specific regulatory gene, rsfA, of Bacillus subtilis; Juan Wu L et al.; Differential gene expression during Bacillus subtilis sporulation is controlled by sigma factors and other regulatory effectors . The first compartmentalized sigma factor, sigma(F), is active specifically in the prespore compartment . During our screening for new chromosome segregation mutants using a sigma(F)-dependent gpr-lacZ reporter as a probe, we identified a new gene (ywfN) required for maximal expression of the reporter and named it rsfA . The product of rsfA has features of gene regulatory proteins, and the protein colocalizes with DNA . The expression of rsfA is under the control of both sigma(F) and sigma(G) . Null mutations in rsfA have different effects on the expression of sigma(F)-dependent genes, suggesting that the RsfA protein is a regulator of transcription that fine-tunes gene expression in the prespore.

J Bacteriol, 2000 Jan, 182(2), 365 - 70
beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis; Choi KH et al.; A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes . We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria . Two genes that were predicted to encode homologs of the beta-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in the Bacillus subtilis genome . Their protein products were expressed, purified, and biochemically characterized . Both B . subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH . bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates . eFabH was not able to accept these CoA thioesters . Reconstitution of a complete round of fatty acid synthesis in vitro with purified E . coli proteins showed that eFabH was the only E . coli enzyme incapable of using branched-chain substrates . Expression of either bFabH1 or bFabH2 in E . coli resulted in the appearance of a branched-chain 17-carbon fatty acid . Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.

J Bacteriol, 2000 Jan, 182(2), 303 - 10
Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis; Jiang M et al.; In the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis, the opposing activities of histidine kinases and aspartyl phosphate phosphatases determine the cell's decision whether to continue with vegetative growth or to initiate the differentiation process . Regulated dephosphorylation of the Spo0A and Spo0F response regulators allows a variety of negative signals from physiological processes that are antithetical to sporulation to impact on the activation level of the phosphorelay . Spo0F approximately P is the known target of two related phosphatases, RapA and RapB . In addition to RapA and RapB, a third member of the Rap family of phosphatases, RapE, specifically dephosphorylated the Spo0F approximately P intermediate in response to competence development . RapE phosphatase activity was found to be controlled by a pentapeptide (SRNVT) generated from within the carboxy-terminal domain of the phrE gene product . A synthetic PhrE pentapeptide could (i) complement the sporulation deficiency caused by deregulated RapE activity of a phrE mutant and (ii) inhibit RapE-dependent dephosphorylation of Spo0F approximately P in in vitro experiments . The PhrE pentapeptide did not inhibit the phosphatase activity of RapA and RapB . These results confirm previous conclusions that the specificity for recognition of the target phosphatase is contained within the amino acid sequence of the pentapeptide inhibitor.

J Bacteriol, 2000 Jan, 182(2), 278 - 85
Membrane topology of the Bacillus subtilis pro-sigma(K) processing complex; Green DH et al.; Activation of the final sporulation-specific transcription factor, sigma(K), is regulated by a signal emanating from the forespore which interacts with the pro-sigma(K) processing complex, comprising SpoIVFA, BofA, and the pro-sigma(K) processing protease, SpoIVFB . Mature sigma(K) then directs late gene expression in the parental compartment of the developing sporangial cell . The nature of this complex and how it is activated to process pro-sigma(K) are not understood . All three proteins are predicted to be integral membrane proteins . Here, we have analyzed the membrane topology of SpoIVFA and SpoIVFB by constructing chimeric forms of spoIVFA and spoIVFB with the complementary reporters phoA and lacZ and analyzing activity in Escherichia coli . SpoIVFA was found to have a single transmembrane-spanning domain, while SpoIVFB was shown to have six transmembrane-spanning domains (6-transmembrane configuration) . Further, SpoIVFA is required to stabilize SpoIVFB in the membrane . SpoIVFB was shown to have a 4-transmembrane configuration when expressed on its own but was found to have a 6-transmembrane configuration when coexpressed with SpoIVFA, while BofA had a positive effect on the assembly of both SpoIVFA and SpoIVFB . The single transmembrane domain of SpoIVFA (approximately residues 73 to 90) was shown to be the principle determinant in stabilizing the 6-transmembrane configuration of SpoIVFB . Although the bofB8 allele, which uncouples the sigma(K) checkpoint, did not appear to promote a conformational change from a 6- to 4-transmembrane configuration of SpoIVFB (apparently ruling out a profound conformational change as the mechanism of activating SpoIVFB proteolytic activity), instability of SpoIVFB may be an important factor in SpoIVFB-mediated processing of pro-sigma(K).

Nucleic Acids Res, 2001 Jan 1, 29(1), 278 - 80
DBTBS: a database of Bacillus subtilis promoters and transcription factors; Ishii T et al.; With the completion of the determination of its entire genome sequence, one of the next major targets of Bacillus subtilis genomics is to clarify the whole gene regulatory network . To this end, the results of systematic experiments should be compared with the rich source of individual experimental results accumulated so far . Thus, we constructed a database of the upstream regulatory information of B.subtilis (DBTBS) . The current version was constructed by surveying 291 references and contains information on 90 binding factors and 403 promoters . For each promoter, all of its known cis-elements are listed according to their positions, while these cis-elements are aligned to illustrate their consensus sequence for each transcription factor . All probable transcription factors coded in the genome were classified with the Pfam motifs . Using this database, we compared the character of B.subtilis promoters with that of Escherichia coli promoters . Our database is accessible at http://elmo.ims.u-tokyo.ac.jp/dbtbs/.

Microbiology, 1999 Dec, 145 ( Pt 12), 3431 - 45
TRAP transporters: an ancient family of extracytoplasmic solute-receptor-dependent secondary active transporters; Rabus R et al.; Tripartite ATP-independent periplasmic transporters (TRAP-T) represent a novel type of secondary active transporter that functions in conjunction with an extracytoplasmic solute-binding receptor . The best characterized TRAP-T family member is from Rhodobacter capsulatus and is specific for C4-dicarboxylates {Forward, J . A., Behrendt, M . C., Wyborn, N . R., Cross, R . & Kelly, D . J . (1997) . J Bacteriol 179, 5482-5493} . It consists of three essential proteins, DctP, a periplasmic C4-dicarboxylate-binding receptor, and two integral membrane proteins, DctM and DctQ, which probably span the membrane 12 and 4 times, respectively . Homologues of DctM, DctP and DctQ were identified in all major bacterial subdivisions as well as in archaea . An orphan DctP homologue in the Gram-positive bacterium Bacillus subtilis may serve as a receptor for a two-component transcriptional regulatory system rather than as a constituent of a TRAP-T system . Phylogenetic data suggest that all present day TRAP-T systems probably evolved from a single ancestral transporter with minimal shuffling of constituents between systems . Homologous TRAP-T constituents exhibit decreasing degrees of sequence identity in the order DctM > DctP > DctQ . DctM appears to belong to a large superfamily of transporters, the ion transporter (IT) superfamily, one member of which can function by either protonmotive force- or ATP-dependent energization . It is proposed that IT superfamily members exhibit the unusual capacity to function in conjunction with auxiliary proteins that modify the transport process by providing (i) high-affinity solute reception, (ii) altered energy coupling and (iii) additional yet to be defined functions.

Microbiology, 1999 Dec, 145 ( Pt 12), 3419 - 29
Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis; Reizer J et al.; Bacillus subtilis can utilize several sugars as single sources of carbon and energy . Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B . subtilis . In this study, an analysis of the complete set of PTS proteins encoded within the B . subtilis genome is presented . Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants . Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified . Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B . subtilis; by contrast only five Enzyme IIA(Glc)-like proteins or domains are encoded within the B . subtilis genome . Consequently, some of the EIIA(Glc)-like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families . In addition, 13 PTS-associated proteins are encoded within the B . subtilis genome . These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction . The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.

Microbiology, 1999 Dec, 145 ( Pt 12), 3409 - 17
Transcriptional analysis of the Bacillus subtilis teichuronic acid operon; Lahooti M et al.; The cell walls of Gram-positive bacteria consist primarily of a macromolecular matrix comprising similar amounts of peptidoglycan and covalently attached anionic polymers . Under most growth conditions the anionic polymers of Bacillus subtilis are principally teichoic acids; in strain 168 these include a polyglycerol teichoic acid and a glucose/galactosamine-containing teichoic acid . However, when cultures are subjected to phosphate stress the bacterium induces a complex series of responses, one of which is the replacement of at least part of the wall teichoic acid with teichuronic acid, a non-phosphate-containing anionic polymer . In this paper the construction of a transcriptional reporter strain that facilitates the monitoring of the promoter region upstream of the tua operon involved in teichuronic acid synthesis and its controlled expression are reported . The expression of the tua operon was monitored in both phosphate-starved, non-growing batch cultures and phosphate-limited continuous cultures . We show that the transcription of the operon correlates well with the anionic polymer composition of the cell walls.

J Biol Chem, 2000 Jan 14, 275(2), 895 - 900
A NAD(P)H oxidase isolated from the archaeon Sulfolobus solfataricus is not homologous with another NADH oxidase present in the same microorganism . Biochemical characterization of the enzyme and cloning of the encoding gene; Arcari P et al.; A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus . The enzyme is a homodimer with M(r) 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit . It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide . The enzyme was resistant against chemical and physical denaturating agents . The temperature for its half-denaturation was 93 and 75 degrees C in the absence or presence, respectively, of 8 M urea . The enzyme did not show any reductase activity . The SsNOX38 encoding gene was cloned and sequenced . It accounted for a product of 36.5 kDa . The translated amino acid sequence was made of 332 residues containing two putative betaalphabeta-fold regions, typical of NAD- and FAD-binding proteins . The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S . solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V . and Bannister, J . V . (1996) Biotechnol . Appl . Biochem . 23, 47-54) . Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase.

J Theor Biol, 2000 Jan 7, 202(1), 87 - 94
A new mathematical approach predicts individual cell growth behavior using bacterial population information; Anderson KR et al.; A theoretical methodology has been developed for studying the growth kinetics of bacterial cells . It utilizes the steady-state cell length distribution in a bacterial population to predict the dependency of growth and division rates on cell length and age . The mathematical model has been applied to the analysis of two bacterial populations, a wild-type strain of Bacillus subtilis, and a minicell-producing strain that carries the divIVB1 mutation . The results show that our model describes the wild-type population very well and that the assumptions typically used in traditional methods are unrealistic . In the case of the minicell-producing mutant we find evidence that the rate of cell division must be a function not only of cell size but also of cell age .

Bioorg Khim, 1999 Sep, 25(9), 716 - 20
{(2E)-4-hydroxy-2-nonenal--an active component of a new natural antimicrobial substance}; Rybin VG et al.; A mixture of water-soluble oxidation products of Sardinops melanosticta sardine oil was found to contain (2E)-4-hydroxy-2-nonenal . This was isolated by column chromatography on silica gel and reversed-phase HPLC . Its structure was elucidated by physicochemical methods . The activity of (2E)-4-hydroxy-2-nonenal against test cultures of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was about 20% of the total antimicrobial activity of the preparation.

FEMS Microbiol Lett, 2000 Jan 15, 182(2), 255 - 8
Acrylonitrile induces autolysis Bacillus subtilis; Reyes GF et al.; Acrylonitrile (AN) is an industrial chemical used in the manufacture of plastics and other polymers . AN has been reported to be an acute toxin and is a known carcinogen in rodents . When AN was mixed with suspensions of Bacillus subtilis, the bacteria began autolysis . It was determined that AN is partially converted to cyanide, a strong protonophore in B . subtilis . Autolytic enzymes in B . subtilis become active when the protonmotive force is dissipated . The amount of cyanide produced from AN, however, was not enough to promote autolysis in exponential B . subtilis . This is the first report showing that AN may induce autolytic reactions in bacteria . It is suggested the autolysis of B . subtilis may be useful in the environmental monitoring of AN . In addition, the metabolism of AN by bacilli may be useful in bioremediation.

Biofactors, 1999, 10(4), 311 - 9
Morphological and biochemical responses of Bacillus subtilis to selenite stress; Garbisu C et al.; When introduced into a chemically defined minimal medium supplemented with 1 mM sodium selenite (79 ppm Se(o)), Bacillus subtilis was found to undergo a series of morphological and biochemical adaptations . The morphological changes included the formation of "round bodies" associated with the detoxification of selenite to elemental selenium . Round bodies observed transiently were not apparent during balanced growth of cells adapted previously to selenite-containing medium . Under balanced growth conditions, cell structures similar to "round bodies", could be produced by treating cells with lysozyme . The selenite-induced structural alterations in cells were accompanied by an increase in the content of thioredoxin and the associated enzyme, NADP-thioredoxin reductase . The results suggest that the biovalence transformation of high levels of selenite may involve a dithiol system.

Appl Environ Microbiol, 2000 Jan, 66(1), 257 - 61
Comparative study of pressure- and nutrient-induced germination of Bacillus subtilis spores; Wuytack EY et al.; Germination experiments with specific germination mutants of Bacillus subtilis, including a newly isolated mutant affected in pressure-induced germination, suggest that a pressure of 100 MPa triggers the germination cascades that are induced by the nutrient germinant alanine (Ala) and by a mixture of asparagine, glucose, fructose, and potassium ions (AGFK), by activating the receptors for alanine and asparagine, GerA and GerB, respectively . As opposed to germination at 100 MPa, germination at 600 MPa apparently short-cuts at least part of the Ala- and AGFK-induced germination pathways . Inhibitors of nutrient-induced germination (HgCl(2) and Nalpha-P-tosyl-L-arginine methyl ester) also inhibit pressure-induced germination at 600 MPa, suggesting that germination at 600 MPa involves activation of a true physiological germination pathway and is therefore not merely a physico-chemical process in which water is forced into the spore protoplast.

Appl Environ Microbiol, 2000 Jan, 66(1), 199 - 205
Artificial and solar UV radiation induces strand breaks and cyclobutane pyrimidine dimers in Bacillus subtilis spore DNA; Slieman TA et al.; The loss of stratospheric ozone and the accompanying increase in solar UV flux have led to concerns regarding decreases in global microbial productivity . Central to understanding this process is determining the types and amounts of DNA damage in microbes caused by solar UV irradiation . While UV irradiation of dormant Bacillus subtilis endospores results mainly in formation of the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine, genetic evidence indicates that an additional DNA photoproduct(s) may be formed in spores exposed to solar UV-B and UV-A radiation (Y . Xue and W . L . Nicholson, Appl . Environ . Microbiol . 62:2221-2227, 1996) . We examined the occurrence of double-strand breaks, single-strand breaks, cyclobutane pyrimidine dimers, and apurinic-apyrimidinic sites in spore DNA under several UV irradiation conditions by using enzymatic probes and neutral or alkaline agarose gel electrophoresis . DNA from spores irradiated with artificial 254-nm UV-C radiation accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, while DNA from spores exposed to artificial UV-B radiation (wavelengths, 290 to 310 nm) accumulated only cyclobutane pyrimidine dimers . DNA from spores exposed to full-spectrum sunlight (UV-B and UV-A radiation) accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, whereas DNA from spores exposed to sunlight from which the UV-B component had been removed with a filter ("UV-A sunlight") accumulated only single-strand breaks and double-strand breaks . Apurinic-apyrimidinic sites were not detected in spore DNA under any of the irradiation conditions used . Our data indicate that there is a complex spectrum of UV photoproducts in DNA of bacterial spores exposed to solar UV irradiation in the environment.

Methods, 2000 Jan, 20(1), 95 - 110
Structure and assembly of the bacterial endospore coat; Henriques AO et al.; Many biological processes are mediated through the action of multiprotein complexes, often assembled at specific cellular locations . Bacterial endospores for example, are encased in a proteinaceous coat, which confers resistance to lysozyme and harsh chemicals and influences the spore response to germinants . In Bacillus subtilis, the coat is composed of more than 20 polypeptides, organized into three main layers: an amorphous undercoat; a lamellar, lightly staining inner structure; and closely apposed to it, a striated electron-dense outer coat . Synthesis of the coat proteins is temporally and spatially governed by a cascade of four mother cell-specific transcription factors . However, the order of assembly and final destination of the coat structural components may rely mainly on specific protein-protein interactions, as well as on the action of accessory morphogenetic proteins . Proteolytic events, protein-protein crosslinking, and protein glycosylation also play a role in the assembly process . These modifications are carried out by enzymes that may themselves be targeted to the coat layers . Coat genes have been identified by reverse genetics or, more recently, by screens for mother cell-specific promoters or for peptide sequences able to interact with certain bait proteins . A role for a given locus in coat assembly is established by a combination of regulatory, functional, morphological, and topological criteria . Because of the amenability of B . subtilis to genetic analysis (now facilitated by the knowledge of its genome sequence), coat formation has become an attractive model for the assembly of complex macromolecular structures during development .

J Mol Biol, 1999 Nov 26, 294(2), 389 - 402
RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT; Declerck N et al.; Transcriptional antiterminators of the BglG/SacY family are regulatory proteins that mediate the induction of sugar metabolizing operons in Gram-positive and Gram-negative bacteria . Upon activation, these proteins bind to specific targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA polymerase from the DNA template . We have previously characterized the RNA-binding domain of SacY from Bacillus subtilis and determined its three-dimensional structure by both NMR and crystallography . In the present study, we have characterized the paralogous domain from LicT and we present the first structural comparison between two BglG/SacY family members . Similar to SacY, the RNA-binding activity of LicT is contained within the 56 N-terminal amino acid residue fragment corresponding to the so-called co-antiterminator (CAT) domain . Surface plasmon resonance affinity measurements show that, compared to SacY-CAT, LicT-CAT binds more tightly and more specifically to its cognate RNA target, with a KD value of about 10(-8) M . The crystal structure of LicT-CAT has been determined at 1.8 A resolution and compared to that of SacY-CAT . Both molecules fold as symmetrical dimers, each monomer comprising a four-stranded antiparallel beta-sheet that stacks against the beta-sheet of the other monomer in a very conserved manner . Comparison of the proposed RNA-binding surfaces shows that many of the conserved atoms concentrate in a central region across one face of the CAT dimer, whereas variable elements are mostly found at the edges . Interestingly, the electrostatic potential maps calculated for the two molecules are quite different, except for the core of the RNA-binding site, which appears essentially neutral in both structures .

Nucleic Acids Res, 2000 Jan 15, 28(2), 552 - 9
Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA; Tapias A et al.; Genetic evidence suggests that the Bacillus subtilis lrpC gene product participates in cell growth and sporulation . The purified LrpC protein, which has a predicted molecular mass of 16.4 kDa, is a tetramer in solution . LrpC binds with higher affinity ( K (app) approximately 80 nM) to intrinsically curved DNA than to non-curved DNA ( K (app) approximately 700 nM) . DNase I footprinting and the supercoiling of relaxed circular plasmid DNA in the presence of topoisomerase I revealed that LrpC induces DNA bending and constrains DNA supercoils in vitro . The LrpC protein cooperatively increases DNA binding of the bona fide DNA-binding and DNA-bending protein Hbsu . LrpC forms inter- and intramolecular bridges on linear and supercoiled DNA molecules, resulting in a large network and DNA compactation . Collectively, these findings suggest that LrpC is an architectural protein and that its activities could provide a means to modulate DNA transactions.

Biochemistry, 1999 Dec 21, 38(51), 16925 - 31
Mechanism of ligand recognition by BmrR, the multidrug-responding transcriptional regulator: mutational analysis of the ligand-binding site; Vazquez-Laslop N et al.; The Bacillus subtilis transcriptional regulator BmrR recognizes dissimilar hydrophobic cations and, in response, activates the expression of a multidrug transporter which expels them out of the cell . The structure of the inducer-binding domain of BmrR, both free and in complex with one of the inducers, tetraphenylphosphonium (TPP), revealed an unusual internal binding site, covered by an amphipathic alpha-helix . Upon unfolding of this helix, the TPP molecule penetrates into the core of the protein, where it contacts six hydrophobic residues and forms an electrostatic bond with a buried glutamate, E134 {Zheleznova et al . (1999) Cell 96, 353-362} . Here, a structure-based mutational analysis was used to understand how BmrR interacts with a wide variety of ligands . We determined the effects of alanine substitutions of each of the seven residues interacting with TPP, and mutations within the amphipathic alpha-helix, on the binding affinities of six different BmrR inducers . The E134A substitution abolished the binding of all but one inducer . Mutations of the hydrophobic residues contacting the ligand, and of the alpha-helix, had more moderate effects, often with the affinity for some inducers increasing and others decreasing as a result of the same substitution . These results indicate that each inducer forms a unique set of contacts within the binding site . The flexible geometry of this site and the lack of involvement of hydrogen bonds in ligand binding are the likely reasons for the extremely broad inducer specificity of BmrR . The similarly broad substrate specificity of multidrug transporters can be governed by the same structural principles.

Biochemistry, 1999 Dec 21, 38(51), 16840 - 6
A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme; Fang X et al.; The folding thermodynamics of the catalytic domain from the Bacillus subtilis RNase P RNA is analyzed using circular dichroism and fluorescence spectroscopies, hydroxyl radical protection, and catalytic activity . Folding of this 255-nucleotide ribozyme can be described with three populated species: unfolded (U), intermediate (I), and native (N) states . The U-to-I transition primarily involves secondary structure formation, whereas the I-to-N transition is dominated by tertiary structure formation . The I-to-N transition is highly cooperative as indicated by the coincidence of the four probes applied here . Two isothermal methods are used to determine the stability of the N state relative to the I state at 10 and 37 degrees C . The first method measures the extent of Mg(2+)-induced folding without urea or at constant urea concentrations . The second method measures the extent of urea-induced unfolding at constant Mg(2+) concentrations . Via application of a cooperative binding analysis, the Mg(2+) transition midpoint (K(Mg)), the Hill constant (n), and the urea-dependent surface burial parameter (m value) determined by both methods are identical, indicating that they report the same, reversible folding event . Three conclusions can be drawn from these results . (i) The folding free energy of a Mg(2+)-dependent tertiary RNA structure can be described by the K(Mg) and n parameters according to a cooperative Mg(2+) binding model . (ii) The Hill constant for this tertiary RNA structure probably represents the differential number of Mg(2+) ions bound in the I-to-N transition . (iii) Under physiological conditions, the stability of this large ribozyme is similar to that of small globular proteins.

Biol Pharm Bull, 1999 Nov, 22(11), 1193 - 201
New 6-O-acyl isoflavone glycosides from soybeans fermented with Bacillus subtilis (natto) . I . 6-O-succinylated isoflavone glycosides and their preventive effects on bone loss in ovariectomized rats fed a calcium-deficient diet; Toda T et al.; Three new 6-O-acylated isoflavone glycosides were isolated from soybeans fermented with Bacillus subtilis (natto) and identified as daidzein 7-O-beta-(6''-O-succinyl)-D-glucoside (1), genistein 7-O-beta-(6''-O-succinyl)-D-glucoside (2), and glycitein 7-O-beta-(6''-O-succinyl)-D-glucoside (3) on the basis of spectral data and chemical transformations . During fermentation, the content of the isoflavone glycosides first decreased and then increased, whereas the corresponding 6''-O-succinyl derivatives first accumulated and then decreased, in either soybeans or soybean cooking solution . These changes suggest that enzymatic interconversion of isoflavone glycosides and the corresponding 6''-O-succinylated derivatives occurs in these media during fermentation . The 6-O-succinylated isoflavone glycosides 1, 2 and 3 accounted for 4.8, 7.2 and 0.6%, respectively, of the total isoflavones in commercial fermented soybeans (Japanese natto) . Oral administration of 1 or 2 alone for 4 weeks at a dose of 50 mg/kg/d prevented bone loss in ovariectomized (ovx) rats fed a calcium-deficient diet, being as effective as the positive controls, daidzin and genistin, respectively . Compound 1 seems to be proestrogenic, like daidzin, which suppresses bone resorption to prevent bone loss after ovariectomy by directly acting on bone sites, while 2 appears to have a different mechanism of action, like that of genistin.

J Agric Food Chem, 1999 May, 47(5), 1870 - 7
Fractionation-reconstitution experiments provide insight into the role of endoxylanases in bread-making; Courtin CM et al.; The impact mechanism of endoxylanases in straight dough bread-making was investigated in fractionation-reconstitution experiments . To this end, two European flours with different bread-making characteristics were separated in gluten, prime starch, a squeegee fraction (SQF), and a water-extractable fraction . Whereas the former fractions contained negligible levels of arabinoxylan (AX), the latter contained, respectively, most of the water-unextractable AX (WU-AX) and all of the water-extractable AX (WE-AX) . In vitro modification with a Bacillus subtilis endoxylanase allowed controlled solubilization of WU-AX from SQF and controlled degradation of solubilized AX and WE-AX from the water-extractables . It followed from bread-making tests with the reconstituted flours that endoxylanases exert positive loaf volume effects in bread-making by lowering the concentration of WU-AX and increasing that of total soluble AX . Limited degradation of WE-AX and significant breakdown of solubilized AX by endoxylanases, on the other hand, resulted in volume losses when compared to their nondegraded counterparts . The volume increasing effects of endoxylanases are therefore related to their ratio of solubilizing to degrading activity and thus to their substrate specificity.

Curr Microbiol, 2000 Feb, 40(2), 137 - 9
Genetic characterization of a new thermotolerant Bacillus licheniformis strain; Mendo SA et al.; A potentially new thermotolerant B . licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B . licheniformis ATCC 10716, producer of bacitracin . Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology . Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern . Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE . The I89 strain holds a 7.6-kb plasmid not present in the reference strain . The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector.

Curr Microbiol, 2000 Feb, 40(2), 119 - 22
Overexpression of the cat-86 gene is associated with thermosensitivity in Bacillus subtilis; Friedman SM et al.; Bacillus subtilis harboring the cat-86 constitutive plasmid pPL708C2 with an ochre mutation at the 9th codon (terc 9) was sensitive to chloramphenicol (Cm(s)) and exhibited relative thermostability when heated at 47 degrees C . Reversion to chloramphenicol resistance (Cm(r)) occurred at a frequency of 5.4 x 10(-8) . All of the plasmid Cm(r) revertants tested were thermosensitive . Similarly, wild-type pPL708C2 present in B . subtilis also rendered the bacterium thermosensitive . When a nonsense mutation is introduced at codon 141, however, this terc 141 variant of pPL708C2 failed to thermosensitize B . subtilis . Another variant of pPL708C2 that produces intact yet catalytically inactive CAT-86 has both His-16 and His-17 at the active site replaced by Pro . Nevertheless, cells of B . subtilis carrying this variant were thermosensitive . Plasmid-free and pPL708C2-bearing strains did not exhibit differences in major heat shock proteins . Electron micrographs revealed a threefold increase of inclusion bodies present in a strain harboring pPL708C2 when compared with those in an isogenic plasmid-free strain.

Protein Sci, 1999 Nov, 8(11), 2428 - 37
A test case for structure-based functional assignment: the 1.2 A crystal structure of the yjgF gene product from Escherichia coli; Volz K; The YER057c/YIL051c/YjgF protein family is a set of 24 full-length homologs, each approximately 130 residues in length, and each with no known function or relationship to proteins of known structure . To determine the function of this family, the structure of one member--the YjgF protein from Escherichia coli--was solved and refined at a resolution of 1.2 A . The YjgF molecule is a homotrimer with exact threefold symmetry . Its tertiary and quaternary structures are related to that of Bacillus subtilis chorismate mutase, although their active sites are completely different . The YjgF protein has an active site curiously similar to protein tyrosine phosphatases, including a covalently modified cysteine, but it is unlikely to be functionally related . The lessons learned from this attempt to deduce function from structure may be useful to future projects in structural genomics.

Biosci Biotechnol Biochem, 1999 Sep, 63(9), 1528 - 34
Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III; Shida T et al.; To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B . subtilis . There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain . An expression plasmid of the ExoA in E . coli was constructed by inserting the exoA gene into the expression vector pKP1500 . The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates . A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized . It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities . Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B . subtilis that is very similar to E . coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E . coli exonuclease III.

Microbiology, 1999 Nov, 145 ( Pt 11), 3205 - 12
Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus; Darbon E et al.; The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified . The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared . The work presented in this paper demonstrates that T . flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes . T . flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr) . These proteins catalyse the phosphorylation of T . flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus . Purified glycerol kinase from T . flavus could also be phosphorylated with enzyme I and HPr from B . subtilis . Similar to enterococcal glycerol kinases, phosphorylated T . flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase . However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T . flavus glycerol kinase caused only a slight increase in enzyme activity.

Microbiology, 1999 Nov, 145 ( Pt 11), 3195 - 204
The Q15H mutation enables Crh, a Bacillus subtilis HPr-like protein, to carry out some regulatory HPr functions, but does not make it an effective phosphocarrier for sugar transport; Martin-Verstraete I et al.; Crh of Bacillus subtilis exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) . Crh can be phosphorylated by ATP at the regulatory Ser-46 and similar to P-Ser-HPr, P-Ser-Crh plays a role in carbon-catabolite repression . The sequence around the phosphorylatable Ser-46 in Crh exhibits strong similarity to the corresponding sequence of HPr of Gram-positive and a few Gram-negative bacteria . In contrast, the catalytic His-15, the site of PEP-dependent phosphorylation in HPr, is replaced with a glutamine in Crh . When Gln-15 was exchanged for a histidyl residue, in vitro PEP-dependent enzyme I-catalysed phosphorylation of the mutant Crh was observed . However, expression of the crhQ15H mutant allele did not restore growth of a ptsH deletion strain on the PTS sugars glucose, fructose or mannitol or on the non-PTS sugar glycerol . In contrast, Q15H mutant Crh could phosphorylate the transcriptional activator LevR as well as LevD, the enzyme IIA of the fructose-specific lev-PTS, which together with enzyme I, HPr and LevE forms the phosphorylation cascade regulating induction of the lev operon via LevR . As a consequence, the constitutive expression from the lev promoter observed in a (delta)ptsH strain became inducible with fructose when the crhQ15H allele was expressed in this strain.

Microbiology, 1999 Nov, 145 ( Pt 11), 3129 - 38
Sequence analysis of three Bacillus cereus loci carrying PIcR-regulated genes encoding degradative enzymes and enterotoxin; Okstad OA et al.; PIcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase . Among the genes regulated by PIcR are: pIcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC); plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC); nhe, encoding the non-haemolytic enterotoxin; hbl, encoding haemolytic enterotoxin BL (HBL); and genes specifying a putative S-layer like surface protein and a putative extracellular RNase . By analysing 37.1 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted . Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified . The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes . Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR . Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.

Microbiology, 1999 Nov, 145 ( Pt 11), 3121 - 7
A novel member of the subtilisin-like protease family from Bacillus subtilis; Valbuzzi A et al.; aprX is a 1326 bp gene of Bacillus subtilis strain 168 that encodes a serine protease, probably intracellular, characterized by significant similarity with subtilisins, thermitases and pyrolysins . Transcription analysis, performed by RT-PCR and primer extension, allowed the localization of the active promoter and showed that aprX is expressed in stationary phase . The pattern of expression of aprX and its dependence on various transition state regulatory genes (degU, degQ, hpr, abrB, sinR), monitored by lacZ transcriptional fusions, are distinctive from those of subtilisin and other degradative enzymes . aprX is not essential for either growth or sporulation.

J AOAC Int, 1999 Nov-Dec, 82(6), 1407 - 12
Determination of fluoroquinolone residues in animal tissues using Escherichia coli as indicator organism; Choi J et al.; Three strains of Escherichia coli (ATCC 128, 10536, and 25922) and one strain of Bacillus subtilis (ATCC 3491) were compared as indicator microorganisms in microbial inhibition tests for their ability to detect fluoroquinolone residues . E . coli strains 128 and 10536 were most susceptible to fluoroquinolone residues, with detection limits of 35-50 micrograms/kg for enrofloxacin . Of the 2 strains, E . coli 10536 was slightly less susceptible . Ciprofloxacin was detected consistently by E . coli 128 at 30 micrograms/kg . Other fluoroquinolone drugs of veterinary interest detected by E . coli 128 were sarafloxacin and difloxacin at 100-250 micrograms/kg concentration . E . coli 25922 yielded 100% sensitivity in detection of enrofloxacin only at the 250 micrograms/kg concentration, and ciprofloxacin and sarafloxacin at 200 micrograms/kg . B . subtilis detected only enrofloxacin 100% of the time at 250 micrograms/kg . The E . coli strains tested were insensitive to other antibacterials commonly used in animals, with the exception of ceftiofur which was detected by E . coli 128 and 10536 at 500 micrograms/kg . The B . subtilis strain was not effective in detecting the fluoroquinolone drugs, whereas the E . coli strains were selective for the fluoroquinolones . E . coli 128 was 100% effective in detecting enrofloxacin and ciprofloxacin in spiked diaphragm homogenate samples at 50 micrograms/kg . Of the microorganisms tested, E . coli strain ATCC 128 was highly suitable as an indicator microorganism in a microbial inhibition assay for selective detection of fluoroquinolone antibacterial residues in animal tissues.

Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14553 - 8
An in vivo membrane fusion assay implicates SpoIIIE in the final stages of engulfment during Bacillus subtilis sporulation; Sharp MD et al.; Shortly after the synthesis of the two cells required for sporulation in Bacillus subtilis, the membranes of the larger mother cell begin to migrate around and engulf the smaller forespore cell . At the completion of this process the leading edges of the migrating membrane meet and fuse, releasing the forespore into the mother cell cytoplasm . We developed a fluorescent membrane stain-based assay for this membrane fusion event, and we isolated mutants defective in the final stages of engulfment or membrane fusion . All had defects in spoIIIE, which is required for translocation of the forespore chromosome across the polar septum . We isolated one spoIIIE mutant severely defective in chromosome translocation, but not in membrane fusion; this mutation disrupts the ATP/GTP-binding site of SpoIIIE, suggesting that ATP binding and hydrolysis are required for DNA translocation but not for the late engulfment function of SpoIIIE . We also correlated relocalization of SpoIIIE-green fluorescent protein from the sporulation septum to the forespore pole with the completion of membrane fusion and engulfment . We suggest that SpoIIIE is required for the final steps of engulfment and that it may regulate or catalyze membrane fusion events.

Int J Biochem Cell Biol, 1999 Oct, 31(10), 995 - 1000
Ferrochelatase; Ferreira GC; Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into protoporphyrin IX . It is encoded by a single gene, and mutations in the human gene are associated with the inherited disorder, erythropoietic protoporphyria . With the development of heterologous overexpression systems and the ready availability of recombinant ferrochelatase, new structural elements have been identified and new aspects of the ferrochelatase-catalyzed reaction mechanism have been unraveled . Namely, a {2Fe-2S} cluster is a prosthetic group in mammalian ferrochelatase, a conserved and essential histidine residue appears to be involved in the binding of the metal substrate and a conserved glutamate residue has been proposed to have a catalytic role . The three-dimensional structure for Bacillus subtilis ferrochelatase, the only known 'water-soluble' ferrochelatase, revealed that the protein contains two similar domains, each of which has a four-stranded beta-sheet flanked by alpha-helices; the active site was modeled to be in a cleft defined by the two domains . The definition of the structure and catalytic mechanism of ferrochelatase should help in the interpretation of the impact caused by erythropoietic porphyria mutations.

Nat Struct Biol, 1999 Dec, 6(12), 1091 - 5
Mg2+-dependent folding of a large ribozyme without kinetic traps; Fang XW et al.; The folding kinetics of the catalytic domain of Bacillus subtilis ribonuclease P is analyzed here by fluorescence and catalytic activity . The folding pathway is apparently free of kinetic traps, as indicated by a decrease in folding rates upon the addition of urea . We apply Mg2+ and urea chevron analysis to fully describe the folding and unfolding kinetics of this ribozyme . A folding scheme containing two kinetic intermediates completely accounts for the free energy, the Mg2+ Hill coefficient and the surface buried in the equilibrium transition . At saturating Mg 2+concentrations, folding is limited by a barrier that is independent of Mg2+ and urea . These results describe the first trap-free folding pathway of a large ribozyme and indicate that kinetic traps are not an obligate feature of RNA folding.

Lett Appl Microbiol, 1999 Oct, 29(4), 228 - 232
Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis; Leuschner RG et al.; Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis . Two germination phases were investigated . The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained . The incubation temperature (37 and 20 degrees C) and heat activation (10 min at 65 degrees C) had a distinct effect on both phases . At 37 degrees C, spores of B . subtilis 604 started to show a decrease in brightness in L-alanine buffer after 3-39 min and needed 10-39 min to complete the phase change . At 20 degrees C, lag times of 10-100 min were observed and the spores needed 30-100 min to reach a constant greylevel . Heat activation and subsequently exposure to L-alanine buffer at 20 degrees C reduced the lag phase to 6-90 min and the phase change was finished after 30-60 min . Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.

EMBO J, 1999 Dec 1, 18(23), 6823 - 31
Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily; Reuter K et al.; The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs . Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis . The structure of the Sfp-CoA complex determined at 1.8 A resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry . This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase . The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule . CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures . The structure reveals regions likely to be involved in the interaction with the PCP substrate.

J Nat Prod, 1999 Nov, 62(11), 1479 - 83
Novel highly oxygenated bisabolane sesquiterpenes from Cremanthodium discoideum; Zhu Y et al.; Five new highly oxygenated bisabolane sesquiterpenes (1-5) were isolated from Cremanthodium discoideum . Their structures were elucidated on the basis of spectroscopic analysis and chemical transformations . The structure and relative stereochemistry of 1 were determined by single-crystal X-ray crystallography on the acetate derivative, 1a . Compound 1 showed antibacterial activity against Bacillus acidilatici and Bacillus subtilis.

Mikrobiologiia, 1999 Jul-Aug, 68(4), 534 - 9
{Sensitivity of soil bacteria isolated from the alienated zone around the Chernobyl Nuclear Power Plant to various stress factors}; Romanovskaia VA et al.; Seventy strains of chemoorganotrophic bacteria isolated by our group in 1993-1994 from soil sampled in the zone around the Chernobyl Nuclear Power Plant (ChNPP) were studied with respect to their sensitivity to various stress factors damaging DNA . Bacillus subtilis, B . cereus (both spores and vegetative cells), Methylobacterium extorquens, M . mesophilicum, and unidentified pigmented bacteria were found to be the most resistant to ultraviolet (UV) radiation, exhibiting LD90 values of 40 to more than 211 J/m2 . The same bacteria, as well as Bacillus polymyxa, were tolerant to hydrogen peroxide (lethal concentrations of H2O2 ranged from 0.3 to 1.0 M); i.e., UV-resistant strains were also tolerant to hydrogen peroxide and vice versa . Fluorescent pseudomonads were the most sensitive to both UV radiation and H2O2, showing LD90 from 6 to 18 J/m2 and a lethal concentration of H2O2 lower than 0.1 M . All of the soil samples collected in the alienated zone around the ChNPP, where the radioactivity of the soil had decreased from 1000 to 2 microCi/kg soil over the period from 1987 to 1995, contained not only resistant bacteria but also a small number of bacteria sensitive to UV radiation and H2O2.

DNA Res, 1999 Oct 29, 6(5), 255 - 64
Identification of yrrU as the methylthioadenosine nucleosidase gene in Bacillus subtilis; Sekowska A et al.; Taking trimethoprim as the selective agent in the presence of thymine, we adapted to Bacillus subtilis a selection procedure depending on the peculiar organisation of the one-carbon metabolism . The corresponding pathways couple synthesis of thymine to tetrahydrofolate consumption as a substrate of the reaction mediated by thymidylate synthase, instead of being a co-enzyme as in the other reactions transferring one-carbon groups . Mutants obtained are thymidylate synthase deficient, and therefore auxotrophic for thymine . This provides positive selection in a first step for gene replacement by a thymidylate synthase cassette, and subsequently against its presence . For systematic recombination of mutations constructed in vitro, we used the property of B . subtilis to grow at high temperature, noting that the thyB gene product is inactive at 46 degrees C, while the product of thyA remains active at this temperature . As the first step, we built up a recipient thyA- background, deleting the gene by in situ recombination . This method was used to investigate the function of the yrrU gene, which is presumably involved in a sulfur recycling pathway associated with polyamine biosynthesis . We showed that yrrU codes for a protein recycling methylthioadenosine, probably a nucleosidase . In addition we observed that B . subtilis can use methylthioribose as a sulfur source, and that it is an efficient sulfur scavenger.

Int J Food Microbiol, 1999 Oct 15, 51(2-3), 183 - 6
The production of 'Kpaye'--a fermented condiment from Prosopis africana (Guill and Perr) Taub . Seeds; Omafuvbe BO et al.; 'Kpaye', a fermented condiment from Prosopis africana seeds was produced using the traditional method . Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus were consistently isolated in the fermentations lasting 120 h . 'Kpaye'production involved a rise in pH, moisture content and total free amino acids while titratable acidity and reducing sugar decreased gradually after 24 h and 72 h of fermentation respectively.

RNA, 1999 Oct, 5(10), 1277 - 89
Probing the TRAP-RNA interaction with nucleoside analogs; Elliott MB et al.; The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2-3 nonconserved spacer nucleotides in trp leader mRNA . To identify chemical groups on the RNA required for stability of the TRAP-RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5'-(UAGCC)-3' repeated 11 times and measured their effect on the TRAP-RNA interaction . Deoxyribonucleoside substitutions revealed that a 2'-hydroxyl group (2'-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP . The remaining hydroxyl groups are dispensable . Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP . In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability . Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP-RNA complex is provided by the AG dinucleotide of the triplet repeats . In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2'-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.

Biochimie, 1999 Aug-Sep, 81(8-9), 847 - 57
Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: involvement of ATPase/helicase activity of PriA for inducible stable DNA replication; Masai H et al.; The E . coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the phiX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks . B . subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E . coli counterpart . We overexpressed and purified BsPriA and examined its biochemical properties . BsPriA binds specifically to both n'-pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'-pas albeit with a specific activity about 30% of that of E . coli PriA . However, it is not capable of supporting n'-pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the phiX174-type primosome . We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance . Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant . K230D was previously reported to be fully functional in assembly of the phiX174-type primosome at a single-stranded n'-pas . Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.

J Bacteriol, 1999 Dec, 181(23), 7346 - 55
Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin; Zheng G et al.; Bacillus subtilis JH642 and a wild strain of B . subtilis called 22a both produce an antilisterial peptide that can be purified by anion-exchange and gel filtration chromatography . Amino acid analysis confirmed that the substance was the cyclic bacteriocin subtilosin . A mutant defective in production of the substance was isolated from a plasmid gene disruption library . The plasmid insertion conferring the antilisterial-peptide-negative phenotype was located in a seven-gene operon (alb, for antilisterial bacteriocin) residing immediately downstream from the sbo gene, which encodes the precursor of subtilosin . An insertion mutation in the sbo gene also conferred loss of antilisterial activity . Comparison of the presubtilosin and mature subtilosin sequences suggested that certain residues undergo unusual posttranslational modifications unlike those occurring during the synthesis of class I (lantibiotic) or some class II bacteriocins . The putative products of the genes of the operon identified show similarities to peptidases and transport proteins that may function in processing and export . Two alb gene products resemble proteins that function in pyrroloquinoline quinone biosynthesis . The use of lacZ-alb and lacZ-sbo gene fusions, along with primer extension analysis, revealed that the sbo-alb genes are transcribed from a major promoter, residing upstream of sbo, that is very likely utilized by the sigma(A) form of RNA polymerase . The sbo and alb genes are negatively regulated by the global transition state regulator AbrB and are also under positive autoregulation that is not mediated by the subtilosin peptide but instead requires one or more of the alb gene products.

J Bacteriol, 1999 Dec, 181(23), 7323 - 30
Protection against 3'-to-5' RNA decay in Bacillus subtilis; Farr GA et al.; A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing . The RNA consisted of a 5'-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3'-proximal end of the ermC transcriptional unit . Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro . Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure . This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase . An abundant RNA with a 3' end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3'-to-5' exonuclease . The finding of impediments to 3'-to-5' degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.

J Bacteriol, 1999 Dec, 181(23), 7314 - 22
Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity; McGee DJ et al.; Arginase of the Helicobacter pylori urea cycle hydrolyzes L-arginine to L-ornithine and urea . H . pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid . Both enzymes are involved in H . pylori nitrogen metabolism . The roles of arginase in the physiology of H . pylori were investigated in vitro and in vivo, since arginase in H . pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium . The H . pylori gene hp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H . pylori strains were made by using two different constructs: 236-2 and rocF::aphA3 . In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium . Compared with WT strain 26695 of H . pylori, the rocF::aphA3 mutant was approximately 1, 000-fold more sensitive to acid exposure . The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of L-arginine, in contrast to the WT, and yielded a approximately 10, 000-fold difference in viability . Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro . Finally, H . pylori mouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively . However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice) . The geometric mean levels of H . pylori recovered from these mice (in log(10) CFU) were 6.1, 5.5, and 4.1, respectively . Thus, H . pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.

J Bacteriol, 1999 Dec, 181(23), 7154 - 60
Bacillus subtilis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression; Yasueda H et al.; The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs . The characteristic enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to exist outside methylotrophs . However, the presumed yckG gene product (YckG) of Bacillus subtilis shows a primary structure similar to that of methylotroph HPS (F . Kunst et al., Nature 390:249-256, 1997) . We have also investigated the sequence similarity between the yckF gene product (YckF) and methylotroph PHI (Y . Sakai, R . Mitsui, Y . Katayama, H . Yanase, and N . Kato, FEMS Microbiol . Lett . 176:125-130, 1999) and found that the yckG and yckF genes of B . subtilis express enzymatic activities of HPS and PHI, respectively . Both of these activities were concomitantly induced in B . subtilis by formaldehyde, with induction showing dependence on the yckH gene, but were not induced by methanol, formate, or methylamine . Disruption of either gene caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system for formaldehyde in B . subtilis . In conclusion, we found an active yckG (for HPS)-yckF (for PHI) gene structure (now named hxlA-hxlB) in a nonmethylotroph, B . subtilis, which inherently preserves the RuMP pathway.

J Agric Food Chem, 1999 Feb, 47(2), 363 - 71
Characteristics and use of okara, the soybean residue from soy milk production--a review; O'Toole DK; Large quantities of okara produced annually pose a significant disposal problem . It contains mostly crude fiber composed of cellulose, hemicellulose, and lignin, about 25% protein, 10-15% oil, but little starch or simple carbohydrates . It is a suitable dietary additive in biscuits and snacks because it reduces calorie intake and increases dietary fiber . The high-quality protein fraction has good water holding and emulsifying qualities and contains a peptide with anti-hypertension effects . The pectic polysaccharides fraction is suitable for thickening acid milk products . Okara fermented with Actinomucor elegans (meitauza), Aspergillus oryzae (koji), Neurospora intermedia (ontjom), and Rhizopus oligosporus (tempe), on consumption, reduces cholesterol level and contains substances that counteract dietary free radicals . Unique and useful products produced by Bacillus subtilis and Penicillium simplicissimum on okara include surfactin and iturin A (fungicidal), okaramines A, B, D-F (D is insecticidal), an oleanane triterpene, and two dihydroquinolinones (one toxic for Artemia salina) . Okara has been used as silkworm food and in the production of ceramics.

Int J Biol Macromol, 1999 Dec 1, 26(4), 243 - 8
New molecular complexes of heterocyclic bis-adducts with bacterial lectins: synthesis and structure-activity relationship studies; Welchinskaya HV et al.; A strongly antitumour effect has been discovered for lectins of Bacillus bacteria {Bacillus subtilis 668(1 + 2)IMV, Bacillus polymyxa 102(1 + 2) KGU} and for their molecular complexes with some heterocyclic bis-adducts of unsubstituted benzimidazole and 6-methyluracile for the first time . These were tested on the tumours: Lymphosarcoma Plissa, Sarcoma 45, Carcinosarcoma Yokera 256 . A new convenient method for the preparation of the heterocyclic bisadducts of imidazole, benzimidazole, uraciles with 1,1,1-trifluoro-2-bromo-2-chloroethane is described . The reactions are catalysed by the 18-crown-6-complex.

Tsitol Genet, 1999 Jul-Aug, 33(4), 3 - 8
{The DNA of recombinant plasmids acquires mutagenic activity in a Bacillus subtilis culture after the insertion of the human genome Alu repeat}; Karpova IS et al.; In our previous work the mutagenic activity of recombinant plasmids (pBR322 carrying the human Alu repeat alone or in combination with preproinsulin or apo AI gene) in competent Bacillus subtilis culture was demonstrated . In present work it was shown that among seven tested plasmids only three revealed mutagenic activity (pBR322 and two Alu repeat-containing constructions) . It seems that mutagenic activity is not inherent to any recombinant molecule in applied test system but depends on its structure . For example, Alu repeat of human genome may attach mutagenic properties to plasmids which either had no such properties, or lost them after gene engineering manipulations.

J Biol Chem, 1999 Nov 19, 274(47), 33601 - 8
Interactions of the major cold shock protein of Bacillus subtilis CspB with single-stranded DNA templates of different base composition; Lopez MM et al.; CspB is a small acidic protein of Bacillus subtilis, the induction of which is increased dramatically in response to cold shock . Although the exact functional role of CspB is unknown, it has been demonstrated that this protein binds single-stranded deoxynucleic acids (ssDNA) . We addressed the question of the effect of base composition on the CspB binding to ssDNA by analyzing the thermodynamics of CspB interactions with model oligodeoxynucleotides . Combinations of four different techniques, fluorescence spectroscopy, gel shift mobility assays, isothermal titration calorimetry, and analytical ultracentrifugation, allowed us to show that: 1) CspB can preferentially bind poly-pyrimidine but not poly-purine ssDNA templates; 2) binding to T-based ssDNA template occurs with high affinity (K(d(25 degrees C)) approximately 42 nM) and is salt-independent, whereas binding of CspB to C-based ssDNA template is strongly salt-dependent (no binding is observed at 1 M NaCl), indicating large electrostatic component involved in the interactions; 3) upon binding each CspB covers a stretch of 6-7 thymine bases on T-based ssDNA; and 4) the binding of CspB to T-based ssDNA template is enthalpically driven, indicating the possible involvement of interactions between aromatic side chains on the protein with the thymine bases . The significance of these results with respect to the functional role of CspB in the bacterial cold shock response is discussed.

Biochem Biophys Res Commun, 1999 Nov 19, 265(2), 305 - 10
Expression of the Csp protein family upon cold shock and production of tetracycline in Streptomyces aureofaciens; Mikulik K et al.; A shift down in temperature causes in Streptomyces aureofaciens a transient repression of polypeptide synthesis . During the acclimation phase 32 proteins were synthesized . The addition of tetracycline (200 microg/ml) to cells from exponential phase of growth leads to induction of 27 novel proteins and 17 upregulated proteins migrated in 2-D gel as proteins expressed upon cold shock . Immunoblot analysis using antibodies raised against CspB, CspC, and CspD of Bacillus subtilis revealed five cross-reactive proteins of the Csp family . Proteins CspB and CspD are predominantly induced at low temperature or by the presence of tetracycline . Expression of Csp proteins during the acclimation phase is regulated on the transcription level . Proteins of the Csp family have been shown to be associated with ribosomes and can be removed by 1 M NH(4)Cl . As expression of Csp proteins differs during development or temperature shift down, these proteins can be considered as trans-acting factors to form contacts with the coding region of specific mRNAs .

J Bacteriol, 1999 Nov, 181(22), 7087 - 97
A mutation in the 3-phosphoglycerate kinase gene allows anaerobic growth of Bacillus subtilis in the absence of ResE kinase; Nakano MM et al.; The Bacillus subtilis ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration . A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated . In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain . The suppressing mutation, which mapped to pgk, the gene encoding 3-phosphoglycerate kinase, failed to suppress a resD mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE . The pgk-1 mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain . The results suggest that accumulation of a glycolytic intermediate, probably 1, 3-diphosphoglycerate, is responsible for the observed effect of the pgk-1 mutation on anaerobiosis of resE mutant cells.

J Bacteriol, 1999 Nov, 181(22), 7065 - 9
Control of synthesis and secretion of the Bacillus subtilis protein YqxM; Stover AG et al.; yqxM is a Bacillus subtilis gene of unknown function residing in an operon with sipW, which encodes a signal peptidase, and tasA, which encodes an antibiotic protein secreted in a sipW-dependent manner . YqxM was undetectable during growth in a variety of rich media, including Luria-Bertani (LB) medium, or in minimal media or under heat shock or ethanol stress conditions but was synthesized and secreted during growth in LB medium supplemented with 1.2 M NaCl . Consistent with the possible involvement of sipW in YqxM secretion, inactivation of sipW prevented YqxM secretion . YqxM was produced and secreted in a sipW-dependent manner during growth in LB medium when the sequences upstream of yqxM were replaced with those of the inducible P(spac) promoter . Coexpression of yqxM and sipW in Escherichia coli resulted in a decrease in the apparent molecular mass of YqxM, consistent with the removal of a signal peptide . These experiments suggest that YqxM production is induced by a high concentration of salt and that YqxM is secreted under the control of SipW . We hypothesize that during most conditions of growth, YqxM is present at very low levels or is not synthesized at all and that this low level or absence is due, at least in part, to posttranscriptional repression.

J Bacteriol, 1999 Nov, 181(22), 7043 - 51
Functional regions of the Bacillus subtilis spore coat morphogenetic protein CotE; Bauer T et al.; The Bacillus subtilis spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment . A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat's outer layer . We have used a series of mutant alleles of cotE to identify regions involved in outer coat protein assembly . We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB . The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing . In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore . These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly.

J Bacteriol, 1999 Nov, 181(22), 7028 - 33
Transcriptional control of the low-temperature-inducible des gene, encoding the delta5 desaturase of Bacillus subtilis; Aguilar PS et al.; The Bacillus subtilis des gene encodes the cold-inducible Delta5 lipid desaturase involved in the formation of unsaturated fatty acids from saturated phospholipid precursors . Here, we describe the expression pattern of the des gene in response to a temperature downshift from 37 to 20 degrees C . We found that the synthesis of des mRNA is undetectable at 37 degrees C but dramatically induced upon the temperature downshift . Decay characteristics of the des transcript as well as the in vivo decay of B . subtilis bulk mRNA were investigated . The results showed that the stability of the des transcript as well as of bulk mRNA lasted substantially longer at 20 degrees C than at 37 degrees C . Functional expression of des at 37 degrees C was achieved by exchanging its promoter with the non-cold shock spac promoter . These data provide the first direct evidence that temperature-mediated control of transcription is the major mechanism regulating the mRNA levels of the B . subtilis desaturase . The present results also demonstrate that the only component of the desaturation system regulated by temperature is the desaturase enzyme.

J Bacteriol, 1999 Nov, 181(22), 7021 - 7
Preprotein translocation by a hybrid translocase composed of Escherichia coli and Bacillus subtilis subunits; Swaving J et al.; Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits . Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B . subtilis SecA-SecYE complex . To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E . coli and B . subtilis secY, secE, and secG genes were expressed in E . coli . Hybrid SecYEG complexes were overexpressed at high levels . Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits . E . coli SecA, but not B . subtilis SecA, supported efficient ATP-dependent translocation of the E . coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E . coli SecY and either E . coli SecE or E . coli SecG were present . Translocation of B . subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase . In contrast to E . coli SecA, B . subtilis SecA binds the SecYEG complexes only with low affinity . These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.

J Bacteriol, 1999 Nov, 181(22), 6996 - 7004
Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis; Tobisch S et al.; The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells . Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated . Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi, pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose . This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis . Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpA mutant . Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes . In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the ccpA mutation . Our data suggest (i) that ccpA mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.

J Bacteriol, 1999 Nov, 181(22), 6889 - 97
Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis; Presecan-Siedel E et al.; In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium . Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose . Inactivation of pta had only a weak inhibitory effect on growth . In contrast to pta and ackA in Escherichia coli, the corresponding B . subtilis genes are not cotranscribed . Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA . The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated . As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant . Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence . This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP) . In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13294 - 9
The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase; Duitman EH et al.; Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic . Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a beta-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration . The gene cluster from B . subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified . The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases . Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype . The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases . MycA represents the first example of a natural hybrid between these enzyme families . (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases . On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics . Comparison of the sequences flanking the mycosubtilin operon of B . subtilis ATCC6633, with the complete genome sequence of B . subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B . subtilis strains.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13074 - 9
Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family; Sinha S et al.; The yabJ gene in Bacillus subtilis is required for adenine-mediated repression of purine biosynthetic genes in vivo and codes for an acid-soluble, 14-kDa protein . The molecular mechanism of YabJ is unknown . YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function . The 1.7-A crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity . The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs . This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c/YjgF/UK114 family of proteins.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 287 - 96
Localisation of the cell wall-associated phosphodiesterase PhoD of Bacillus subtilis; Muller JP et al.; The localisation of phosphate-starvation-induced phosphodiesterase PhoD from Bacillus subtilis was studied by analysing processing, release and immunogold labelling of the sections . Although the processing of the pre-protein was extremely slow, the major fraction of PhoD could be detected at the surface of the cell wall . The results indicate that inefficient processing of the translocated pre-protein keeps PhoD in a cell wall-associated location . The uncleaved signal peptide might function as a membrane anchor.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 133 - 9
Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme; Park T et al.; The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli . The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence . The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues . Its molecular mass was determined to be 54 inverted question mark omitted inverted question mark760 Da . On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E . coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases . High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E . coli alkaline phosphatase . Comparison of Tca alkaline phosphatase and E . coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites . Heat-stable Tca alkaline phosphatase activity was detected in E . coli YK537, harboring pJRAP.

Nucleic Acids Res, 1999 Dec 1, 27(23), 4541 - 6
Adenines at -11, -9 and -8 play a key role in the binding of Bacillus subtilis Esigma(A) RNA polymerase to -10 region single-stranded DNA; Qiu J et al.; The sigma subunit of RNA polymerase interacts with the promoter DNA in at least two regions: the -35 and the -10 consensus elements . The latter contacts are involved both in recognition and in melting of the promoter DNA to form the transcriptionally-competent open complex . RNA polymerase holoenzyme, but neither core nor sigma alone, binds with high selectivity to single-stranded DNA (ssDNA) containing the non-template -10 consensus sequence . We have used equilibrium competition to assess the specificity of holoenzyme binding to a 19 base oligonucleotide containing a -10 consensus element, TATAAT . Analysis of all 18 possible single point mutations in the -10 consensus sequence reveals that binding by Bacillus subtilis Esigma(A)holoenzyme depends critically upon adenine at position -11 and, unexpectedly, is strongly affected by substitutions of the poorly conserved adenines at -9 and -8 . Similarly, ssDNA binding by Escherichia coli Esigma(70)holoenzyme is most strongly affected by substitutions of adenines within the -10 region consensus . The critical role of -11A in binding ssDNA supports a key role for this base in the nucleation of DNA melting . A novel role for -9A and -8A is proposed in the context of recent models of promoter melting.

New Microbiol, 1999 Oct, 22(4), 315 - 22
Effect of glutathione L-cystein and L-djenkolic acid in the synthesis and mutagenicity of azide metabolite in Bacillus subtilis ATCC 6633 strain; Elbetieha A et al.; The Bacillus subtilis ATCC 6633 strain synthesizes a mutagenic metabolite from sodium azide and O-acetylserine . Mutagenicity of azide was decreased in growth media containing 10(-4) M glutathione, L-cysteine or L-djenkolic acid whereas dithiothritol (DTT) added at the same concentration did not reduce the mutagenicity of azide . Likewise, glutathione, L-cysteine, L-djenkolic acid, and DTT were found to have no effect in reducing the mutagenicity of the in vitro produced metabolite using bacterial cell-free extract . These results suggest that O-acetyl-serine sulfhydrylase catalyzes the reaction of azide and O-acetylserine to form a mutagenic metabolite, which is ninhydrin positive and migrates in TLC to an Rf value similar to that of azidoalanine in both acidic and basic solvent systems.

Hautarzt, 1999 Oct, 50(10), 701 - 5
{Measurement and evaluation of natural and artificial UV radiation}; Krins A et al.; Natural and artificial UV radiation are environmental factors with both beneficial and harmful biological effects . This article will explain the physical measurement quantities and their relation to the biologically effective dose and will summarize the present technical state of the art of personal UV monitoring . In practical use are dosimeters based on polysulphone, a polymer which undergoes changes in its optical properties upon irradiation with UV . Other systems determine the UV dose by quantifying damage induced in Bacillus subtilis spores upon UV exposure . An electronic UV sensor represents a new and interesting development . Personal UV dosimeters will become an useful tool in both clinical and scientific areas within dermatology.

J Biol Chem, 1999 Nov 12, 274(46), 32810 - 7
A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168; Azarkina N et al.; The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases . The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase . The cytochrome bd content of LUH27 can be varied by using different growth conditions . LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D . The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O . The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B . subtilis . The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily . The genome sequence of B . subtilis 168 contains gene clusters for four respiratory oxidases . Two of these clusters, cta and qox, are deleted in LUH27 . The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively . Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase . It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e . cytochrome b(558)b(595)b'.

Clin Diagn Lab Immunol, 1999 Nov, 6(6), 930 - 3
Detection of borreliacidal antibodies in Lyme borreliosis patient sera containing antimicrobial agents; Jobe DA et al.; The borreliacidal-antibody test has been used for the serological detection and confirmation of Lyme borreliosis . However, the presence of antimicrobial agents in serum can confound the accurate detection of borreliacidal antibodies . In this study, we developed a Bacillus subtilis agar diffusion bioassay to detect small concentrations of antimicrobial agents in serum . We also used XAD-16, a nonionic polymeric resin, to adsorb and remove high concentrations of amoxicillin, cefotaxime, ceftriaxone, cefuroxime, doxycycline, and erythromycin without significantly affecting even small concentrations of immunoglobulin M (IgM) or IgG borreliacidal antibodies . High concentrations of penicillin could also be removed by adding 1 U of penicillinase without significantly influencing the levels of borreliacidal antibodies . These simple procedures greatly enhance the clinical utility of the borreliacidal-antibody test.

Phytother Res, 1999 Nov, 13(7), 616 - 8
Antimicrobial screening of some Indian spices; De M et al.; In India, spices have been traditionally used since ancient times, for the preservation of food products as they have been reported to have antiseptic and disinfectant properties . In this respect, a preliminary screening for antimicrobial activities of 35 different Indian spices has been carried out . Of the spices surveyed, the results indicate that clove, cinnamon, bishop's weed, chilli, horse raddish, cumin, tamarind, black cumin, pomegranate seeds, nutmeg, garlic, onion, tejpat, celery, cambodge, have potent antimicrobial activities against the test organisms Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 10536) and Saccharomyces cerevisiae (ATCC 9763) . The results also establish the traditional use of spices as food preservatives, disinfectants and antiseptics .

J Mol Biol, 1999 Nov 12, 293(5), 997 - 1004
Quaternary re-arrangement analysed by spectral enhancement: the interaction of a sporulation repressor with its antagonist; Scott DJ et al.; The protein/protein interaction between SinI and SinR has been studied by analytical ultracentrifugation and gel electrophoresis in an attempt to understand how these proteins contribute to developmental control of sporulation in Bacillus subtilis . SinR was found to be tetrameric, while SinI was found to exist as monomers and dimers in a rapidly reversible equilibrium . Labelling of SinR by incorporating the tryptophan analogue 7-azatryptophan (7AW) into the protein in place of tryptophan shifts the UV absorbance spectrum, thus allowing selective monitoring of 7AWSinR at 315 nm using the UV absorption optics of the analytical ultracentrifuge.Selective monitoring of SinR in mixtures of SinR and SinI enables the binding and stoichiometry of the interaction to be investigated quantitatively and unambiguously . We demonstrate that the oligomeric forms of SinR and SinI re-arrange to form a tight 1:1 SinR:SinI complex, with no stable intermediate species . A fragment of SinR, SinR(1-69), which contains only the DNA-binding domain, was found to be monomeric, showing that the protein appears not to oligomerise in a similar manner to the Cro repressor, a protein with which it shares a marked structural similarity .

Biochem Cell Biol, 1999, 77(4), 343 - 7
Aminoacyl-tRNA synthetase genes of Bacillus subtilis: organization and regulation; Pelchat M et al.; In Bacillus subtilis, 14 of the 24 genes encoding aminoacyl-tRNA synthetases (aaRS) are regulated by tRNA-mediated antitermination in response to starvation for their cognate aminoacid . Their transcripts have an untranslated leader mRNA of about 300 nucleotides, including alternative and mutually exclusive terminator-antiterminator structures, just upstream from the translation initiation site . Following antitermination, some of these transcripts are cleaved leaving at the 5'-end of the mature mRNAs, stable secondary structures that can protect them against degradation . Although most B . subtilis aaRS genes are expressed as monocistronic mRNAs, the gltX gene encoding the glutamyl-tRNA synthetase is cotranscribed with cysE and cysS encoding serine acetyl-transferase and cysteinyl-tRNA synthetase, respectively . Transcription of gltX is not controlled by a tRNA, but tRNA(CyS)-mediated antitermination regulates the elongation of transcription into cysE and cysS . The full-length gltX-cysE-cysS transcript is then cleaved into a monocistronic gltX mRNA and a cysE-cysS mRNA.

Arch Biochem Biophys, 1999 Nov 15, 371(2), 191 - 201
Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits; Kahler AE et al.; Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subunits, PyrDI (M(r) = 33,094) and PyrDII (M(r) = 28,099) . The two subunits were overexpressed jointly and individually and purified . PyrDI was an FMN-containing flavoprotein with an apparent native molecular mass of 85,000 . Overexpressed PyrDII formed inclusion bodies and was purified by refolding and reconstitution . Refolded PyrDII bound 1 mol FAD and 1 mol {2Fe-2S} per mol PyrDII . Coexpression and purification of PyrDI and PyrDII yielded a native holoenzyme complex with an apparent native molecular mass of 114,000 that indicated a heterotetramer (PyrDI(2)PyrDII(2)) . The holoenzyme possessed dihydroorotate:NAD(+) oxidoreductase activity and could also reduce menadione and artificial dyes . Purified PyrDI also possessed DHOD activity but could not reduce NAD(+) . Compared to PyrDI, the holoenzyme had a more than 20-fold smaller K(m) value for dihydroorotate, an approximately 50-fold smaller K(i) value for orotate, and approximately 500-fold greater catalytic efficiency . Dihydroorotate:NAD(+) oxidoreductase activity could be recovered by mixing the purified subunits . Recovered activity showed a clear dependence on FAD reconstitution of PyrDII but not on reconstitution with FeS clusters . PyrDII had a strong preference for FAD over FMN and bound it with an estimated K(d) value of 4.9 +/- 0.8 nM . pyrDII mutants containing alanine substitutions of the cysteine ligands to the {2Fe-2S} cluster failed to complement the pyr bradytrophy of a DeltapyrDII strain, indicating a requirement for the FeS cluster in PyrDII for normal function in vivo .

Biochemistry, 1999 Nov 2, 38(44), 14638 - 43
Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase; Zhang YW et al.; Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II . Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I . Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity . To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis . Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme . The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay . Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length . These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.

J Biol Chem, 1999 Nov 5, 274(45), 32318 - 24
Studies on the ADP-ribose pyrophosphatase subfamily of the nudix hydrolases and tentative identification of trgB, a gene associated with tellurite resistance; Dunn CA et al.; Four Nudix hydrolase genes, ysa1 from Saccharomyces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E . coli . The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose . Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily . In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases . On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase . In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E . coli, increased the transformant's tolerance to potassium tellurite.

J Mol Biol, 1999 Nov 5, 293(4), 795 - 805
Specific interaction of the RNA-binding domain of the bacillus subtilis transcriptional antiterminator GlcT with its RNA target, RAT; Langbein I et al.; Expression of the Bacillus subtilis ptsGHI operon is controlled by transcriptional antitermination mediated by the antiterminator protein GlcT . The antiterminator is inactivated in the absence of glucose, presumably by phosphorylation . A conditional terminator in the ptsG mRNA leader region has been identified . Mutations in this terminator resulted in constitutive expression of the operon . The terminator is overlapped by an inverted repeat (called ribonucleic-antiterminator, RAT) which is thought to form a stem-loop structure upon binding of the antiterminator protein GlcT . The N-terminal 60 amino acid residues of GlcT are able to bind to the RAT and prevent transcriptional termination in vivo . Sequence-specific interaction between the RNA-binding domain and the RAT was demonstrated by surface plasmon resonance analysis . Mutations affecting the RNA-binding domain were isolated and will be discussed with respect to their consequences for dimerization and RNA binding .

Microbiology, 1999 Oct, 145 ( Pt 10), 2957 - 66
Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm-pupG operon; Schuch R et al.; In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized . The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase . The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here . When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity . Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon . Both enzymes are induced when nucleosides are present in the growth medium . Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate . Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression . Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells . The nucleoside-catabolizing system in B . subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source . When added alone, inosine barely supports growth of B . subtilis . This slow nucleoside catabolism contrasts with that of E . coli, which grows rapidly on a nucleoside as a carbon source . When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B . subtilis . The findings of this study therefore indicate that the B . subtilis system for nucleoside catabolism differs greatly from the well-studied system in E . coli.

Microbiology, 1999 Oct, 145 ( Pt 10), 2923 - 30
Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells; Triccas JA et al.; The identification of Mycobacterium tuberculosis genes expressed within host cells would contribute greatly to the development of new strategies to combat tuberculosis . By combining the natural fluorescence of the Aequoria victoria green fluorescent protein (GFP) with the counterselectable property of the Bacillus subtilis SacB protein, M . tuberculosis promoters displaying enhanced in vivo activity have been isolated . Macrophages were infected with recombinant Mycobacterium bovis bacille Calmette-Guerin containing a library of M . tuberculosis promoters controlling gfp and sacB expression, and fluorescent bacteria recovered by fluorescence-activated cell sorting . The expression of sacB was used to eliminate clones with strong promoter activity outside the macrophage, resulting in the isolation of seven clones containing M . tuberculosis promoters with greater activity intracellularly . The gene products identified displayed similarity to proteins from other organisms whose functions include nutrient utilization, protection from oxidative stress and defence against xenobiotics . These proposed functions are consistent with conditions encountered within the host cell and thus suggest that the augmented activity of the isolated promoters/genes may represent strategies employed by M . tuberculosis to enhance intracellular survival and promote infection.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 437 - 9
Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity; Kacena MA et al.; The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir . Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase . Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B . subtilis and E . coli cultures, respectively, than in ground cultures . Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

Infect Immun, 1999 Nov, 67(11), 6213 - 6
P48 major surface antigen of Mycoplasma agalactiae is homologous to a malp product of Mycoplasma fermentans and belongs to a selected family of bacterial lipoproteins; Rosati S et al.; A major surface antigenic lipoprotein of Mycoplasma agalactiae, promptly recognized by the host's immune system, was characterized . The mature product, P48, showed significant similarity and shared conserved amino acid motifs with lipoproteins or predicted lipoproteins from Mycoplasma fermentans, Mycoplasma hyorhinis, relapsing fever Borrelia spp., Bacillus subtilis, and Treponema pallidum.

Biochem Biophys Res Commun, 1999 Oct 22, 264(2), 380 - 7
Cytochrome c-553 from the alkalophilic bacterium Bacillus pasteurii has the primary structure characteristics of a lipoprotein; Vandenberghe IH et al.; The complete sequence of Bacillus pasteurii cytochrome c-553 was determined by standard methods of Edman degradation of overlapping peptides combined with mass spectrometry . The protein contains 92 residues and a single heme-binding site . It is most similar to Bacillus licheniformis, Bacillus PS3, and Bacillus subtilis cytochromes c-551, which are lipoproteins that are partially solubilized through proteolytic cleavage of the N-terminal diacyl-glyceryl-cysteine membrane anchor . The high yield of the B . pasteurii cytochrome c-553, together with evidence that shorter forms of the cytochrome occur in the mixture of otherwise pure protein, suggests that the membrane anchor is very susceptible to proteolysis and that the soluble form of the cytochrome is therefore released from the membrane upon cell breakage . A sequence-based calculation of the protein secondary structure suggests the presence of a typical cytochrome helical fold with a random-coil N-terminus tail .

Lipids, 1999 Aug, 34(8), 841 - 6
Characterization of the ybdT gene product of Bacillus subtilis: novel fatty acid beta-hydroxylating cytochrome P450; Matsunaga I et al.; We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis . A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis . In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product . The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450 . In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce beta-hydroxymyristic acid and alpha-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry . The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture . The amount of beta-hydroxyl product was slightly higher than that of alpha-hydroxyl product at all times during the reaction . However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used . HPLC analysis with a chiral column showed that the beta-hydroxyl product was nearly enantiomerically pure R-form . These results suggest that this P450 enzyme is involved in a novel biosynthesis of beta-hydroxy fatty acid.

Radiat Environ Biophys, 1999 Sep, 38(3), 175 - 84
The track structures of ionizing particles and their application to radiation biophysics . I . A new analytical method for investigating two biophysical models; Briden PE et al.; A new approach to the interpretation of the effects of radiation on cells is described, in which sample particle tracks are constructed using a Monte Carlo computer program and the exposure of cellular targets to these tracks is simulated using a second program known as BIOPHYS . Data on the shapes and DNA contents of the cell nuclei are obtained from the literature . It is assumed that the sensitive material is DNA, and that the target is divided into cubes of approximately 2 nm (the diameter of the DNA helix) per side; the numbers of these cubes containing different numbers of ionizations are derived . Two different methods of analysing the output of BIOPHYS are described . In the first, it is assumed that lethality is caused by the occurrence of a number of ionizations equal to or greater than a certain threshold in one cube; in the second method, it is assumed that only two ionizations are required, in different parts of the cube, but that only some fraction of the cube is sensitive . These models have been applied to the interpretation of the variation of radiosensitivity with a linear energy transfer (LET) of spores of Bacillus subtilis exposed wet and dry, and good fits to the published experimental data were obtained using both models . Fits to experimental data for a range of other cell lines will be presented in a second paper.

Methods, 1999 Sep, 19(1), 156 - 62
Vaccine entrapment in liposomes; Gregoriadis G et al.; The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment . Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines . Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines . On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used . When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles . A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores . On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size .

Gene, 1999 Sep 3, 237(1), 45 - 52
Identification of new sigma K-dependent promoters using an in vitro transcription system derived from Bacillus subtilis; Fujita M; In Bacillus subtilis, the genes that depend on sigma K-RNA polymerase for their transcription are expressed in the mother cell compartment at later stages of sporulation . More than a dozen genes belonging to the sigma K regulon have been identified . Here I describe the identification of two additional promoters under the control of sigma K-RNA polymerase . Using a set of histidine-tagged RNA polymerases prepared from cells harvested at various times during the course of growth and sporulation (Fujita, M., Sadaie, Y., 1998 . Gene 221, 185-190), transcription initiated from putative promoter sequences on a number of DNA fragments, as inferred from genome sequencing, was examined in vitro . One of these showed sigma K-dependent transcription . For further characterization of transcription initiated from this site, in vitro transcription analysis was performed using RNA polymerase holoenzyme reconstituted from purified sigma K and core RNA polymerase . Two sigma K-dependent promoters, yfhP P1 and yfhP P2, separated by a distance of about 15 bp, were thereby identified . These promoters are located immediately upstream of the yfhP gene that encodes a protein of unknown function consisting of 327 amino acids residues . The promoter strength, the rate of open complex formation and the RNA polymerase binding affinity were examined for these two promoters in comparison with other known sigma K-dependent promoters, gerE and cotD . The promoter strength displayed was in the order of gerE > cotD > yfhP P2 > yfhP P1.

EMBO J, 1999 Oct 15, 18(20), 5675 - 82
Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation; Elias-Arnanz M et al.; The outcome of collisions between Bacillus subtilis phage Phi29 DNA polymerase and oppositely oriented transcription complexes has been studied in vitro . We found that the replication fork was unable to go past a transcription ternary complex stalled head-on . However, head-on collisions did not lead to a deadlock . Both DNA and RNA polymerase remained bound to the template and, when the halted transcription complex was allowed to move, the replication machinery resumed normal elongation . These results suggested that a replication fork that encounters an RNA polymerase head-on whose movement is not impeded would bypass the transcription machinery . Our results for head-on collisions between concurrently moving replication and transcription complexes are indeed consistent with the existence of a resolving mechanism . The ability of Phi29 DNA polymerase to resolve head-on collisions with itself during symmetrical replication of Phi29 DNA in vivo is likely to be related to its ability to pass a head-on oriented RNA polymerase.

DNA Seq, 1998, 9(3), 149 - 61
The division and cell wall gene cluster of Enterococcus hirae S185; Duez C et al.; A chromosomal 10355-bp segment of Enterococcus hirae S185 contains nine orfs which occur in the same order as the MraW-, FtsL-, PBP3-, MraY-, MurD-, MurG-, FtsQ-, FtsA- and FtsZ-encoding genes of the division and cell wall clusters of Escherichia coli and Bacillus subtilis . The E . hirae DNA segment lacks the genes which in E . coli encode the ligases Ddl, MurC, MurE and MurF and the integral membrane protein FtsW . The encoded E . hirae and E . coli proteins share 25% to 50% identity except FtsL and FtsQ (approximately = 14% identity).

Nucleic Acids Res, 1999 Nov 1, 27(21), 4298 - 304
Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites; Mobley EM et al.; Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain . The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates . In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module . New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt . At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions . The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold . Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate . These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate.

Nucleic Acids Res, 1999 Nov 1, 27(21), 4143 - 50
NMR-derived solution structure of a 17mer hydroxymethyluracil-containing DNA; Vu HM et al.; Incorporation of 5-(hydroxymethyl)-2'-deoxyuridine into DNA in place of thymine by SPO1, a Bacillus subtilis bacteriophage, allows the viral DNA to bind selectively to transcription factor 1 . We have synthesized a TF1-binding site: d(5'-ACCHACHCHHHGHAGGT-3')-d(5'-ACCHACAAAGAGHAGGT-3') and studied this molecule using NMR spectroscopy . The chemical shifts of exchangeable and non-exchangeable protons were sequentially assigned . Absence of corresponding NOEs in the imino-imino region suggested that the end base pairs did not form Watson-Crick hydrogen bond . Restrained molecular dynamics calculation yielded a family of B-DNA structures whose r.m.s.d . was 0.66 A (all atoms) for the internal 15 bp . The helical twist was 38.5 degrees per step . The base pairs were situated directly on the helix axis (X-displacement = -0.2 A) . All sugars exhibited C2'-endo puckering with P = 167.3 degrees and upsilon(max)= 38.2 degrees . The OH groups of all hmU bases resided on the 3' side of the base plane and may affect the base orientation relative to the sugar plane as the average chi value for all hmU was 4 degrees more positive than that of other nucleosides (258 degrees versus 254 degrees ) . Positive roll angles (rho) and small flanking twists (omega) at hmU suggested that the two hmU-A base pair steps open toward the minor grooves.

Mol Gen Genet, 1999 Sep, 262(2), 351 - 4
Regulation of the expression of the cold shock proteins CspB and CspC in Bacillus subtilis; Kaan T et al.; The small acidic proteins CspB and CspC are the major cold shock-induced proteins of Bacillus subtilis . Analysis of mRNA revealed a transient four-fold increase in the transcription level of both genes during cold shock . The cspB and cspC mRNAs are dramatically stabilised after a temperature downshift from 37 degrees C to 15 degrees C . The data in this study support the idea that the expression of CspB and CspC in B . subtilis during cold shock is regulated mainly at the post-transcriptional level, as is also the case with CspA in Escherichia coli.

J Bacteriol, 1999 Oct, 181(20), 6230 - 7
Characterization of a new sigma-K-dependent peptidoglycan hydrolase gene that plays a role in Bacillus subtilis mother cell lysis; Nugroho FA et al.; Bacillus subtilis produces a 30-kDa peptidoglycan hydrolase, CwlH, during the late sporulation phase . Disruption of yqeE led to a complete loss of CwlH formation, indicating the identity of yqeE with cwlH . Northern blot analysis of cwlH revealed a 0.8-kb transcript after 6 to 7.5 h for the wild-type strain but not for the sigma(F), sigma(E), sigma(G), and sigma(K) mutants . Expression of the sigma(K)-dependent cwlH gene depended on gerE . Primer extension analysis also suggested that cwlH is transcribed by Esigma(K) RNA polymerase . CwlH produced in Escherichia coli harboring a cwlH plasmid is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and exhibited an optimum pH of 7.0 and high-level binding to the B . subtilis cell wall . A cwlC cwlH double mutation led to a lack of mother cell lysis even after 7 days of incubation in DSM medium, but the single mutations led to mother cell lysis after 24 h.

J Antibiot (Tokyo), 1999 Jul, 52(7), 613 - 9
Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives; Kracht M et al.; Inactivation of enveloped viruses (VSV, SFV, and SHV-1) by surfactin lipopeptides was dependent on the hydrophobicity, i.e . the number of carbon atoms of the fatty acid, and on the charge of the peptide moiety as well as on the virus species . Surfactins with fatty acid chains of 13 carbon atoms showed very low antiviral activity in comparison to C14 and C15 isoforms . C15 surfactin monomethyl ester also inactivated SFV which was resistant to the mixture of surfactin isoforms as produced by Bacillus subtilis . In contrast, the dimethyl ester showed no virus-inactivation capacity . Disintegration of viral structures as determined by electron microscopy after inactivation of VSV and SFV was comparable to the titer reduction . The effect of the surfactin isoforms and methyl esters on erythrocyte hemolysis correlated with the virus-inactivation capacity . Surfactins with a fatty acid chain moiety of 15 carbon atoms and one negative charge showed the highest antiviral activity.

Biochem Biophys Res Commun, 1999 Oct 5, 263(3), 646 - 51
Isolation and characterization of a novel antifungal peptide from Aspergillus niger; Gun Lee D et al.; A novel antifungal peptide (termed as Anafp) was isolated from the culture supernatant of the filamentous fungi, Aspergillus niger . The whole amino acid sequence of Anafp was determined and the peptide was found to be composed of a single polypeptide chain with 58 amino acids including six cysteine residues . The peptide shows some degree of sequence homology to a cysteine-rich antifungal peptides reported from the seeds of Sinapis alba and Arabidopsis thaliana or the extracellular media of Aspergillus giganteus and Penicillium chrysogenumsome . Cysteine-spacing pattern of Anafp was similar to that of the antifungal peptide from Penicillium chrysogenum . The Anafp exhibited potent growth inhibitory activities against yeast strains as well as filamentous fungi at a range from 4 to 15 microM . In contrast, Anafp did not show antibacterial activity against Escherichia coli and Bacillus subtilis even at 50 microM .

Structure Fold Des, 1999 Sep 15, 7(9), 1113 - 24
A prototypical cytidylyltransferase: CTP:glycerol-3-phosphate cytidylyltransferase from bacillus subtilis; Weber CH et al.; BACKGROUND: The formation of critical intermediates in the biosynthesis of lipids and complex carbohydrates is carried out by cytidylyltransferases, which utilize CTP to form activated CDP-alcohols or CMP-acid sugars plus inorganic pyrophosphate . Several cytidylyltransferases are related and constitute a conserved family of enzymes . The eukaryotic members of the family are complex enzymes with multiple regulatory regions or repeated catalytic domains, whereas the bacterial enzyme, CTP:glycerol-3-phosphate cytidylyltransferase (GCT), contains only the catalytic domain . Thus, GCT provides an excellent model for the study of catalysis by the eukaryotic cytidylyltransferases . RESULTS: The crystal structure of GCT from Bacillus subtilis has been determined by multiwavelength anomalous diffraction using a mercury derivative and refined to 2.0 A resolution (R(factor) 0.196; R(free) 0.255) . GCT is a homodimer; each monomer comprises an alpha/beta fold with a central 3-2-1-4-5 parallel beta sheet . Additional helices and loops extending from the alpha/beta core form a bowl that binds substrates . CTP, bound at each active site of the homodimer, interacts with the conserved (14)HXGH and (113)RTXGISTT motifs . The dimer interface incorporates part of a third motif, (63)RYVDEVI, and includes hydrophobic residues adjoining the HXGH sequence . CONCLUSIONS: Structure superpositions relate GCT to the catalytic domains from class I aminoacyl-tRNA synthetases, and thus expand the tRNA synthetase family of folds to include the catalytic domains of the family of cytidylyltransferases . GCT and aminoacyl-tRNA synthetases catalyze analogous reactions, bind nucleotides in similar U-shaped conformations, and depend on histidines from analogous HXGH motifs for activity . The structural and other similarities support proposals that GCT, like the synthetases, catalyzes nucleotidyl transfer by stabilizing a pentavalent transition state at the alpha-phosphate of CTP.

Curr Opin Microbiol, 1999 Oct, 2(5), 524 - 8
Codon usage and lateral gene transfer in Bacillus subtilis; Moszer I et al.; Bacillus subtilis possesses three classes of genes, differing by their codon preference . One class corresponds to prophages or prophage-like elements, indicative of the existence of systematic lateral gene transfer in this organism . The nature of the selection pressure that operates on codon bias is beginning to be understood.

Appl Environ Microbiol, 1999 Oct, 65(10), 4652 - 8
Characteristics of two forms of alpha-amylases and structural implication; Ohdan K et al.; Complete (Ba-L) and truncated (Ba-S) forms of alpha-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined . The amino acid sequence deduced from the nucleotide sequence of the alpha-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region . The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B . subtilis X-23 cells . Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L . An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as alpha-amylase.

J Biol Chem, 1999 Oct 8, 274(41), 29115 - 21
The interactions of histidine-containing amphipathic helical peptide antibiotics with lipid bilayers . The effects of charges and pH; Vogt TC et al.; The alpha-helix of the designed amphipathic peptide antibiotic LAH(4 )(KKALLALALHHLAHLALHLALALKKA-NH(2)) strongly interacts with phospholipid membranes . The peptide is oriented parallel to the membrane surface under acidic conditions, but transmembrane at physiological pH (Bechinger, B . (1996) J . Mol . Biol . 263, 768-775) . LAH(4) exhibits antibiotic activities against Escherichia coli and Bacillus subtilis; the peptide does not, however, lyse human red blood cells at bacteriocidal concentrations . The antibiotic activities of LAH(4) are 2 orders of magnitude more pronounced at pH 5 when compared with pH 7.5 . Although peptide association at low pH is reduced when compared with pH 7.5, the release of the fluorophore calcein from large unilamellar 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles is more pronounced at pH values where LAH(4) adopts an orientation along the membrane surface . The calcein release experiments thereby parallel the results obtained in antibiotic assays . Despite a much higher degree of association, calcein release activity of LAH(4) is significantly decreased for negatively charged membranes . Pronounced differences in the interactions of LAH(4) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine membranes also become apparent when the mechanisms of dye release are investigated . The results presented in this paper support models in which antibiotic activity is caused by detergent-like membrane destabilization, rather than pore formation by helical peptides in transmembrane alignments.

Biochemistry, 1999 Sep 28, 38(39), 12629 - 38
The track of the pre-tRNA 5' leader in the ribonuclease P ribozyme-substrate complex; Christian EL et al.; The ribonuclease P (RNase P) ribozyme is an endonuclease that binds precursor tRNAs and catalyzes the removal of 5' leader nucleotides . Biochemical and photo-cross-linking studies have identified sites of contact between the mature tRNA domain of pre-tRNA and the ribozyme; however, relatively little is known about the location of the 5' leader in the ribozyme-substrate complex . To investigate the local three-dimensional environment of the 5' leader, we employed the short-range photo-cross-linking agent 4-thiouridine (s(4)U) . The s(4)U photoagent was incorporated into a series of pre-tRNA substrates containing unique uridine residues in the 5' leader sequence at positions -1, -3, -5, -7, or -10 . The modified substrates formed high-affinity complexes with the ribozyme and produced discrete intermolecular cross-links to RNase P RNA from Bacillus subtilis . Locations of the cross-linked nucleotides in the ribozyme and pre-tRNA were determined by reverse transcriptase primer extension . Photoagents incorporated into the 5' leader detected discrete elements of ribozyme structure in a progression from J18/2 to L15 to P3 . Importantly, all of the cross-linked species retained the ability to cleave the covalently attached pre-tRNA, indicating that the cross-links reflect the native structure of the ribozyme-substrate complex . Together with available structural and biochemical data, the cross-linking results suggest a model for the position of the 5' leader within the ground-state ribozyme-substrate complex.

Mol Gen Genet, 1999 Aug, 262(1), 173 - 9
The katX gene of Bacillus subtilis is under dual control of sigmaB and sigmaF; Petersohn A et al.; The gene katX, which encodes a catalase in Bacillus subtilis, is transcribed by EsigmaF in the pre-spore . This catalase is responsible for the resistance to hydrogen peroxide shown by germinating and outgrowing spores . We demonstrated that katX is also a sigmaB-dependent general stress gene, since it is strongly induced by heat, salt and ethanol stress, as well as by energy depletion . The -10 and -35 sequences of the sigmaB- and sigmaF-dependent promoters of katX overlap, and the transcriptional start points used by EsigmaB and EsigmaF differ by only one nucleotide . Our results indicate that the level of KatX level in outgrowing spores depends mainly on EsigmaF, because sigB mutants show normal KatX activity in dormant and outgrowing spores . katX mutants also develop the non-specific resistance to oxidative stress that is typical of glucose-starved cells . Therefore, the physiological role of sigmaB-dependent katX expression remains obscure . The results indicate an overlap between the sigmaB regulon and the sigmaF regulon, and the physiological implications of this overlap are discussed.

Biosci Biotechnol Biochem, 1999 Aug, 63(8), 1494 - 6
Antimicrobial activity of Monascus pilosus IFO 4520 against contaminant of Koji; Kono I et al.; Antimicrobial activity of Monascus pilosus IFO 4520 was examined to prevent contamination during beni-koji making in the open air . The antibacterial effect of the beni-koji prepared with this strain occured with 30 mg/ml of beni-koji extract in combination with 0.5% lactic acid against two contaminants of koji, Micrococcus varians and Bacillus subtilis . There were two compounds, antibacterial and antiyeast substances, in the beni-koji extract . These results suggest a possibility of inhibiting the growths of contaminants during beni-koji making using beni-koji extract and lactic acid.

J Bacteriol, 1999 Oct, 181(19), 6205 - 9
Spore peptidoglycan structure in a cwlD dacB double mutant of Bacillus subtilis; Popham DL et al.; Bacillus subtilis cwlD and dacB mutants produce spore peptidoglycan (PG) with increased cross-linking but with little change in spore core hydration compared to the wild type . A cwlD dacB double mutant produced spores with a two- to fourfold greater increase in PG cross-linking and novel muropeptides containing glycine residues but no significant changes in spore resistance or core hydration.

J Bacteriol, 1999 Oct, 181(19), 6171 - 5
The "pro" sequence of the sporulation-specific sigma transcription factor sigma(E) directs it to the mother cell side of the sporulation septum; Ju J et al.; sigma(E), a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma(E)) . Activation of sigma(E) occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-sigma(E) amino terminus . This reaction is believed to take place at the mother cell-forespore septum . Using a chimera of pro-sigma(E) and green fluorescent protein (GFP) to visualize the intracellular location of pro-sigma(E) by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-sigma(E)::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment . Thus, pro-sigma(E)::GFP had been sequestered to the mother cell side of the septum . This segregation of pro-sigma(E)::GFP, and presumably pro-sigma(E), to the mother cell is likely to be the reason why sigma(E) activity is restricted to that compartment.

J Bacteriol, 1999 Oct, 181(19), 6081 - 91
Three asparagine synthetase genes of Bacillus subtilis; Yoshida K et al.; Three asparagine synthetase genes, asnB, asnH, and asnO (yisO), were predicted from the sequence of the Bacillus subtilis genome . We show here that the three genes are expressed differentially during cell growth . In a rich sporulation medium, expression of asnB was detected only during exponential growth, that of asnH was drastically elevated at the transition between exponential growth and stationary phase, and that of asnO was seen only later in sporulation . In a minimal medium, both asnB and asnH were expressed constitutively during exponential growth and in stationary phase, while the expression of asnO was not detected in either phase . However, when the minimal medium was supplemented with asparagine, only the expression of asnH was partially repressed . Transcription analyses revealed that asnB was possibly cotranscribed with a downstream gene, ytnA, while the asnH gene was transcribed as the fourth gene of an operon comprising yxbB, yxbA, yxnB, asnH, and yxaM . The asnO gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E . Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of an Escherichia coli strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase . In B . subtilis, deletion of asnO or asnH, singly or in combination, had essentially no effect on growth rates in media with or without asparagine . In contrast, deletion of asnB led to a slow-growth phenotype, even in the presence of asparagine . A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that B . subtilis might have yet another asparagine synthetase, not recognized by sequence analysis . The strains lacking asnO failed to sporulate, indicating an involvement of this gene in sporulation.

J Bacteriol, 1999 Oct, 181(19), 6053 - 62
The ripX locus of Bacillus subtilis encodes a site-specific recombinase involved in proper chromosome partitioning; Sciochetti SA et al.; The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli . XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication . Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains . The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure . This result is consistent with RipX having a role in the resolution of chromosome dimers in B . subtilis . Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination . The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B . subtilis . In addition to RipX, the CodV protein of B . subtilis shows extensive similarity to XerC and XerD . The RipX and CodV proteins were shown to bind in vitro to DNA containing the E . coli dif site . Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site . Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.

J Mol Biol, 1999 Sep 24, 292(3), 581 - 8
Oligomeric structures of the phage phi29 histone-like protein p6; Abril AM et al.; Protein p6 of Bacillus subtilis phage phi29 has been described as a histone-like protein, playing a role in genome organization and compaction, on the basis of its high intracellular abundance, its pleiotropic effect, and its ability to bind and highly compact the whole phi29 DNA in vitro . Protein p6 forms large multimeric nucleoprotein complexes in which a right-handed superhelical DNA wraps toroidally around the protein core . Analytical ultracentrifugation analysis, at the concentration estimated in vivo (at least 1 mM), showed that protein p6 self-associates into elongated oligomers, suggesting that, in the absence of DNA, the protein could form a scaffold for DNA binding . In this work we have studied the structure of these oligomers by transmission electron microscopy and image processing . The results show that protein p6 aggregates into crooked-shaped oligomers, compatible with a helical structure . The oligomers could interact head-to-tail to form doughnut-shaped structures or they could grow into right-handed double-helical filaments by a nucleation-dependent polymerization process . The dimensions of the crooked-shaped structures are in agreement with that of the DNA in the nucleoprotein complex previously described . We propose that the crooked-shaped structures could act as a scaffold imposing the right-handed path followed by the DNA, and thus it could be considered a non-transient DNA chaperone .

Mol Gen Mikrobiol Virusol, 1999, (3), 3 - 7
{PO-independent termination of transcription of catabolite operons in Escherichia coli and Bacillus subtilis}; Gershanovich VN; The review discusses the Po-independent antitermination in E . coli and B . subtilis . The functional role of antiterminators BglG (in E . coli), SacY, SacT, BglG, LicT, and GlcT (in B . subtilis) is described . Special attention is paid to the role of antiterminators in connection with the phosphoenolpyruvate-dependent phosphotransferase system involved in the carbohydrate transport in bacteria.

Mikrobiologiia, 1999 May-Jun, 68(3), 304 - 11
{Expression of genes for guanyl-specific ribonucleases in Bacillus intermedius and Bacillus pumilus is regulated by the PhoP-PhoR two-component signal transduction system of PHO regulon of Bacillus subtilis}; Znamenskaia LV et al.; Promoters of the genes of guanyl-specific ribonucleases of Bacillus intermedius (binase) and Bacillus pumilus (RNase Bp) were found to contain sequences homologous to those recognizable by the regulatory protein PhoP in the promoters of the PHO regulon of B . subtilis, as well as regions partially homologous to the binding sites of another regulatory protein, PhoB, in the promoters of the PHO regulon of Escherichia coli . The role of the two-component regulatory systems PhoP-PhoR and PhoB-PhoR in the regulation of expression of the genes of binase and RNase Bp in recombinant strains of B . subtilis and E . coli was studied by using mutant strains . It was established that the expression of these genes in recombinant B . subtilis cells is stringently controlled by the PhoP-PhoR two-component regulatory system, whereas the expression of these genes in E . coli cells is not controlled by the regulatory proteins PhoB or PhoR . Presumably, regulatory systems of the response to phosphate starvation, analogous to the PHO regulon of B . subtilis, also function in other representatives of the genus Bacillus.

Electrophoresis, 1999 Aug, 20(11), 2225 - 40
Dual channel imaging of two-dimensional electropherograms in Bacillus subtilis; Bernhardt J et al.; The allocation of proteins to stimulons and regulons is an essential step towards the understanding of the global regulation of the expression of entire genomes . The computer-aided evaluation and matching of two-dimensional protein gels loaded with radioactively labeled proteins from exponentially growing or stressed cells is a useful but time-consuming procedure for the description of stimulons and regulons . This paper describes the dual-channel image analysis that offers the opportunity to visualize the content and synthesis rate of a whole set of bacterial proteins on a single electropherogram . By pulse-labeling with L-{35S}methionine, the protein synthesis pattern (red color) can be directly compared with the protein level pattern (green color) . Because matching of other gels can be avoided, this new technique is useful for the rapid search for proteins that belong to different stimulons or regulons . This approach was tested for the identification of proteins of heat stress or oxidative stress stimulons . Proteins that were induced by heat or oxidative stress colored red while proteins whose synthesis was switched off by the stress factor colored green . Proteins that were continuously synthesized before and after the imposition of stress retained their yellow color . The advantages and possible pitfalls of the technique are discussed.

J Radiat Res (Tokyo), 1999 Jun, 40(2), 115 - 24
Base substitution spectra of nalidixylate resistant mutations induced by monochromatic soft X and 60Co gamma-rays in Bacillus subtilis spores; Takahashi N et al.; Bacillus subtilis spores were exposed to three types of photons, monochromatic soft X-rays with the energy corresponding to the absorption peak of phosphorus K-shell electron (2,153 eV) and with the slightly lower energy (2,147 eV), and 60Co gamma-rays . From the irradiated spores, 233 mutants exhibiting nalidixic acid resistance were isolated, and together with 94 spontaneous mutants, the sequence changes in the 5'-terminal region of the gyrA gene coding for DNA gyrase subunit A were determined . Among eighteen alleles of the gyrA mutations, eight were single-base substitutions, nine were tandem double-base substitutions, and one was a double substitution skipping a middle base pair . About 6% of the radiation-induced mutations were tandem double-base substitutions, whereas none was observed among the spontaneous ones . Among spontaneous mutations, A:T and G:C pairs were equally subjected to mutations, whereas the substitutions from G:C pairs and those to A:T pairs predominated among those induced with soft X-rays . The peak-energy X-rays were more effective in killing and causing mutations than the low-energy X-rays, however, there seemed no base-change events uniquely attributable to phosphorus K-shell absorption.

Biochem J, 1999 Oct 1, 343 Pt 1, 107 - 14
Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver; Bladergroen BA et al.; CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis . As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver . The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa . The deduced amino acid sequence is very similar to that of human ET (89% identity) . Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase . Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein . Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET . The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane . There was no prediction for an amphipathic alpha-helix . Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot . Determination of the ET activity during liver development showed a 2 . 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly . Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA . Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx . 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development.

Eur J Biochem, 1999 Oct 1, 265(1), 308 - 17
The phosphotransferase system (PTS) of Streptomyces coelicolor identification and biochemical analysis of a histidine phosphocarrier protein HPr encoded by the gene ptsH; Parche S et al.; HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria . We have purified HPr from Streptomyces coelicolor cell extracts . The N-terminal sequence matched the product of an S . coelicolor orf, designated ptsH, sequenced as part of the S . coelicolor genome sequencing project . The ptsH gene appears to form a monocistronic operon . Determination of the evolutionary relationship revealed that S . coelicolor HPr is equally distant to all known HPr and HPr-like proteins . The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well-conserved . HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein . Histidine-tagged HPr was purified to homogeneity . HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively . This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine . Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose . HPr-P could also phosphorylate enzyme IIGlucose of B . subtilis, enzyme IILactose of S . aureus, and IIAMannitol of E . coli . ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B . subtilis . These results present the first identification of a gene of the PTS complement of S . coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.

Eur J Biochem, 1999 Sep, 264(3), 724 - 35
Effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein; Jekow P et al.; Bacteriophage SPP1 portal protein is a large cyclical homo-oligomer composed of 13 subunits . The solution structure and assembly behavior of this protein with high-point rotational symmetry was characterized . The purified protein was present as a monodisperse population of 13-mers, named gp6H, at univalent salt concentrations in the hundred millimolar range (>/= 250 mM NaCl) or in the presence of bivalent cations in the millimolar range (>/= 5 mM MgCl2) . Gp6H had a slightly higher sedimentation coefficient, a smaller shape-dependent frictional ratio, and a higher rate of intersubunit cross-linking in the presence of magnesium than in its absence . In the absence of bivalent cations and at univalent salt concentrations below 250 mM, the 13-mer molecules dissociated partially into stable monomers, named gp6L . The monomer had a somewhat different shape from the subunit present in the 13-mer, but maintained a defined tertiary structure . The association-dissociation equilibrium was mainly between the monomer and the 13-mer with a minor population of intermediate oligomers . Their interconversion was strongly influenced by the ionic environment . Under physiological conditions, the concentration of Mg2+ found in the Bacillus subtilis cytoplasm (10-50 mM) probably promotes complete association of gp6 into 13-mer rings with a compact conformation.

Mol Biol Evol, 1999 Sep, 16(9), 1125 - 34
Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis; Garcia-Vallve S et al.; Glycosyl hydrolase (GH) genes from Escherichia coli and Bacillus subtilis were used to search for cases of horizontal gene transfer . Such an event was inferred by G + C content, codon usage analysis, and a phylogenetic congruency test . The codon usage analysis used is a procedure based on a distance derived from a Pearson linear correlation coefficient determined from a pairwise codon usage comparison . The distances are then used to generate a distance-based tree with which we can define clusters and rapidly compare codon usage . Three genes (yagH from E . coli and xynA and xynB from B . subtilis) were determined to have arrived by horizontal gene transfer and were located in E . coli CP4-6 prophage, and B . subtilis prophages 6 and 5, respectively . In this study, we demonstrate that with codon usage analysis, the proposed horizontally transferred genes can be distinguished from highly expressed genes.

FEBS Lett, 1999 Aug 20, 457(1), 112 - 6
Positive regulation of Bacillus subtilis sigD by C-terminal truncated LacR at translational level; Ogura M et al.; DegR is a positive regulator for degradative enzyme synthesis in Bacillus subtilis . The degR gene is transcribed by RNA polymerase containing delta D, and the level of its expression is low in a mecA-deficient mutant . In a search for suppressors of the mecA effect through mini-Tn10 transposon mutagenesis, a lacR mutation designated lacR288 was discovered . The B . subtilis lacR gene encodes the repressor for lacA which specifies beta-galactosidase, and therefore, inactivation of the lacR gene results in overproduction of the enzyme . In the lacR288 mutant, however, the expression of lacA was at a negligible level, indicating that the repressor activity was not destroyed by the mutation . The putative gene product of the lacR288-containing gene is a 288-amino acid protein lacking the C-terminal 42 amino acids of intact LacR and carries no extra amino acids derived from the transposon sequence . The suppression by lacR288 of the decreased degR expression in the mecA background was found to be caused by an increase in the delta D level as shown by Western blot analysis . Furthermore, the increase was due to post-transcriptional regulation of sigD, the gene encoding delta D, as revealed by using both transcriptional and translational sigD-lacZ fusions . The lacR288 mutation had no effect on the stability of the delta D protein . Based on these results we conclude that the lacR288 mutation stimulates sigD expression at the translational level.

Biotechnol Bioeng, 1999 Nov 5, 65(3), 291 - 7
Effect of inactivation of nuo and ackA-pta on redistribution of metabolic fluxes in Escherichia coli; Yang YT et al.; The nuoA-N gene cluster encodes a transmembrane NADH:ubiquinone oxidoreductase (NDH-I) responsible for coupling redox chemistry to proton-motive force generation . Interactions between nuo and the acetate-producing pathway encoded by ackA-pta were investigated by examining the metabolic patterns of several mutant strains under anaerobic growth conditions . In an ackA-pta strain, the flux to acetate was decreased dramatically, whereas flux to lactate was increased significantly when compared with its parent strain; the fluxes to pyruvate and ethanol also increased slightly . In addition, pyruvate was excreted . A strain carrying the nuo mutation showed metabolic flux distribution similar to the wild type . The ackA-pta-nuo strain showed a different metabolic pattern . It not only exhibited reduced acetate accumulation but also significantly lower ethanol and formate synthesis . Metabolic flux distribution analysis suggests that the excessive carbon flux was redirected at the pyruvate node through the lactate dehydrogenase pathway for lactate formation rather than the pyruvate formate-lyase (PFL) pathway for acetyl-CoA and formate production . The diminished capacity through the formate and ethanol (ADH) pathways was not the result of genetic disruption of functional PFL or ADH production . The introduction of a Bacillus subtilis acetolactate synthase gene returned formate, ethanol, and lactate levels to those of the wild type (ackA(+)pta(+)nuo(+)) strain . Furthermore, transfer of a lactate dehydrogenase mutation yielded a strain producing ethanol as the sole fermentation product . As confirmation of the nuo effect, cultures of the ackA-pta strain, supplemented with an NDH-I inhibitor, produced intermediary levels of flux to ethanol and formate . Mutations in both ackA-pta and nuo are required to significantly reduce the flux through the PFL pathway .

Extremophiles, 1999 Aug, 3(3), 227 - 33
Genetic analysis of the chromosome of alkaliphilic Bacillus halodurans C-125; Takami H et al.; Seventeen Sse8387I linking clones isolated from the chromosome of Bacillus halodurans C-125 for the purpose of constructing a physical map were sequenced and analyzed by comparison with the BSORF database and the nonredundant protein databank . The orientations of Sse8387I or AscI linking clones serving to join adjacent fragments were determined by southern blot analysis using specific DNA probes . One-third of the open reading frames (ORFs) identified in the Sse8387I linking clones showed no significant similarity to any protein so far reported . The ORFs showing significant similarities to those of Bacillus subtilis were mapped in the chromosome of strain C-125, and the locations of the putative genes on the map were not well conserved between B . halodurans C-125 and B . subtilis.

J Bacteriol, 1999 Sep, 181(18), 5860 - 4
Synthetic lethal phenotypes caused by mutations affecting chromosome partitioning in Bacillus subtilis; Britton RA et al.; We investigated the genetic interactions between mutations affecting chromosome structure and partitioning in Bacillus subtilis . Loss-of-function mutations in spoIIIE (encoding a putative DNA translocase) and smc (involved in chromosome structure and partitioning) caused a synthetic lethal phenotype . We constructed a conditional mutation in smc and found that many of the spoIIIE smc double-mutant cells had a chromosome bisected by a division septum . The growth defect of the double mutant was exacerbated by a null mutation in the chromosome partitioning gene spo0J . These results suggest that mutants defective in nucleoid structure are unable to move chromosomes out of the way of the invaginating septum and that SpoIIIE is involved in repositioning these bisected chromosomes during vegetative growth.

J Bacteriol, 1999 Sep, 181(18), 5742 - 9
A 5' RNA stem-loop participates in the transcription attenuation mechanism that controls expression of the Bacillus subtilis trpEDCFBA operon; Sudershana S et al.; The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation . Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats . TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach the trp structural genes . In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5' end of the trp leader transcript . Deletion of this structure resulted in a dramatic increase in expression of a trpE'-'lacZ translational fusion and a reduced ability to regulate expression in response to tryptophan . By introducing a series of point mutations in the 5' stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels . Our results indicate that the 5' stem-loop functions primarily through the TRAP-dependent regulatory pathway . Gel shift results demonstrate that the 5' stem-loop increases the affinity of TRAP for trp leader RNA four- to fivefold, suggesting that the 5' structure interacts with TRAP . In vitro transcription results indicate that this 5' structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough . An oligonucleotide complementary to a segment of the 5' stem-loop was used to demonstrate that formation of the 5' structure is required for proper attenuation control of this operon.

J Bacteriol, 1999 Sep, 181(18), 5718 - 24
Identification of sigma(B)-dependent genes in Bacillus subtilis using a promoter consensus-directed search and oligonucleotide hybridization; Petersohn A et al.; A consensus-directed search for sigma(B) promoters was used to locate potential candidates for new sigma(B)-dependent genes in Bacillus subtilis . Screening of those candidates by oligonucleotide hybridizations with total RNA from exponentially growing or ethanol-stressed cells of the wild type as well as a sigB mutant revealed 22 genes that required sigma(B) for induction by ethanol . Although almost 50% of the proteins encoded by the newly discovered sigma(B)-dependent stress genes seem to be membrane localized, biochemical functions have so far not been defined for any of the gene products . Allocation of the genes to the sigma(B)-dependent stress regulon may indicate a potential function in the establishment of a multiple stress resistance . AldY and YhdF show similarities to NAD(P)-dependent dehydrogenases and YdbP to thioredoxins, supporting our suggestion that sigma(B)-dependent proteins may be involved in the maintenance of the intracellular redox balance after stress.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 153 - 60
A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker; Oswald W et al.; Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools . Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A . pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A . pleuropneumoniae chromosome . A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A . pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A . pleuropneumoniae . The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive . A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A . pleuropneumoniae mutants that had lost the km(r)-sacB cassette.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 43 - 7
Anionic polymers of Bacillus subtilis cell wall modulate the folding rate of secreted proteins; Chambert R et al.; In order to characterize the dynamics of the interaction between the emergent membrane translocated exoprotein and the components of Bacillus subtilis cell wall, we examined the kinetics of the in vitro refolding of levansucrase and alpha-amylase, at pH 7 and 37 degrees C, in the presence of polyphosphates (polyP) of various chain lengths (2</=n</=65) . These soluble anionic polymers are considered here to mimic the role of teichoic acids . Even in the absence of calcium, levansucrase rapidly refolded in the presence of polyP of n>/=16 . In contrast, polyP modulate indirectly the rate of alpha-amylase refolding via their affinity for calcium . These differential effects might explain that the rate of the cell wall translocation of alpha-amylase secretion was found to be half that of levansucrase.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 31 - 6
Molecular characterization of a flagellar (fla) operon in the oral spirochete Treponema denticola ATCC 35405; Stamm LV et al.; A Treponema denticola 9.6-kb motility locus containing 11 genes was identified, sequenced and analyzed . The genes were designated tap1, flgD, flgE, orf4, motA, motB, fliL, fliM, fliY, orf10 and fliP . The order of these genes is identical to that of the corresponding region of the Treponema pallidum fla operon . Seven of the deduced Fla proteins share significant homology with both Escherichia coli and Bacillus subtilis proteins associated with flagellar structure and function . Reverse transcription-PCR analysis indicated that the T . denticola fla genes are transcribed as a single unit . A putative sigma(28)-like promoter, virtually identical to the T . pallidum fla promoter, was identified upstream of tap1 . These results showed that the T . denticola and T . pallidum fla operons are highly conserved, supporting the proposed phylogenetic relatedness of these spirochetes.

FEBS Lett, 1999 Sep 17, 458(2), 145 - 50
Thermodynamics of nucleotide binding to NBS-I of the Bacillus subtilis preprotein translocase subunit SecA; den Blaauwen T et al.; SecA is the dissociatable nucleotide and preprotein binding subunit of the bacterial translocase . The thermodynamics of nucleotide binding to soluble SecA at nucleotide binding site I were determined by isothermal titration calorimetry . Binding of ADP and non-hydrolyzable ATPgammaS is enthalpy-driven (DeltaH(0) of -14.44 and -5.56 kcal/mol, respectively), but is accompanied by opposite entropic contributions (DeltaS(0) of -18.25 and 9.55 cal/mol/K, respectively) . ADP binding results in a large change in the heat capacity of SecA (DeltaC(p)=-780 cal/mol/K) . It is suggested that ADP binding promotes the interaction between the two thermodynamically discernible domains of SecA which is accompanied by a shielding of hydrophobic surface from solvent.

Nucleic Acids Res, 1999 Oct 1, 27(19), 3811 - 20
Cloning and biochemical characterization of Bacillus subtilis YxiN, a DEAD protein specifically activated by 23S rRNA: delineation of a novel sub-family of bacterial DEAD proteins; Kossen K et al.; DEAD, DEAH and DExH proteins are involved in almost every facet of RNA biochemistry . Members of these protein families exhibit an RNA-dependent ATPase activity and some possess an ATP-dependent RNA helicase activity . Although genetic studies have identified specific functions for certain DEx(D)/(H)proteins from which an RNA substrate can be reasonably inferred, only DbpA from Escherichia coli has been shown to exhibit significant RNA specificity in vitro . Here we describe the characterization of YxiN from Bacillus subtilis, the second DEx(D)/(H)protein to show significant RNA specificity as an isolated, homogenous protein . The ATPase activity of YxiN, like that of DbpA, is stimulated by a 154 nt fragment of 23S rRNA . YxiN has a 2 nM apparent binding constant for this fragment, yet its ATPase activity shows 1800-fold RNA specificity . Along with the conserved motifs shared among all DEAD proteins, YxiN and DbpA have a conserved C-terminal extension . This extension is highly conserved in several additional DEAD proteins . We propose that the C-terminus identifies a protein sub-family whose members bind 23S rRNA and that proteins of this family are likely to function in rRNA maturation/ribosome biogenesis or an unappreciated aspect of translation.

Can J Vet Res, 1999 Jul, 63(3), 193 - 200
Pharmacokinetics of enrofloxacin given by the oral, intravenous and intramuscular routes in broiler chickens; Bugyei K et al.; Enrofloxacin was given to broiler chickens, 3 groups of 6 birds each, at a dose of 5 mg/kg . Routes of administration were intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) and blood samples were collected from the jugular vein for determination of serum drug levels over a 54-hour period after administration . Drug levels were determined using Bacillus subtilis spore suspension on Meuller-Hinton antibiotic medium . Intravenous administration produced drug levels which followed a bi-exponential decay according to the model C = 101e(-1.84(t)) + 1.30e(-0.06(t)) . After i.m . administration, the mean Cmax observed (2.01 microg/mL) occurred at 1 h and levels were detected for up to 48 h . The mean time to maximum concentration (Tmax) for the birds occurred at 0.79 h . The model describing serum concentrations after i.m . administration was C = 1.35e(-0.48(t)) + 1.27e(-0.07(t)) - 2.06e(-2.1(t)) . Serum concentrations after oral administration were lower and the mean +/- standard error of mean, of the maximum concentrations (Cmax) was 0.99 microg/mL at 2 h after administration . The mean residence times after the 3 routes of administration were not significantly different and ranged from 12.5-13.7 h . Bioavailability by the oral route was 80.1% . Dialysis of chicken plasma vs saline indicated that the protein binding was 22.7%.

Mol Microbiol, 1999 Sep, 33(5), 1015 - 26
Characterization of a morphological checkpoint coupling cell-specific transcription to septation in Bacillus subtilis; Feucht A et al.; Early in the process of spore formation in Bacillus subtilis, asymmetric cell division produces a large mother cell and a much smaller prespore . Differentiation of the prespore is initiated by activation of an RNA polymerase sigma factor, sigmaF, specifically in that cell . sigmaF is controlled by a regulatory cascade involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor, SpoIIAA, and a membrane-bound phosphatase, SpoIIE, which converts the inactive, phosphorylated form of SpoIIAA back to the active form . SpoIIE is required for proper asymmetric division and much of the protein is sequestered into the prespore during septation . Importantly, activation of sigmaF is dependent on formation of the asymmetric septum . We have now characterized this morphological checkpoint in detail, using strains affected in cell division and/or spoIIE function . Surprisingly, we found that significant dephosphorylation of SpoIIAA occurred even in the absence of septation . This shows that the SpoIIE phosphatase is at least partially active independent of the morphological event and also that cells can tolerate significant levels of unphosphorylated SpoIIAA without activating sigmaF . We also describe a spoIIE mutant in which the checkpoint is bypassed, probably by an increase in the dephosphorylation of SpoIIAA . Taken together, the results support the idea that sequestration of SpoIIE protein into the prespore plays an important role in the control of sigmaF activation and in coupling this activation to septation.

FEMS Microbiol Lett, 1999 Aug 15, 177(2), 279 - 88
Analysis of a ptsH homologue from Streptomyces coelicolor A3(2); Butler MJ et al.; A ptsH homologue of Streptomyces coelicolor A3(2) was identified in the emerging genome sequence, cloned in Escherichia coli and the S . coelicolor HPr over-produced and purified . The protein was phosphorylated in vitro in a phosphoenolpyruvate (PEP)-dependent manner by purified enzyme I (EI) from Bacillus subtilis, and much less efficiently in an ATP-dependent manner by purified HPr kinase, also from B . subtilis . There was no indication of ATP-dependent phosphorylation of the purified protein by cell extracts of either S . coelicolor or Streptomyces lividans . Deletion of the ptsH homologue from the S . coelicolor and S . lividans chromosomes had no effect on growth when fructose was supplied as sole carbon source, and in S . coelicolor it had no effect on glucose repression of agarase and galactokinase synthesis, suggesting that the HPr encoded by this gene does not play an essential role in fructose transport nor a general role in carbon catabolite repression.

J Biol Chem, 1999 Sep 10, 274(37), 25990 - 4
Reevaluation of the nucleotide cofactor specificity of the RecA protein from Bacillus subtilis; Steffen SE et al.; The RecA protein from the Gram-positive bacterium, Bacillus subtilis, has been reported to catalyze dATP hydrolysis and to promote strand exchange in the presence of dATP but to have no ATP hydrolysis or ATP-dependent strand exchange activity (Lovett, C . M., Jr., and Roberts, J . W . (1985) J . Biol . Chem . 260, 3305-3313) . The well characterized RecA protein from Escherichia coli, in contrast, catalyzes the hydrolysis of ATP and dATP at similar rates and can use either ATP or dATP as a cofactor for the strand exchange reaction . To explore this reported difference in nucleotide cofactor specificity in detail, we developed an overexpression system for the B . subtilis RecA protein and purified the protein to greater than 95% homogeneity . Contrary to the previous report, we find that the B . subtilis RecA protein catalyzes the hydrolysis of both dATP and ATP and can perform strand exchange using either dATP or ATP as a cofactor . Our results suggest that the inability of previous investigators to detect the ATP hydrolysis and ATP-dependent strand exchange activities of the B . subtilis RecA protein may have been due to the particular assay conditions that were used in the earlier study.

Appl Environ Microbiol, 1999 Sep, 65(9), 4288 - 91
Characterization of two Bacillus probiotics; Green DH et al.; Bacillus subtilis is currently used as an oral probiotic . We examined two commercial B . subtilis probiotic preparations, Enterogermina and Biosubtyl . Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B . subtilis.

Appl Environ Microbiol, 1999 Sep, 65(9), 4255 - 60
Bacterial spores survive treatment with commercial sterilants and disinfectants; Sagripanti JL et al.; This study compared the activity of commercial liquid sterilants and disinfectants on Bacillus subtilis spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material . Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization . These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied . Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.

Appl Environ Microbiol, 1999 Sep, 65(9), 4040 - 8
Identification of an ATP-driven, osmoregulated glycine betaine transport system in Listeria monocytogenes; Ko R et al.; The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine . Previously we showed that glycine betaine transport was the result of Na(+)-glycine betaine symport . In this report, we identify a second glycine betaine transporter from L . monocytogenes which is osmotically activated but does not require a high concentration of Na(+) for activity . By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated . DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters . The three open reading frames are closely spaced, suggesting that they are arranged in an operon . Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU . One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L . monocytogenes appear to be TTG . That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na(+) is an indication that only the ATP-dependent transporter and the Na(+)-glycine betaine symporter occur in L . monocytogenes.

Antimicrob Agents Chemother, 1999 Sep, 43(9), 2183 - 92
The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production; Tsuge K et al.; Bacillus subtilis YB8 produces the lipopeptide antibiotic plipastatin . B . subtilis MI113, which is a derivative of strain 168, was converted into a new plipastatin producer, strain 406, by competence transformation with the chromosomal DNA of YB8 . Transposon mini-Tn10 insertional mutagenesis was applied to strain 406, which revealed that lpa-8 (sfp) (encoding 4'-phosphopantetheinyl transferase) and the pps operon (located between 167 and 171 degrees ) are essential for plipastatin production . The pps operon was previously suggested to encode putative peptide synthetases (A . Tognoni, E . Franchi, C . Magistrelli, E . Colombo, P . Cosmina, and G . Grandi, Microbiology 141:645-648, 1995) and was thought to be the fengycin operon (V . Tosato, A . M . Albertini, M . Zotti, S . Sonda, and C . V . Bruschi, Microbiology 143:3443-3450, 1997) . We claim that the pps operon is the pli operon, encoding plipastatin synthetase . By using a new high-performance liquid chromatography system, we revealed that strain 168 expressing only lpa-8 can also produce plipastatin, although the yield is very low . However, the introduction of the pleiotropic regulator degQ of strain YB8 into strain 168 expressing lpa-8 resulted in a 10-fold increase in the production of plipastatin.

J Biochem (Tokyo), 1999 Sep, 126(3), 461 - 9
Differential and additive effects of the three conserved isoleucine residues in the promoter -10 binding region on Bacillus subtilis sigma(A) structure and function; Liao CT et al.; The promoter -10 binding region of the Bacillus subtilis sigma(A) factor forms an amphiphilic alpha-helix with three conserved isoleucines located at four-residue intervals . To investigate the structural and functional roles of the three isoleucine residues, we constructed a series of sigA mutants with single and double Ile-to-Ala substitutions on the hydrophobic face of this alpha-helix and isolated intragenic revertants with either same-site or second-site suppressor that partially restores the structural stability and transcription activity of the mutant sigma(A) factors . Our data show that the three conserved isoleucine residues (Ile-194, Ile-198, and Ile-202) are involved in the hydrophobic core packing of sigma(A); they affect differentially and additively the structure and function of sigma(A), with the central isoleucine residue (Ile-198) playing the most important role . By analogy with the crystal structure of a sigma(70) peptide, it is apparent that interdigital interactions exist between the three conserved isoleucine residues and certain hydrophobic amino acids in region 2 . 1 of sigma(A) . They include at least the van der Waals contacts between Ile-194 and both Leu-145 and Ile-149, between Ile-198 and both Ile-149 and Tyr-153, as well as between Ile-202 and Tyr-153 . The same-site suppressors, Val-194 and Val-198, restore the structural stability and transcription activity of sigma(A) by repacking the hydrophobic core of sigma(A) . The second-site suppressor (S291F) appears to be allele-specific, but it is not as effective as the same-site suppressors in restoring sigma(A) structure and function.

Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10412 - 7
Bacillus subtilis aconitase is an RNA-binding protein; Alen C et al.; The aconitase protein of Bacillus subtilis was able to bind specifically to sequences resembling the iron response elements (IREs) found in eukaryotic mRNAs . The sequences bound include the rabbit ferritin IRE and IRE-like sequences in the B . subtilis operons that encode the major cytochrome oxidase and an iron uptake system . IRE binding activity was affected by the availability of iron both in vivo and in vitro . In eukaryotic cells, aconitase-like proteins regulate translation and stability of iron metabolism mRNAs in response to iron availability . A mutant strain of B . subtilis that produces an enzymatically inactive aconitase that was still able to bind RNA sporulated 40x more efficiently than did an aconitase null mutant, suggesting that a nonenzymatic activity of aconitase is important for sporulation . The results support the idea that bacterial aconitases, like their eukaryotic homologs, are bifunctional proteins, showing aconitase activity in the presence of iron and RNA binding activity when cells are iron-deprived.

Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10290 - 5
An enhancer element located downstream of the major glutamate dehydrogenase gene of Bacillus subtilis; Belitsky BR et al.; The rocG gene of Bacillus subtilis, encoding a catabolic glutamate dehydrogenase, is transcribed by SigL (sigma(54))-containing RNA polymerase and requires for its expression RocR, a member of the NtrC/NifA family of proteins that bind to enhancer-like elements, called upstream activating sequences (UAS) . Unlike the case for other sigma(54)-dependent genes, rocG has no UAS; instead, its expression depends on a sequence located 1.5 kilobases downstream of the rocG promoter, beyond the end of the rocG coding region . The same sequence also serves as the UAS for the downstream rocABC operon and can activate rocG if moved upstream of its promoter . Furthermore, the activating sequence can be moved as far as 15 kilobases downstream of the rocG promoter and still retain partial activity.

J Bacteriol, 1999 Sep, 181(17), 5476 - 81
Regulation of synthesis of the Bacillus subtilis transition-phase, spore-associated antibacterial protein TasA; Stover AG et al.; Previously, we identified a novel component of Bacillus subtilis spores, called TasA, which possesses antibacterial activity . TasA is made early in spore formation, as cells enter stationary phase, and is secreted into the medium as well as deposited into the spore . Here, we show that tasA expression can occur as cells enter stationary phase even under sporulation-repressing conditions, indicating that TasA is a transition-phase protein . tasA and two upstream genes, yqxM and sipW, likely form an operon, transcription of which is under positive control by the transition-phase regulatory genes spo0A and spo0H and negative control by the transition phase regulatory gene abrB . These results are consistent with the suggestion that yqxM, sipW, and tasA constitute a transition phase operon that could play a protective role in a variety of cellular responses to stress during late-exponential-phase and early-stationary-phase growth in B . subtilis.

J Bacteriol, 1999 Sep, 181(17), 5384 - 8
Role of the sporulation protein BofA in regulating activation of the Bacillus subtilis developmental transcription factor sigmaK; Resnekov O; During sporulation, the Bacillus subtilis transcription factor sigmaK is activated by regulated proteolytic processing . I have used a system that facilitates the analysis of the contributions of a modified form of the processing enzyme, SpoIVFB-GFP, and the regulatory proteins BofA and SpoIVFA to the conversion of pro-sigmaK to sigmaK . The results show that in the presence of BofA, SpoIVFA levels increase by greater than 20-fold, SpoIVFA is substantially stabilized, and pro-sigmaK processing is inhibited . In addition, enhanced accumulation of the SpoIVFA protein in the absence of BofA (achieved through the use of an ftsH null mutation) substantially inhibits pro-sigmaK processing . These results suggest that during growth, increased accumulation of the SpoIVFA protein inhibits the activity of SpoIVFB-GFP and regulates the activation of sigmaK.

J Bacteriol, 1999 Sep, 181(17), 5341 - 9
Cold shock response of Bacillus subtilis: isoleucine-dependent switch in the fatty acid branching pattern for membrane adaptation to low temperatures; Klein W et al.; Bacillus subtilis has developed sophisticated mechanisms to withstand fluctuations in temperature . Membrane fatty acids are the major determinants for a sufficiently fluid membrane state to ensure the membrane's function at all temperatures . The fatty acid profile of B . subtilis is characterized by a high content of branched fatty acids irrespective of the growth medium . Here, we report on the importance of isoleucine for B . subtilis to survive cold shock from 37 to 15 degrees C . Cold shock experiments with strain JH642 revealed a cold-protective function for all intermediates of anteiso-branched fatty acid biosynthesis . Metabolites related to iso-branched or straight-chain fatty acid biosynthesis were not protective . Fatty acid profiles of different B . subtilis wild-type strains proved the altered branching pattern by an increase in the anteiso-branched fatty acid content and a concomitant decrease of iso-branched species during cold shock . There were no significant changes in the fatty acid saturation or acyl chain length . The cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine correlated with their inability to synthesize more anteiso-branched fatty acids, as shown by the fatty acid profile . The switch to a fatty acid profile dominated by anteiso-C(15:0) and C(17:0) at low temperatures and the cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine focused our attention on the critical role of anteiso-branched fatty acids in the growth of B . subtilis in the cold.

J Bacteriol, 1999 Sep, 181(17), 5193 - 200
An autoregulatory circuit affecting peptide signaling in Bacillus subtilis; Lazazzera BA et al.; The competence and sporulation factor (CSF) of Bacillus subtilis is an extracellular pentapeptide produced from the product of phrC . CSF has at least three activities: (i) at low concentrations, it stimulates expression of genes activated by the transcription factor ComA; at higher concentrations, it (ii) inhibits expression of those same genes and (iii) stimulates sporulation . Because the activities of CSF are concentration dependent, we measured the amount of extracellular CSF produced by cells . We found that by mid-exponential phase, CSF accumulated to concentrations (1 to 5 nM) that stimulate ComA-dependent gene expression . Upon entry into stationary phase, CSF reached 50 to 100 nM, concentrations that stimulate sporulation and inhibit ComA-dependent gene expression . Transcription of phrC was found to be controlled by two promoters: P1, which precedes rapC, the gene upstream of phrC; and P2, which directs transcription of phrC only . Both RapC and CSF were found to be part of autoregulatory loops that affect transcription from P1, which we show is activated by ComA approximately P . RapC negatively regulates its own expression, presumably due to its ability to inhibit accumulation of ComA approximately P . CSF positively regulates its own expression, presumably due to its ability to inhibit RapC activity . Transcription from P2, which is controlled by the alternate sigma factor sigma(H), increased as cells entered stationary phase, contributing to the increase in extracellular CSF at this time . In addition to controlling transcription of phrC, sigmaH appears to control expression of at least one other gene required for production of CSF.

J Food Prot, 1996 Mar, 59(3), 261 - 7
Potential Bacillus subtilis alpha-amylase-based time-temperature integrators to evaluate pasteurization processes; Van Loey A et al.; Thermal inactivation kinetics of Bacillus subtilis alpha-amylase (BSA) in different environmental conditions was studied by performing isothermal experiments . As a response property, residual enzymic activity and residual heat of enzyme deterioration were chosen . A comparison of processing values determined from the read-out of a system with actual integrated processing values revealed the potentials of these systems as time-temperature integrators to be used in the pasteurization domain (temperatures of 70 to 100 degrees C) for target attributes with z-values ranging from 6 to 12 degrees C.

Microbiology, 1999 Aug, 145 ( Pt 8), 2171 - 80
Transcription of genes near the sspE locus of the Bacillus subtilis genome; Yamamoto H et al.; The yfhP, yfhQ (mutY homologue), yfhS and yfhR (oxidoreductase homologue) genes, which are located upstream of the sspE locus, have been identified in the Bacillus subtilis genome . Transcriptional analysis showed that yfhP, yfhQ and yfhR are transcribed during the exponential growth phase, and sspE is monocistronically transcribed in the late sporulation phase and co-transcribed with yfhQ and/or yfhR during exponential growth . However, SspE was not translated during this period . Northern blot and primer extension analyses indicated that yfhS is transcribed by E sigma E during sporulation . No significant difference between wild-type and yfhS mutant strains was found in the rate of sporulation or germination, the heat tolerance of spores or the transcription of the sspE locus during sporulation . The transcription of the yfhP and yfhQ-yfhR-sspE loci increased 2.5- and 5.3-fold in a yfhP-deficient strain compared to the wild-type strain at t-2 (2 h before initiation of sporulation) . In addition, transcription corresponding to the yfhR-sspE loci increased more than twofold with maximum values observed at t-1.5 . These results suggest that YfhP may act as a negative regulator for the transcription of yfhQ, yfhR, sspE and yfhP.

Arch Biochem Biophys, 1999 Sep 1, 369(1), 1 - 10
Cytochrome P450 and the individuality of species; Nelson DR; The P450 superfamily is expanding rapidly on many fronts . Arabidopsis genomic sequencing is producing about 2 to 3 novel P450s per week, with some clusters containing 9-14 genes . Bacterial genomes also carry surprises, such as the 20 P450s found in Mycobacterium tuberculosis and the 7 in Bacillus subtilis . The race to finish the human genome has already identified the majority of human P450s, some by expressed sequence tags only . The rapid discovery of new genes is being complemented by detailed analysis of our human genes to identify and characterize the complete set of human P450 polymorphisms and disease-causing mutations, one aspect of our "chemical individuality." Phylogenetic trees are included for plant, fungal, animal, and bacterial P450s . Emphasis is given to the higher order nomenclature of P450 clans, as a tool to see the larger picture of P450 evolution . Arabidopsis is the current record holder in P450 genes, with 186 named genes and a prediction of 350 in the total genome to be completed next year . The biosynthesis of cholesterol in bacteria is discussed in relation to CYP51 as a lanosterol 14 alpha-demethylase . This enzyme may have been the first eukaryotic P450 .

J Inorg Biochem, 1999 Jun 15, 75(2), 123 - 33
Antimicrobial and mutagenic activity of some carbono- and thiocarbonohydrazone ligands and their copper(II), iron(II) and zinc(II) complexes; Bacchi A et al.; Several mono- and bis- carbono- and thiocarbonohydrazone ligands have been synthesised and characterised; the X-ray diffraction analysis of bis(phenyl 2-pyridyl ketone) thiocarbonohydrazone is reported . The coordinating properties of the ligands have been studied towards Cu(II), Fe(II), and Zn(II) salts . The ligands and the metal complexes were tested in vitro against Gram positive and Gram negative bacteria, yeasts and moulds . In general, the bisthiocarbonohydrazones possess the best antimicrobial properties and Gram positive bacteria are the most sensitive microorganisms . Bis(ethyl 2-pyridyl ketone) thiocarbonohydrazone, bis(butyl 2-pyridyl ketone)thiocarbonohydrazone and Cu(H2nft)Cl2 (H2nft, bis(5-nitrofuraldehyde)thiocarbonohydrazone) reveal a strong activity with minimum inhibitory concentrations of 0.7 microgram ml-1 against Bacillus subtilis and of 3 micrograms ml-1 against Staphylococcus aureus . Cu(II) complexes are more effective than Fe(II) and Zn(II) ones . All bisthiocarbono- and carbonohydrazones are devoid of mutagenic properties, with the exception of the compounds derived from 5-nitrofuraldehyde . On the contrary a weak mutagenicity, that disappears in the copper complexes, is exhibited by monosubstituted thiocarbonohydrazones.

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