J Bacteriol, 1996 Apr, 178(7), 1980 - 9 Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms; Mendelson NH et al.; Factors governing the morphogenesis of Bacillus subtilis colonies as well as the spatial-temporal pattern of expression of a reporter gene during colony development were examined by systematically varying the initial nutrient levels and agar concentrations (wetness), the relative humidity throughout incubation, and the genotype of the inoculum . A relationship between colony form and reporter gene expression pattern was found, indicating that cells respond to local signals during colony development as well as global conditions . The most complex colony forms were produced by motile strains grown under specific conditions such that cells could swim within the colony but not swarm outward uniformly from the colony periphery . The wetness of the growth environment was found to be a critical factor . Complex colonies consisted of structures produced by growth of finger-like projections that expanded outward a finite distance before giving rise to a successive round of fingers that behaved in a similar fashion . Finger tip expansion occurred when groups of cells penetrated the peripheral boundary . Although surfactin production was found to influence similar colony forms in other B . subtilis strains, the strains used here to study reporter gene expression do not produce it . The temporal expression of a reporter gene during morphogenesis of complex colonies by motile strains such as M18 was investigated . Expression arose first in cells located at the tips of fingers that were no longer expanding . The final expression pattern obtained reflects the developmental history of the colony. J Bacteriol, 1996 Apr, 178(7), 1971 - 9 LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family; Schnetz K et al.; Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization . Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family . Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family . The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t . The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold . This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t . However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization. Biochemistry, 1996 Mar 19, 35(11), 3636 - 40 Peptidyl-prolyl cis-trans isomerase of Bacillus subtilis: identification of residues involved in cyclosporin A affinity and catalytic efficiency; Gothel SF et al.; The 17-kDa peptidyl-prolyl cis-trans-isomerase from Bacillus subtilis (PPiB) is a member of the cyclophilin family and shows strong homology to PPIases of eukaryotic origin (40%) and less identify to PPIase sequences of Gram-negative bacteria (27-32%) . Although the majority of residues that form the PPIase active site are highly conserved, three residues, V52, H90, and H109 in the sequence of the B.subtilis PPIase, were found to differ from the sequences found in human (hCyP) and Escherichia coli (eCyP) . Also, the binding affinity of cyclosporin A (CsA) to the different PPIases varies in IC(50) values from 6 nM for human PPIase hCyPA and 84 nM for the human hCyPB to over 120 nM for B . subtilis and 3000 nM for E . coli . In addition, a variety of k(cat)/K(m) values, ranging from 1.1 mM(-1) s(-1) for the B . subtilis PPIase to over 10 mM(-1) s(-1) for human and 13 mM(-1) s(-1) for E . coli, were detected using the common substrate suc-Ala-Ala-Pro-Phe-pNA . Through site-specific mutagenesis we demonstrate that the differences in the three mentioned residues are mainly responsible for the variations in IC(50) and k(cat)/K(m) values . Replacement of H90 to N90, or H109 to W109, resembling the amino acid sequence of human hCyPA, resulted in more efficient CsA binding (IC(50) value for H90N, 60 nM, and for H109W, 95 nm), whereas replacement of H90 to R90, or H109 to F109, resembling the amino acid sequence of E . coli eCyP, resulted in less efficient CsA binding (IC(50) value for H90R, 2000nM, and for H109F, 5000 nM) . In addition to lower CsA affinity, mutant protein H109F shows a k(cat)/K(m) value of 10.5 mM(-1) s(-1), comparably high to that of the wild-type E . coli protein . In contrast, other mutants like C57F, H90N, H90R, and H109W do not differ significantly in k(cat)/K(m) values from wild-type PPiB . Replacement of V52 to M52, which is conserved in E . coli and all known eukaryotic PPIases, does not show any effect in CsA binding affinity (IC(50) value for V52M, 120 nM), but it raises the catalytic efficiency by 12-fold to k(cat)/K(m) of 14 mM(-1) s(-1) . In conclusion, our studies suggest that the unique histidine residues H90 and H109 in B . subtilis PPIase are, at least in part, responsible for its intermediate CsA affinity and that the v52 residue confers the low conversion rate. FEMS Microbiol Lett, 1996 Mar 15, 137(1), 13 - 8 Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines; Ghim SY et al.; The pyrimidine nucleotide biosynthetic (pyr) operon in Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which PyrR, a bifunctional pyr RNA-binding attenuation protein/uracil phosphoribosyltransferase, plays a crucial role . A convenient procedure for isolation of pyrR mutants with defects in the regulation of pyr operon expression is described . The selection is based on the selection of spontaneous mutations that convert the pyrimidine-sensitive growth of cpa strain (lacking arginine-repressible carbamyl phosphate synthetase) to pyrimidine resistance . Twelve such mutants were isolated and sequenced . All resulted from point mutations in the pyrR gene. Eur J Biochem, 1996 Mar 15, 236(3), 843 - 6 Aminopeptidase from Streptomyces griseus: primary structure and comparison with other zinc-containing aminopeptidases; Maras B et al.; The aminopeptidase from Streptomyces griseus is a calcium-activated metalloenzyme, which contains 2 mol tightly bound zinc/mol protein . This aminopeptidase rapidly hydrolyzes peptide bonds formed by N-terminal hydrophobic amino acids, such as leucine, methionine and phenylalanine . We have determined the complete primary structure of the protein, which contains 284 amino acid residues, yielding a molecular mass of 29723 Da . A search in the Swiss-Prot database for sequence similarities revealed a low degree of identity (26-34%) to Saccharomyces cerevisiae aminopeptidase Y, Aeromonas proteolytica aminopeptidase, and a hypothetical 49.5-kDa protein from Bacillus subtilis, which is supposed to belong to the aminopeptidase Y family . In all these proteins, the residues that are known to be involved in zinc coordination are conserved. Biochim Biophys Acta, 1996 Mar 15, 1289(2), 217 - 20 A simple solid-state NMR method for determining peptidoglycan crosslinking in Bacillus subtilis; Shenouda NS et al.; The crosslink index of the peptidoglycan of intact cell walls of Bacillus subtilis grown in media containing L-{15N}aspartic acid has been determined by a combination of cross-polarization magic-angle spinning 15N NMR and gas chromatography/mass spectrometry . The crosslink index of 62% decreased to 52% when the bacteria were exposed to the antibiotic cephalothin. Biochemistry, 1996 Mar 5, 35(9), 2926 - 33 A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates; Zapf JW et al.; Transient phosphorylation at an aspartate residue on the Spo0F protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli . The response regulator Spo0F-P is stable to hydrolysis (t1/2 > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated forms of Spo0F . Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate Spo0F, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors . Rapid hydrolysis of Spo0F-P generated with phosphoramidate by proteins downstream in the phosphorelay (Spo0B and Spo0A) is consistent with phosphorylation at the active site of Spo0F . The initial rate of Spo0F-P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as Spo0F, function as phosphoryl transferase enzymes (McCleary et al., 1993) . The results establish that Spo0F functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation. Mikrobiol Z, 1996 Mar-Apr, 58(2), 46 - 55 {The effect of live cultures of Bacillus subtilis on body nonspecific resistance}; Kudriavtsev VA et al.; Alive cultures of Bacillus subtilis, antagonists of pyogenic microflora, have been studied for their effect on functional activity of macrophagal cells and induction of endogenic serum alpha-interferon in the female mice of the SBA line . It is established that even a single intravaginal and intraperitoneal introduction of bacilli in a dose of 1 milliard cells in 0.1 ml of cultural fluid stimulates migration, absorption and especially bactericidal activity of macrophages of peritoneal exudate . Introduction of alive cultures of aerobic bacilli essentially stimulates the in vivo production of the serum and alpha-interferon induced in vitro by the Newcastle disease virus . The highest immune-stimulating effect under a single introduction of B . subtilis is achieved 66 h later and has a tendency to gradual decrease. Mol Microbiol, 1996 Mar, 19(5), 941 - 8 Three two-component signal-transduction systems interact for Pho regulation in Bacillus subtilis; Sun G et al.; The Pho regulon of Bacillus subtilis is controlled by three two-component signal-transduction systems: PhoP/PhoR, ResD/ResE, and the phosphorelay leading to the phosphorylation of SpoOA . Two of these systems act as positive regulators, while the third is involved in negative regulation of the Pho regulon . Under phosphate-starvation-induction conditions, the response regulator (RR) PhoP, and the histidine protein kinase (HK) PhoR, are involved in the induction of Pho-regulon genes including the phoPR operon and genes encoding the major vegetative alkaline phosphatases, phoA and phoB . ResD (the RR) and ResE (the HK) are positive regulators of both aerobic and anaerobic respiration in B . subtilis . Current data suggest that they are also positive regulators of the Pho regulon, as is the transition-state regulatory protein AbrB . Data presented reveal that ResDE and AbrB are involved in activation of the Pho regulon through separate regulatory pathways . SpoOA approximately P (RR) exerts a negative effect on the Pho regulon through its repression of AbrB, and possibly through repression of ResDE . Both pathways converge to regulate transcription of the phoPR operon. Mol Microbiol, 1996 Mar, 19(5), 1047 - 60 Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly; Barak I et al.; Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor sigma F, which directs early forespore-specific gene expression . We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B . subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium . We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa . Each of five point-mutation alleles, which have traditionally defined the locus, and two transposon-generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression . The amino-terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane-spanning segments . The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous-phase exposed domains . An in-frame fusion joining the 3' end of the B . megaterium spoIIE coding sequence to the 5' end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation-specific fluorescent signal localized to the sites of sporulation septum assembly . We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development. Mol Microbiol, 1996 Mar, 19(6), 1295 - 306 Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20; Meijer WJ et al.; We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions . Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B . subtilis chromosomal replication terminator . Here, we demonstrate that this segment is part of a functional replication terminator . This newly identified element, designated TerLS20, is the first replication terminator identified on a theta plasmid from a Gram-positive bacterium . TerLS20 is distinct from other known replication terminators in the sense that it is functional in both orientations . The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry. Mol Microbiol, 1996 Mar, 19(6), 1151 - 7 Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in Bacillus subtilis; Perego M et al.; Bacteria use two-component signal transduction systems to sense and respond to their environment . A sensor kinase and a response-regulator transcription factor work in concert by phosphorylation/dephosphorylation through kinase and phosphatase activities to maintain a level of phosphorylated response regulator commensurate with the level of signal input . Signal input can be accommodated through stimulation of the kinase activity or the phosphatase activity of the two-component system . With some notable exceptions, the sensor kinases recognize a single stimulatory ligand . A new dimension in the regulation of two-component signal transduction systems was discovered in the Rap phosphatases which dephosphorylate the SpoOF response-regulator of Bacillus subtilis independent of the sensor kinases . This family of phosphatases is encoded by at least six chromosomal genes . Although not all of the phosphatases of the family have activity on phosphorylated SpoOF, the two best-characterized members, RapA and RapB, prevent sporulation by dephosphorylating this response regulator component of the phosphorelay . Phosphatase activity of RapA is regulated by a gene, phrA, in the same transcriptional unit, that encodes a peptide secreted from the cell which may serve as a quorum sensor . Most of the Rap phosphatase operons have a gene coding for a protein with some similarity to PhrA in their transcription units, but it is uncertain whether all of these play a role in regulation . The Rap phosphatases are postulated to be a mechanism for allowing signals other than those that affect the sensor kinases to regulate the signal transduction pathway . They may have been recruited to help regulate sporulation because the multiple signals regulating this process may outstrip the recognition capacity of the kinases. Phytochemistry, 1996 Mar, 41(4), 1191 - 5 Antibacterial hydroperoxysterols from Xanthosoma robustum; Kato T et al.; Three new hydroperoxysterols, 24-hydroperoxy-4 alpha, 14 alpha- dimethylcholesta-8,25-dien-3 beta-ol, 25-hydroperoxy-4 alpha, 14 alpha-dimethylcholesta-8,23-dien-3 beta-ol and 25-hydroperoxycycloart-23-en- 3 beta-ol, have been isolated from the aerial parts of Xanthosoma robustum, besides 24-hydroperoxycycloart-25-en-3 beta-ol, 4 alpha, 14 alpha-dimethylcholesta-8,24-dien-3 beta-ol and cycloartenol . Additionally, the two new diols, 4 alpha, 14 alpha-dimethylcholesta-8,25-dien-3 beta, 24-diol and 4 alpha, 14 alpha-dimethylcholesta-8,23-dien-3 beta, 25-diol were obtained from the first two hydroperoxysterols, respectively, by reduction with triphenylphosphine . The structures have been defined by chemical and spectroscopic studies . The four hydroperoxysterols exhibited antibacterial activities against Escherichia coli, Bacillus subtilis and Micrococcus luteus. Phytochemistry, 1996 Mar, 41(4), 1041 - 6 Antimicrobial steroids from the fungus Fomitopsis pinicola; Keller AC et al.; Phytochemical examination of the dichloromethane extract of the European fungus Fomitopsis pinicola led to the isolation of a new lanostanoid derivative identified from spectral and chemical evidences as 3 alpha-(4-carboxymethyl-3-hydroxy-3-methylbutanoyloxy)-lanosta++ +-8,24-dien-21-oic acid . In addition, seven known triterpenes, polyporenic acid C, 3 alpha-acetyloxylanosta-8,24-dien-21-oic acid, ergosta-7,22-dien-3 beta-ol,21-hydroxylanosta-8,24-dien-3-one, pinicolic acid A, trametenolic acid B and pachymic acid, were also isolated . Antimicrobial activity against Bacillus subtilis in a TLC bioassay was observed for five of the isolated steroids. Prikl Biokhim Mikrobiol, 1996 Mar-Apr, 32(2), 231 - 6 {Hydrolyzing ability of yeast proteases in relation to protein substrates}; Nekliudov AD et al.; The possibility of use of brewer's yeast as multienzymatic preparations for hydrolyzing of animal blood proteins was demonstrated . The kinetic characteristics of hydrolysis of blood proteins by activated brewer's yeast mass and neutral proteinase from Bacillus subtilis 102 and the activation energies were estimated . Brewer's yeast biomass may be used as a source of enzymes of broad substrate specificity and noncontiguous amino acids, in particular, isoleucine, to increase the biological efficiency of blood proteins by 15-30%. Prikl Biokhim Mikrobiol, 1996 Mar-Apr, 32(2), 194 - 202 {Some patterns in formation of antibody-antigen-antibody complexes on a solid phase: experimental study and mathematical modeling}; Zherdev AV et al.; Equilibrium and kinetic constants were determined for interactions between alpha-amylase from Bacillus subtilis and polyclonal antibodies (with immobilization of either reagent) . The effects of desorption of immobilized molecules and intermolecular complexes on the immunochemical reaction were studied . Models of sandwich enzyme immunoassay, whereby complexes of immobilized antibody-determined antigen-labeled antibody were formed, were developed from these results . The results obtained from the model and experiments were compared . The desorption was shown to cause the hook-effect, that is, a decrease in the label binding at high concentrations of the antigen. Vet Med (Praha), 1996 Mar, 41(3), 93 - 5 High-voltage electrophoretic identification of residual antibiotics in milk; Krcmar P et al.; Residual antibiotics in milk were identified by high-voltage electrophoresis in 1% agarose gel and bioautographic detection . The test strain Bacillus subtilis BGA was used for the detection, pH of the culture medium was 8.0 . An electrophoretic identification map of 13 selected antibiotics has been set up and the following minimal inhibition concentrations (MIC), expressed in microgram per 1 g, have been established: benzylpenicillin 0.06; ampicillin 0.25; streptomycin 0.5; dihydrostreptomycin 0.2; spectinomycin 40; gentamycin 0.06; neomycin 0.15; oxytetracycline 5; tetracycline 2.5; chlortetracycline 1; erythromycin 0.01; tylosin 1; chloramphenicol 20 . The established values of MIC were compared with the Maximum Residue Limits (MRL) as defined currently in the legislation of the European Union . The condensation of samples by freeze-drying increased the sensitivity of the method which was used for the identification of residual antibiotics detected by microbiological screening techniques in milk. J Bacteriol, 1996 Mar, 178(5), 1473 - 5 The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain; Markham PN et al.; Rhodamine and tetraphenylphosphonium, the substrates of the Bacillus subtilis multidrug efflux transporter Bmr, induce the expression of Bmr through direct interaction with its transcriptional activator BmrR . Here we show that the C-terminal domain of BmrR, expressed individually, binds both these compounds and therefore can be used as a model for molecular analysis of the phenomenon of multidrug recognition. J Bacteriol, 1996 Mar, 178(5), 1374 - 85 Regulators of aerobic and anaerobic respiration in Bacillus subtilis; Sun G et al.; Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration . The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD . They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially . ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex) . The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins . resD null mutations are more deleterious to the cell than those of resE . resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate . A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation . The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis. J Mol Biol, 1996 Mar 1, 256(3), 436 - 48 The Bacillus subtilis response regulator Spo0A stimulates transcription of the spoIIG operon through modification of RNA polymerase promoter complexes; Bird TH et al.; Sporulation in Bacillus subtilis is dependent on the response regulator Spo0A, which both represses and activates transcription in vitro . The activity of Spo0A is increased by phosphorylation . We previously demonstrated that the phosphorylation increased the ability of Spo0A to stimulate in vivo transcription from the promoter for the spoIIG operon, one of the operons known to be regulated by Spo0A in vivo . In the work reported here we have examined the kinetics of transcription initiation at the spoIIG operon promoter using a single round transcription assay and the kinetics of formation of spoIIG promoter-RNA polymerase complexes using DNase I footprinting . Both the kinetic assays and the footprint assays indicated that the initial binding of the polymerase to the template was not dependent on the presence of Spo0A . The phosphorylated form of Spo0A stimulated the rate of initiation by affecting a step that occurred after the initial interaction of the polymerase with the template . Phosphorylation of Spo0A may stimulate transcription by modifying preinitiation complexes containing the polymerase and the promoter. FEBS Lett, 1996 Feb 26, 381(1-2), 29 - 31 A functional Spo0A is required for maximal aprE expression in Bacillus subtilis; Olmos J et al.; The initiation of sporulation in Bacillus subtilis is under control of the transcriptional factor Spo0A . Most Spo0A mutants fail to initiate the sporulation process and all the sporulation initiated processes such as the synthesis of subtilisin . However, the product of spo0A9V, one of the several spo0A mutants characterized, distinguishes itself in the fact that, while it appears to effectively repress abrB, it fails to activate the spoIIA operon . The aim of this study was to examine the effect of the spo0A9V mutation on aprE expression and we found that in different genetic backgrounds, the spo0A9V mutation has a negative effect on aprE::lacZ expression. J Mol Biol, 1996 Feb 23, 256(2), 301 - 18 The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination; Ayora S et al.; Inactivation of Bacillus subtilis orf1177 in an otherwise Rec+ strain reduced genetic exchange and DNA repair . When the mutation was transferred into a set of recombination-deficient and repair-deficient strains, the DNA repair and recombination ability of the double or triple mutant strains was drastically reduced . B . subtilis Orf1177 protein shares substantial homology with the Escherichia coli Mdf, RecG and UvrB proteins . In vivo analysis of UV-induced mutations suggests that Orf1177 is necessary for strand-specific DNA repair, as is the case for the E . coli MFD protein . Therefore, orf1177 and Orf1177 were termed mfd gene and Mfd protein, respectively . The purified Mfd protein has a native molecular mass of 140 kDa (expected molecular mass 133 kDa) . The Mfd protein is a sequence-independent DNA binding protein with weak ATPase activity . The Mfd protein was able to displace in vitro B . subtilis or E . coli RNA polymerase stalled at a lesion . Therefore, Mfd protein appears to target the transcribed strand for repair by recognizing a stalled RNA polymerase and dissociating it from the DNA . In addition, the strong recombination-deficient phenotype of mfd- rec- strains suggest that Mfd protein is involved in homologous DNA recombination. Gene, 1996 Feb 22, 169(1), 17 - 23 Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region; Suh JW et al.; We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs) . The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17 . The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons . This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36 . Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16 . Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters . The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required . Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization . Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs. Proc Natl Acad Sci U S A, 1996 Feb 20, 93(4), 1549 - 53 Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis; Perego M et al.; Rap phosphatases are a recently discovered family of protein aspartate phosphatases that dephosphorylate the Spo0F--P intermediate of the phosphorelay, thus preventing sporulation of Bacillus subtilis . They are regulators induced by physiological processes that are antithetical to sporulation . The RapA phosphatase is induced by the ComP-ComA two-component signal transduction system responsible for initiating competence . RapA phosphatase activity was found to be controlled by a small protein, PhrA, encoded on the same transcript as RapA . PhrA resembles secreted proteins and the evidence suggests that it is cleaved by signal peptidase I and a 19-residue C-terminal domain is secreted from the cell . The sporulation deficiency caused by the uncontrolled RapA activity of a phrA mutant can be complemented by synthetic peptides comprising the last six or more of the C-terminal residues of PhrA . Whether the peptide controls RapA activity directly or by regulating its synthesis remains to be determined . Complementation of the phrA mutant can also be obtained in mixed cultures with a wild-type strain, suggesting the peptide may serve as a means of communication between cells . Importation of the secreted peptide required the oligopeptide transport system . The sporulation deficiency of oligopeptide transport mutants can be suppressed by mutating the rapA and rapB genes or by introduction of a spo0F mutation Y13S that renders the protein insensitive to Rap phosphatases . The data indicate that the sporulation deficiency of oligopeptide transport mutants is due to their inability to import the peptides controlling Rap phosphatases. FEMS Microbiol Lett, 1996 Feb 15, 136(2), 151 - 6 Permeability of dormant spores of Bacillus subtilis to gramicidin S; Tanimoto Y et al.; Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis, had a partial inhibitory effect on L-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth . The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly . Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores . An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region . These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B . subtilis dormant spores. FEMS Microbiol Lett, 1996 Feb 15, 136(2), 117 - 22 Characterization of iturin synthetase in the wild-type Bacillus subtilis strain producing iturin and in an iturin deficient mutant; Feignier C et al.; The multi-enzyme system responsible for the biosynthesis of iturin, an antifungal lipopeptide of Bacillus subtilis, was partially purified by chromatography on different affigels . In the wild-type strain, two subunits of the iturin synthetase (ITs and ITagp) were characterized: ITs activated only L-Ser, one of the iturin amino acid components, and ITagp activated L-Asn, D-Asn, L-Gln and L-Pro, amino acids corresponding to a partial sequence of iturin . In an iturin deficient mutant, the activity of the ITagp subunit was modified. Experientia, 1996 Feb 15, 52(2), 175 - 9 Unusual antimicrobial compounds from Aeollanthus buchnerianus; Dellar JE et al.; Using bioassay guided isolation, three novel 12 carbon polyoxygenated fatty acids and a novel abietane diterpene have been isolated from the chloroform extract of aerial parts of Aeollanthus buchnerianus (Lamiaceae) . Rigorous spectroscopic methods were used for compound identification . (Z,Z)-8zeta-acetoxy-5zeta-hydroxydodeca-2,6-dienoic acid and (Z,Z)-5zeta, 8zeta-dihydroxydodeca-2,6-dienoic acid inhibited the spore germination of Cladosporium cucumerinum (both with Minimum Inhibitory Dose (MID) values of 1 microgram) and Aspergillus niger (MID 5 and 25 microgram respectively) . Further, they also reduced the hyphal growth of Pythium ultimum . (Z)-5zeta-hydroxy-6zeta,7zeta,8zeta-triacetox ydodeca-2-dienoic acid exhibited short term inhibition of the growth of Cladosporium cucumerinum . The novel abietane diterpenoid, (rel)-14alpha-acetoxyabiet-7-en-18-oic acid inhibited the growth of the gram positive bacteria Bacillus subtilis, Staphylococcus aureus and Streptomyces scabies (MIC values 80, 20 and 20 micrograms ml(-1) respectively). Biochem Biophys Res Commun, 1996 Feb 15, 219(2), 463 - 8 Secretion of active subtilisin YaB by a simultaneous expression of separate pre-pro and pre-mature polypeptides in Bacillus subtilis; Chang YC et al.; Alkaline elastase YaB, produced by alkalophilic Bacillus YaB, is an extracellular serine protease having 55% homology to subtilisin BPN' and thus could be called subtilisin YaB . It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease . To examine if the pro-peptide of subtilisin YaB functions in trans to guide the folding of secreted subtilisin YaB in vivo, we made genes encoding the pre-pro, pro and pre-mature portions and placed them under the control of the spac-1 promoter on a multi-copy plasmid . When simultaneous expression in Bacillus subtilis of both the pre-pro and pre-mature genes was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG), protease activity was detected in the medium . On the other hand, we could not detect protease activity when the expression of either the pre-mature gene alone or both the pro and pre-mature genes was induced . From these results, we concluded that the pro region functions in trans and outside the cells for the proper folding and activation of the enzyme. Nucleic Acids Res, 1996 Feb 15, 24(4), 628 - 39 Sequence analysis of 56 kb from the genome of the bacterium Mycoplasma pneumoniae comprising the dnaA region, the atp operon and a cluster of ribosomal protein genes; Hilbert H et al.; To sequence the entire 800 kilobase pair genome of the bacterium Mycoplasma pneumoniae, a plasmid library was established with contained the majority of the EcoR1 fragments from M.pneumoniae . The EcoR1 fragments were subcloned from an ordered cosmid library comprising the complete M.pneumoniae genome . Individual plasmid clones were sequenced in an ordered fashion mainly by primer walking . We report here the initial results from the sequence analysis of -56 kb comprising the dnaA region as a potential origin of replication, the ATPase operon and a region coding for a cluster of ribosomal protein genes . The data were compared with the corresponding genes/operons from Bacillus subtilis, Escherichia coli, Mycoplasma capricolum and Mycoplasma gallisepticum. Genes Dev, 1996 Feb 15, 10(4), 478 - 88 Transcription factor Spo0A switches the localization of the cell division protein FtsZ from a medial to a bipolar pattern in Bacillus subtilis; Levin PA et al.; Entry into sporulation by the Gram-positive bacterium Bacillus subtilis is governed by two transcription factors, Spo0A and sigma H, and involves a switch in the site of division from a medial to a polar location . We report that at the onset of sporulation, assembly of the cell division protein FtsZ shifts from midcell to potential division sites near both poles . The switch to a bipolar pattern of FtsZ localization is dependent on Spo0A . Additionally, synthesis of an activated form of Spo0A during growth artificially activates the switch in FtsZ localization and results in the formation of polar septa . The sigma H factor, on the other hand, is dispensable for the switch in the position of the FtsZ assembly site, although it is required for formation of the polar septum . Our results suggest that during the transition from growth to sporulation, Spo0A induces the expression of genes that suppress FtsZ assembly at the midcell site and activate sites at both poles, whereas sigma H induces genes required for a subsequent step in cytokinesis. Mol Gen Genet, 1996 Feb 5, 250(2), 230 - 6 A T7 promoter-specific, inducible protein expression system for Bacillus subtilis; Conrad B et al.; The adaptation and application of the Escherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression in Bacillus subtilis is reported . The expression cassette used in Bacillus subtilis was tightly regulated and T7 RnA polymerase (T7 RNAP)appeared 30 minutes after induction . The efficiency of T7 promoter-specific gene expression in B.subtilis was studied using one secretory and two cytosolic proteins of heterologous origin . The accumulation of E . coli beta-galactosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter brockii in B . subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity . The alpha-amylase of Thermactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10-20 h after T7 RNAP induction, but was also deposited in cellular fractions . The addition of rifampicin inhibited chi-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation. Gene, 1996 Feb 2, 168(1), 61 - 5 Cloning and sequencing of a new holin-encoding gene of Bacillus licheniformis; Kyogoku K et al.; A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec) . Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptides of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively) . Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1-G2, respectively, exhibited higher extra-cellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398 . The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin-encoding gene . Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of beta-galactosidase into the extracellular fraction. Gene, 1996 Feb 2, 168(1), 55 - 60 Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase; Fischer C et al.; We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169 degrees on the chromosome . An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment . This fragment contained an incomplete and two complete open reading frames (ORFs) . The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25038 Da), was found to be responsible for the observed reduced mannitol fermentation . The 3' part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identify . However, the function of these two proteins remains unknown . The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72889 Da . PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases . Several of the essential aa in the loops known to form the active site of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA . After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue. Gene, 1996 Feb 2, 168(1), 123 - 4 Sequence of a gene encoding a putative primary sigma factor from Borrelia burgdorferi strain B31; Tsai CP et al.; Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced . Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa) . Comparison with Escherichia coli sigma 70 and Bacillus subtilis sigma 43 showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding. J Biol Chem, 1996 Feb 2, 271(5), 2621 - 6 Analysis of abrB mutations, mutant proteins, and why abrB does not utilize a perfect consensus in the -35 region of its sigma A promoter; Xu K et al.; The Bacillus subtilis global regulator AbrB is a DNA-binding protein composed of six identical monomers of 96 amino acids that shows specificity to the promoter regions of its target genes including its own . We have sequenced thirteen previously uncharacterized abrB mutations . Four mutant AbrB proteins were purified, and their DNA-binding properties and multimeric structures were examined . AbrB23 (R25S) had no appreciable DNA binding activity but retained a hexameric structure, indicating that Arg25 is important in DNA interactions . Three other mutant proteins, AbrB1 (C56Y), AbrB19 (Gln83-->termination codon), and AbrB100 (L69P), showed decreased DNA binding and altered multimeric interactions . Analysis of the expression and AbrB binding affinities of mutant abrB promoters demonstrated that a consensus -35 region is incompatible with proper autoregulation of the abrB gene. Mol Divers, 1996 Feb, 1(2), 121 - 4 Antibiotic activity of polyketide products derived from combinatorial biosynthesis: implications for directed evolution; Fu H et al.; A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria . A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified . Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized . When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively . We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways . If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation . Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules . They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties. Biosci Biotechnol Biochem, 1996 Feb, 60(2), 271 - 6 Bacillus stearothermophilus cell shape determinant gene, mreC and mreD, and their stimulation of protease production in Bacillus subtilis; Kubo M et al.; Protease production stimulating genes were isolated from a soybean protein degrading bacterium, Bacillus stearothermophilus HA19 . The cloned fragment stimulated production of a 37-kDa protease in B . subtilis . The nucleotide sequence of the genes and their flanking regions were identical to the B . subtilis cell shape determinant genes mreC and mreD {J . Bacteriol., 176, 6729-6742 (1992); J . Bacteriol., 176, 6717-6728 (1992)} . The mreC and mreD genes in B . subtilis stimulate secretion of a neutral protease (37-kDa), and the protease activity in the culture medium reached 2500 U per ml (approximately 10 times higher than the host strain) after 24 h of cultivation in L broth, suggesting the mreCD genes regulate protease expression and the protease is related to the cell shape determination in Bacilli . The protease productions in B . subtilis carrying mreC or mreD deletion plasmids were not elevated, so the 37-kDa protease stimulation requires both mreC and mreD genes . The extracellular protease was purified, and the molecular mass of the enzyme was 37,000 Da by SDS-polyacrylamide gel electrophoresis and gel filtration . The optimum pH and temperature for the enzyme activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable at pH 7-10 . The enzyme was inactivated by EDTA, but not by phenylmethyl sulfonyl fluoride and diisopropyl fluorophosphate. Orig Life Evol Biosph, 1996 Feb, 26(1), 47 - 59 Response of Bacillus subtilis spores to dehydration and UV irradiation at extremely low temperatures; Dose K et al.; Spores of Bacillus subtilis have been exposed to the conditions of extreme dehydration (argon/silica gel; simulated space vacuum) for up to 12 weeks at 298 K and 80 K in the dark . The inactivation has been correlated with the production of DNA-double strand-breaks . The temperature-dependence of the rate constants for inactivation or production of DNA-double strand-breaks is surprisingly low . Controls kept in the frozen state at 250 K for the same period of time showed no sign of deterioration . In another series of experiments the spores have been UV irradiated (253.7 nm) at 298 K, 200 K and 80 K after exposure to dehydrating conditions for 3 days . Fluence-effect relationships for inactivation, production of DNA-double strand-breaks and DNA-protein cross-links are presented . The corresponding F37-values for inactivation and production of DNA lesions are significantly increased only at 80 K (factor of 4 to 5) . The data indicate that the low temperatures that prevail in the outer parts of the Solar System or at the nightside of Mars or the Moon are not sufficiently low to crucially inhibit inactivation by dehydration . Our data place further constraints on the panspermia hypothesis. Appl Microbiol Biotechnol, 1996 Feb, 44(6), 746 - 52 Purification and characterization of a truncated Bacillus subtilis alpha-amylase produced by Escherichia coli; Marco JL et al.; A Bacillus subtilis amylase gene was inserted into a plasmid which was transferred to Escherichia coli . During cloning, a 3' region encoding 171 carboxy-terminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein . The transformed cells produced substantial amylolytic activity . The active protein was purified to apparent homogeneity . Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of the B . subtilis complete alpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa) . This truncated enzyme form hydrolysed starch with a Km of 3.845 mg/ml . Activity was optimal at pH 6.5 and 50 degrees C, and the purified enzyme was stable at temperatures up to 50 degrees C . While Hg2+, Fe3+ and Al+3 were effective in inhibiting the truncated enzyme, Mn2+ and Co2+ considerably enhanced the activity. Mol Microbiol, 1996 Feb, 19(3), 587 - 98 Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome; Morel-Deville F et al.; To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of approximately 4 kb . The replicon was derived from a broad-host-range plasmid, pAM beta 1, which replicates by a unidirectional theta mechanism and its thermosensitive . The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194 . Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats . The integrated replicon was active at the permissive temperature, and approximately 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin . Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats . We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication. Mol Microbiol, 1996 Feb, 19(4), 901 - 7 Establishing differential gene expression in sporulating Bacillus subtilis: phosphorylation of SpoIIAA (anti-anti-sigmaF) alters its conformation and prevents formation of a SpoIIAA/SpoIIAB/ADP complex; Magnin T et al.; Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Bacillus subtilis . Its activity is controlled by an anti-sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA . We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated . Upon gel filtration in the presence of ADP, SpoIIAA-P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was coeluted with SpoIIAB, indicating the formation of a protein/protein complex . The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer . Its dissociation constant was 13 mu M . Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA-P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA-P was not a phosphorylated dimer of SpoIIAA . Our tentative conclusion, that SpoIIAA and SpoIIAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin. Proteins, 1996 Feb, 24(2), 238 - 46 Overexpression of Bacillus subtilis phosphoribosylpyrophosphate synthetase and crystallization and preliminary X-ray characterization of the free enzyme and its substrate-effector complexes; Bentsen AK et al.; Bacillus subtilis phosphoribosylpyrophosphate (PRPP) synthetase has been expressed to high levels in an Escherichia coli host strain devoid of endogenous PRPP synthetase . A rapid and efficient purification protocol has been developed allowing production of enzyme preparations with purity conforming to the stringent criteria required for crystallization . Crystallization experiments, in combination with dynamic light scattering studies, have led to the production of three crystal forms of the enzyme . These forms include the free enzyme, the enzyme in a binary complex with the substrate adenosine triphosphate (ATP), and the enzyme in a quaternary complex with the substrate analog alpha, beta-methylene adenosine triphosphate (mATP), the substrate ribose-5-phosphate (Rib-5-P), and the allosteric inhibitor adenosine diphosphate (ADP) . Diffraction data showed that all three crystal forms are suitable for structure determination . They crystallize in the same hexagonal space group, P6(3), with virtually identical unit cell dimensions of a = b = 115.6 angstroms, c = 107.8 angstrom, and with two molecules in the asymmetric unit . The self-rotation function showed the existence of a non-crystallographic twofold axis perpendicular to the c axis . The availability of the different complexes should allow questions regarding the molecular mechanisms of catalysis and allostery in PRPP synthetase to be addressed. Planta Med, 1996 Feb, 62(1), 67 - 9 Antifungal and antibacterial chalcones from Myrica serrata; Gafner S et al.; The dichloromethane extract of the leaves of Myrica serrata inhibits growth of Cladosporium cucumerinum, Bacillus subtilis, and Escherichia coli on TLC plates . Activity-guided fractionation led the isolation of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (1), 2',4'-dihydroxy-6'-methoxy-5'-methylchalcone (aurentiacin A) (2), 2',6'-dihydroxy-4'-methoxy-3',5'-dimethyldihydrochalcone (3), 2'-hydroxy-4',6'-dimethoxy-3'-methyldihydrochalcone (4), and 2', 6'-dihydroxy-4'-methoxy-3'-methyldihydrochalcone (5) . In addition, the flavanones demethoxymatteucinol (6) and cryptostrobin (7) were also identified. Curr Opin Struct Biol, 1996 Feb, 6(1), 53 - 61 RNA-protein complexes; Nagai K; The three commonly found RNA-binding domains, the ribonucleoprotein (RNP) domain, the double stranded RNA binding domain (dsRBD) and the K homology (KH) domain, have now been shown to have an alpha/beta fold similar to that found in many ribosomal proteins . Crystal structures of two hairpin RNA-protein complexes have been determined recently: the U1A spliceosomal protein bound to hairpin II of U1 small nuclear RNA, and MS2 bacteriophage capsid protein bound to a hairpin present at the ribosomal binding site of MS2 replicase mRNA . The crystal structure of the tryptophan operon RNA binding attenuation protein from Bacillus subtilis shows a novel structure with 11 monomers arranged in a doughnut-shaped ring that binds 11 copies of (U/G)AG triplets presented in the leader sequence of the tryptophan operon polycistronic message. Curr Biol, 1996 Feb 1, 6(2), 111 - 4 Bacterial differentiation: sizing up sporulation; Jenal U et al.; New results on Bacillus subtilis sporulation suggest that size differences between the post-septation compartments trigger differential gene expression, which is then coordinated by communication between the nascent mother cell and forespore compartments. Exp Cell Res, 1996 Feb 1, 222(2), 345 - 59 Affinity purification and comparative analysis of two distinct human uracil-DNA glycosylases; Caradonna S et al.; Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells . We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells . This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by the Bacillus subtilis bacteriophage, PBS2 . Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS-PAGE and silver staining . In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations . Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell . Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1 . With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment . Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes . Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently post-translationally processed into a lower molecular weight form . This is substantiated by mitochondrial import studies which reveal that in vitro expressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria . Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria . Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000 . SDS-PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell . Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus . Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA . These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme. Appl Environ Microbiol, 1996 Feb, 62(2), 545 - 51 Comparative sporicidal effects of liquid chemical agents; Sagripanti JL et al.; We compared the effectiveness of glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate (plus a sublethal amount of hydrogen peroxide), sodium hypochlorite, and phenol to inactivate Bacillus subtilis spores under various conditions . Each chemical agent was distinctly affected by pH, storage time after activation, dilution, and temperature . Only three of the preparations (hypochlorite, peracetic acid, and cupric ascorbate) studied here inactivated more than 99.9% of the spore load after a 30-min incubation at 20 degrees C at concentrations generally used to decontaminate medical devices . Under similar conditions, glutaraldehyde inactivated approximately 90%, and hydrogen peroxide, formaldehyde, and phenol produced little killing of spores in suspension . By kinetic analysis at different temperatures, we calculated the rate of spore inactivation (k) and the activation energy of spore killing (delta E) for each chemical agent . Rates of spore inactivation had a similar delta E value of approximately 20 kcal/mol (ca.83.68 kJ/mol) for every substance tested . The variation among k values allowed a quantitative comparison of liquid germicidal agents. J Bacteriol, 1996 Feb, 178(4), 1197 - 9 Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria; Pelicic V et al.; Expression in mycobacteria of the structural gene sacB, which encodes the Bacillus subtilis levansucrase, was investigated . sacB expression is lethal to Mycobacterium smegmatis and Mycobacterium bovis BCG in the presence of 10% sucrose . sacB could thus be used as a counterselectable marker in mycobacteria. J Bacteriol, 1996 Feb, 178(4), 1178 - 86 Identification of a membrane protein involved in activation of the KinB pathway to sporulation in Bacillus subtilis; Dartois V et al.; The initiation of sporulation in Bacillus subtilis is dependent on the phosphorylation of the Spo0A transcription factor mediated by the phosphorelay and by two major kinases, KinA and KinB . Temporal expression of these kinases was analyzed, and an assessment of their respective contributions to the production of Spo0A-P was undertaken . The results show that KinB is expressed and activated prior to KinA; i.e., the two kinases are solicited sequentially in the sporulation process and are thought to be activated by different signaling pathways . A strategy was developed to isolate mutations specifically affecting the KinB pathway, using the newly improved mini-Tn10 delivery vector pIC333 . Several mutants were obtained, one of which carried a transposon in a gene coding for a small integral membrane protein, named KbaA . Inactivation of the kbaA gene appeared to affect KinB activity but not transcription of kinB . A Spo+ suppressor (kinB45) of the kbaA null mutation was isolated in the promoter region of kinB . An eightfold increase of kinB expression levels over wild-type levels was observed in the kinB45 mutant . Thus, overexpression of the kinB-kapB operon was sufficient to overcome the sporulation defect caused by inactivation of kbaA in a KinA- strain . Transcription of kinB was found to be repressed by SinR, while the kinB45 mutant was no longer sensitive to SinR regulation . Implications of these observations on the transcriptional regulation of kinB and the role of KbaA in KinB activation are discussed. J Bacteriol, 1996 Feb, 178(4), 1088 - 93 hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes; Schulz A et al.; Whereas in Escherichia coli only one heat shock regulon is transiently induced by mild heat stress, for Bacillus subtilis three classes of heat shock genes regulated by different mechanisms have been described . Regulation of class I heat shock genes (dnaK and groE operons) involves an inverted repeat (CIRCE element) which most probably serves as an operator for a repressor . Here, we report on the analyses of an hrcA null mutant (delta hrcA), in which hrcA, the first gene of the dnaK operon, was deleted from the B . subtilis chromosome . This strain was perfectly viable at low and high temperatures . Transcriptional analysis of the deletion mutant revealed a high level of constitutive expression of both the dnaK and groE operons even at a low temperature . A further increase in the amount of groE transcript was observed after temperature upshift, suggesting a second induction mechanism for this operon . Overproduction of HrcA protein from a second copy of hrcA derived from a plasmid (phrcA+) in B . subtilis wild-type and delta hrcA strains prevented heat shock induction of the dnaK and groE operons at the level of transcription almost completely and strongly reduced the amounts of mRNA at a low temperature as well . Whereas the wild-type strain needed 4 h to resume growth after temperature upshift, the delta hrcA strain stopped growth only for about 1 h . Overproduction of HrcA protein prior to a heat shock almost completely prevented growth at a high temperature . These data clearly demonstrate that the hrcA product serves as a negative regulator of class I heat shock genes. J Bacteriol, 1996 Feb, 178(3), 854 - 61 Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3-dihydroxybenzoate; Rowland BM et al.; Bacillus subtilis has duplicate isochorismate synthase genes, menF and dhbC . Isochorismate synthase is involved in the biosynthesis of both the respiratory chain component menaquinone (MK) and the siderophore 2,3-dihydroxybenzoate (DHB) . Several menF and dhbC deletion mutants were constructed to identify the contribution made by each gene product to MK and DHB biosynthesis . menF deletion mutants were able to produce wild-type levels of MK and DHB, suggesting that the dhbC gene product is able to compensate for the lack of MenF . However, a dhbC deletion mutant produced wild-type levels of MK but was DHB deficient, indicating that MenF is unable to compensate for the lack of DhbC . A menF dhbC double-deletion mutant was both MK and DHB deficient . Transcription analysis showed that expression of dhbC, but not of menF, is regulated by iron concentration . A dhbA'::lacZ fusion strain was constructed to examine the effects of mutations to the iron box sequence within the dhb promoter region . These mutations abolished the iron-regulated transcription of the dhb genes, suggesting that a Fur-like repressor protein exists in B . subtilis. J Bacteriol, 1996 Feb, 178(3), 846 - 53 Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis; Nygaard P et al.; The isolation of mutants defective in adenine metabolism in Bacillus subtilis has provided a tool that has made it possible to investigate the role of adenine deaminase in adenine metabolism in growing cells . Adenine deaminase is the only enzyme that can deaminate adenine compounds in B . subtilis, a reaction which is important for adenine utilization as a purine and also as a nitrogen source . The uptake of adenine is strictly coupled to its further metabolism . Salvaging of adenine is inhibited by the stringent response to amino acid starvation, while the deamination of adenine is not . The level of adenine deaminase was reduced when exogenous guanosine served as the purine source and when glutamine served as the nitrogen source . The enzyme level was essentially the same whether ammonia or purines served as the nitrogen source . Reduced levels were seen on poor carbon sources . The ade gene was cloned, and the nucleotide sequence and mRNA analyses revealed a single-gene operon encoding a 65-kDa protein . By transductional crosses, we have located the ade gene to 130 degrees on the chromosomal map. J Bacteriol, 1996 Feb, 178(3), 768 - 73 The permeability of the wall fabric of Escherichia coli and Bacillus subtilis; Demchick P et al.; To study the overall structure of the peptidoglycan fabric of the sacculi of gram-negative and gram-positive walls, actively growing cultures of Escherichia coli and Bacillus subtilis were treated with boiling sodium dodecyl sulfate solutions . The sacculi were then treated with enzymes to eliminate proteins and nucleic acids . These intact saccoli were probed with fluorescein-labeled dextrans with a range of known molecular weights . The penetration of the probes could be monitored by the negative-staining appearance in the fluorescence microscope . At several chosen times, the molecular weight fraction that allowed barely observable entry of the fluorescein-labeled probe and the molecular weight fraction that penetrated to achieve almost, but not quite, the concentration of probe in the solution external to the sacculi were determined . From three pairs of times and molecular weights that met one or the other of these two criteria, the effective pore size could be calculated . The minimum size of protein molecule that could diffuse through the pores was also calculated . Two mathematical models, which gave essentially the same results, were used to interpret the experimental data: one for the permeation of random coils through a surface containing holes and the other for rigid spheres diffusing through water-filled cylindrical pores . The mean estimate of the effective hole radius in walls from E . coli is 2.06 nm, and that of the effective hole size in walls from B . subtilis is 2.12 nm . These results are supported by experiments in which the loss of preloaded cells was monitored . Various fluorescein-labeled dextran samples were mixed with samples of intact cell walls, held for a long time, and then diluted . The efflux of the dextrans was monitored . Neither large nor small dextrans stained under these conditions . Only with dextran samples of a sufficiently small size were the sacculi filled during the preincubation period, and only with the largest of these could the probe not escape quickly . From the pore (or mesh) size, it can be concluded that the wall fabric of both organisms has few imperfections and that the major passageway is through the smallest possible pore, or "tessera," formed by the maximal cross-linking of the peptides from glycan chain to glycan chain compatible with the degree of rotational flexibility of the chains of repeating disaccharides of N-acetyl muramic acid and N-acetyl glucosamine . A tessera is composed of two chains of eight saccharides cross-linked by two octapeptides . The size of a globular hydrophilic molecule, if it did not bind to wall components, that could pass freely through the meshwork of an unstretched sacculus of either organism is roughly 25 kDa . We stress that this is only a rough estimate, and it may be possible for proteins of less than 50 kDa to pass through the native wall during normal growth conditions. J Bacteriol, 1996 Feb, 178(3), 705 - 13 Transcriptional regulation of the Bacillus subtilis menp1 promoter; Qin X et al.; The Bacillus subtilis men genes encode biosynthetic enzymes for formation of the respiratory chain component menaquinone . The menp1 promoter previously was shown to be the primary cis element for menFD gene expression . In the present work, it was found that either supplementation with nonfermentable carbon sources or reutilization of glycolytic end products increased menp1 activity in the late postexponential phase . The effect on menp1 activity by a particular end product (such as acetoin or acetate) was prevented by blocking the corresponding pathway for end product utilization . Alteration of a TGAAA motif within the promoter region resulted in unregulated menp1 activity throughout the culture cycle, irrespective of the carbon source added. Nucleic Acids Res, 1996 Jan 15, 24(2), 282 - 8 Distamycin-induced inhibition of formation of a nucleoprotein complex between the terminase small subunit G1P and the non-encapsidated end (pacL site) of Bacillus subtilis bacteriophage SPP1; Chai S et al.; The small subunit of the Bacillus subtilis bacteriophage SPP1 terminase (G1P) forms a sequence-specific nucleoprotein complex with the SPP1 non-encapsidated end (pacL site) during initiation of DNA encapsidation . Gel mobility shift assay was used to study the G1P-pacL interaction . Distamycin, a minor groove binder that induces local distortion of the DNA, inhibits G1P-pacL complex formation . The competition of G1P with distamycin for DNA binding at the pacL site is independent of the order of addition of the reactants . Other minor groove binders, such as spermine or Hoechst 33258, which do not distort DNA, failed to compete with G1P for pacL DNA binding . Cationic metals, which generate a repertoire of DNA structures different from that caused by the minor groove binders, can partially reverse the distamycin-induced inhibition of G1P binding to pacL DNA . The major groove binder methyl green, which does not distort sequence-directed bending of pacL DNA, competes with G1P for binding at the pacL site . Our data suggest that the natural sequence-directed bend that exists within the pacL site is the architectural element that facilitates assembly of a nucleoprotein complex and hence initiation of DNA encapsidation by bacteriophage SPP1. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 347 - 51 Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity; Seok YJ et al.; The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr) . The isolated amino-terminal domain (EIN) of E . coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr . An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein . A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield . Intact E . coli EI is effective in phosphotransfer from PEP to HPr from E . coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum . Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E . coli or M . capricolum requires the intermediacy of HPr . The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E . coli, B . subtilis, and M . capricolum as well as to EIAglc from E . coli . These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E . coli HPr. FEBS Lett, 1996 Jan 2, 378(1), 98 - 100 Crystallisation of the Bacillus subtilis sporulation inhibitor SinR, complexed with its antagonist, SinI; Lewis RJ et al.; The transcription factor SinR, a pleiotropic regulator of late growth processes in Bacillus subtilis, has been crystallised as a complex with its antagonist SinI, in a form suitable for structural analysis . The SinI:SinR crystals diffract X-rays generated from a rotating copper anode source to 2.3 A spacing and a complete native dataset has been collected to this resolution limit . The space group of the crystals is P3(1)21 (or its enantiomorph P3(2)21) with cell dimensions a = b = 60.76 A, c = 87.79 A . Assuming that there is a single SinI:SinR heterodimer in the asymmetric unit, the crystals have a Vm of 2.53 A3.Da-1. Biochimie, 1996, 78(11-12), 1017 - 24 Some novel transcription attenuation mechanisms used by bacteria; Yanofsky C et al.; A variety of transcription attenuation mechanisms are used by bacteria to regulate gene and operon expression . This review summarizes previous and current studies designed to elucidate the features of the specific attenuation mechanisms that regulate expression of the tryptophanase (tna) operon of Escherichia coli and the tryptophan (trp) operon of Bacillus subtilis . Initiation of transcription in the tna operon is regulated by catabolite repression . Once initiated, transcription is regulated by tryptophan-induced inhibition of Rho-mediated transcription termination in the leader region of the operon . An operon-encoded leader peptide, TnaC, containing a crucial tryptophan residue, plays an essential role in induction . This peptide appears to act in cis on the ribosome translating tnaC to inhibit its release at the tnaC stop codon . The stalled ribosome would block Rho's access to the tna transcript, thereby preventing termination . Transcription of the trp operon of B subtilis is regulated by an attenuation mechanism that responds to a tryptophan-activated eleven subunit RNA-binding regulatory protein, called TRAP . Activated TRAP binds to repeated GAG sequences in the leader segment of the trp operon transcript, disrupting an RNA antiterminator and promoting formation of a terminator . Activated TRAP also regulates translation of trpG in the folate operon by binding to repeat GAG sequences surrounding the trpG ribosome binding site . A temperature sensitive tryptophanyl-tRNA synthetase (trpS) mutant was previously observed to overexpress the trp operon and trpG, when grown at elevated temperatures in the presence of tryptophan . We have found that the trpS defect increases trp operon and trpG expression by interfering with TRAP's ability to act . We suggest that either accumulation of uncharged tRNA(Trp) or overproduction of a TRAP-binding transcript reduces the level of functional TRAP in the trpS mutant. Acta Microbiol Immunol Hung, 1996, 43(4), 289 - 99 The development of a Bacillus subtilis 168 culture condition for enhanced and accelerated beta-mannanase production; el-Helow ER et al.; A shaken flask cultivation condition for enhanced and accelerated beta-mannanase formation by Bacillus subtilis 168 was achieved . Among five examined fermentation media a formula that supported enzyme generation and retarded biomass yield and sporulation was selected . The deficiency of biomass production in this medium was mastered by choosing a seed culture medium that accelerated growth and initiation of beta-mannanase synthesis . With respect to enzyme production, the optimum pH and temperature were 7 and 37 degrees C, respectively . The biosynthesis of the enzyme was extremely influenced by the cell growth state as a modulator, glucose as a catabolite repressor, and galactomannan as an inducer . A galactomannan concentration of 4 g l-1 induced a beta-mannanase activity level of 17.5 U ml-1 after 24 h of incubation at the experimental condition . Higher inducer concentrations supported growth rather than enzyme production . The influence of inoculum size was so remarkable that, at optimum, a crude filtrate with an enzyme activity of 33U ml-1 was yielded within 4 hours . It appears that this is among the highest rates reported for beta-mannanase production . We have also demonstrated that blocking of the sporulation process at stage II do not affect enzyme production significantly . This would allow an extended enzyme production phase especially in a continuous culture. Rocz Panstw Zakl Hig, 1996, 47(4), 439 - 44 {The effect of growth media on recovery of test microorganisms after exposure to saturated steam under pressure}; Krzywicka H et al.; The aim of this study was to find out which growth media give the best condition for the development of test bacteria after exposure to saturated steam under pressure . The test organisms were strains of Bacillus subtilis NCTC 3610 and Bacillus stearothermophilus NCTC 8923 . The test prepared from spore suspension were exposed to saturated steam under pressure 0.2 atn-B.subtilis, and 0.7 atn-B . stearothermophilus with various length of exposure /sublethal conditions/ . After the exposure the tests were placed in growth media . The obtained results show that the compositions of the medium in which spore-forming bacteria are grown after the exposure under sublethal conditions to saturated steam under pressure affects the recovery of the test organism . The media with glucose, tryptose and L-alanine provided the best conditions for growth. Protein Eng, 1996 Jan, 9(1), 77 - 83 Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide; You L et al.; Sequential rounds of error-prone PCR to introduce random mutations and screening of the resultant mutant libraries have been used to enhance the total catalytic activity of subtilisin E significantly in a non-natural environment, aqueous dimethylformamide (DMF) . Seven DNA substitutions coding for three new amino acid substitutions were identified in a mutant isolated after two additional generations of directed evolution carried out on 10M subtilisin E, previously "evolved' to increase its specific activity in DMF . A Bacillus subtilis-Escherichia coli shuttle vector was developed in order to increase the size of the mutant library that could be established in B.subtilis and the stringency of the screening process was increased to reflect total as well as specific activity . This directed evolution approach has been extremely effective for improving enzyme activity in a non-natural environment: the resulting-evolved 13M subtilisin exhibits specific catalytic efficiency towards the hydrolysis of a peptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in 60% DMF solution that is three times that of the parent 10M and 471 times that of wild type subtilisin E . The total activity of the 13M culture supernatant is enhanced 16-fold over that of the parent 10M. Annu Rev Genet, 1996, 30, 297 - 41 Molecular genetics of sporulation in Bacillus subtilis; Stragier P et al.; The process of sporulation in the bacterium Bacillus subtilis proceeds through a well-defined series of morphological stages that involve the conversion of a growing cell into a two-cell-chamber sporangium within which a spore is produced . Over 125 genes are involved in this process, the transcription of which is temporally and spatially controlled by four DNA-binding proteins and five RNA polymerase sigma factors . Through a combination of genetic, biochemical, and cell biological approaches, regulatory networks have been elucidated that explicitly link the activation of these sigma factors to landmark events in the course of morphogenesis and to each other through pathways of intercellular communication . Signals targeting proteins to specific subcellular localizations and governing the assembly of macromolecular structures have been uncovered but their nature remains to be determined. Biochimie, 1996, 78(6), 381 - 9 Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis; Condon C et al.; In this review, we summarize progress on the regulation of the aminoacyl-tRNA synthetase genes in Bacillus subtilis . Most of the genes encoding this set of enzymes in B subtilis are members of a large family of Gram-positive genes and operons controlled by a novel antitermination mechanism that uses their cognate uncharged tRNA as the effector . A subset of these genes is, in addition, likely to be controlled at the level of mRNA processing and degradation . We describe the key experiments leading to these conclusions. Biotechnol Prog, 1996 Jan-Feb, 12(1), 31 - 9 Intracellular expression of Vitreoscilla hemoglobin (VHb) enhances total protein secretion and improves the production of alpha-amylase and neutral protease in Bacillus subtilis; Kallio PT et al.; In an attempt to alleviate oxygen limitation during batch cultivations, a heterologous bacterial hemoglobin gene (vhb) of Vitreoscilla was introduced into Bacillus subtilis . Biochemically active VHb, as demonstrated by immunoblot analysis and carbon monoxide binding assay, was intracellularly expressed in B . subtilis from an inducible promoter-repressor (spac-lacI) . Expression of VHb in oxygen-limited B . subtilis batch bioreactor cultivations enhanced cell growth, decreased accumulation of acetate, and increased the total protein secreted into the culture medium by approximately 1.5-fold . In addition, VHb-expressing B . subtilis cultures exhibited increases of approximately 30% and 5-17% in neutral protease activity and alpha-amylase activity, respectively, relative to the parental, VHb-free strain. Mol Microbiol, 1996 Jan, 19(2), 343 - 56 Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis; Luttinger A et al.; comR (pnpA) is a newly identified gene in Bacillus subtilis that is necessary for the expression of late competence genes . Transformability of a comR (pnpA) mutant is 1-5% of that seen in comR+ strains . Cloning and sequencing identified ComR as polynucleotide phosphorylase (PNPase) . The PNPase amino acid sequence has 50% identity and 67% similarity with the Escherichia coli enzyme . Enzymatic assays show that this is the only PNPase activity in B . subtilis . comR (pnpA) is necessary for comG-lacZ and comK-lacZ expression, but this requirement is bypassed by a mecA disruption . In B . subtilis, the loss of PNPase has little effect on expression from a fusion of the srfA promoter directly to lacZ, but is necessary for normal expression from certain srfA-lacZ fusions that include portions of the normal srfA transcript . When a srfA-lacZ translational fusion is tested in isogenic pnpA+ and pnpA derivatives of E . coli, lower expression is seen in the pnpA mutant . Since expression from lacZ fusions to comA, sinR, and mecA appeared similar in the B . subtilis pnpA and pnpA+ strains, the loss of PNPase does not have a strong general effect on gene expression . These results suggest that PNPase may be necessary for modification of the srfA transcript in order to activate translation or stabilize the transcript, and that this may be necessary for competence development . This is the first evidence of post-transcriptional effects on the development of competence in B . subtilis. Mol Microbiol, 1996 Jan, 19(2), 329 - 42 In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase; Mitra S et al.; A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose . Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B . subtilis PNPase gene (Luttinger et al., 1996--accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase) . Processive 3'-to-5' exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site . Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure . A similar stall site was observed for an RNA that comprised the intergenic region between the B . subtilis rpsO and pnpA genes . The ability to initiate degradation of a substrate that had a stem structure at its 3' end differed for the B . subtilis and Escherichia coli PNPase enzymes. Mol Microbiol, 1996 Jan, 19(2), 319 - 28 A dual role for the Bacillus subtilis glpD leader and the GlpP protein in the regulated expression of glpD: antitermination and control of mRNA stability; Glatz E et al.; The Bacillus subtilis glpD gene encodes glycerol-3-phosphate dehydrogenase . This gene is preceded by a leader region containing an inverted repeat which acts as a transcription terminator . Expression of glpD is controlled by antitermination of transcription at the inverted repeat . Antitermination is effected by the glpP gene product in conjunction with glycerol-3-phosphate and, consequently, GlpP mutants fail to grow on glycerol as a sole carbon and energy source . We have isolated a number of glycerol-positive revertants of GlpP mutants . Most of these revertants have mutations in the inverted repeat of the glpD leader and produce glycerol-3-phosphate dehydrogenase constitutively . Unlike wild-type bacteria, they are not sensitive to glucose repression of glpD . A few of the revertants are temperature sensitive, i.e . they grow on glycerol at 32 degrees C but not at 45 degrees C and produce glycerol-3-phosphate dehydrogenase only at 32 degrees C . Northern blot analyses demonstrated that the temperature-sensitive expression of glpD is due to destabilization of glpD mRNA . Furthermore, introduction of the wild-type glpP gene into the revertants stabilized the glpD mRNA . This is probably a result of a direct interaction between the GlpP protein and the leader of glpD mRNA . Besides its function in antitermination of transcription of glpD, it is suggested that GlpP is also involved in controlling glpD mRNA stability . Introduction of the glpP gene into the revertants also restored glucose repression, indicating that this repression is mediated by the GlpP protein. Biosci Biotechnol Biochem, 1996 Jan, 60(1), 111 - 6 A regulatory mechanism for the balanced synthesis of membrane phospholipid species in Escherichia coli; Saha SK et al.; The mechanism that assures the balanced synthesis of zwitterionic (phosphatidylethanolamine) and acidic phospholipids (phosphatidylglycerol and cardiolipin) in Escherichia coli has been examined by genetically manipulating the two enzymes at the biosynthetic branch point, i.e., phosphatidylglycerophosphate synthase, encoded by pgsA, and phosphatidylserine synthase, encoded by pssA . A mutant in which the most part of the pssA gene was replaced with a drug resistance gene lacked phosphatidylserine synthase and phosphatidylethanolamine and required divalent metal ions for growth, as did a previously reported insertion-inactivated pssA mutant . When this mutant harbored a plasmid containing a Bacillus subtilis gene that encodes membrane-bound phosphatidylserine synthase, the phosphatidylethanolamine content was dependent on its activity, in contrast to that with the soluble E . coli counterpart . A defective mutation, pgsA3, caused reductions not only in acidic-phospholipid synthesis but also in phosphatidylethanolamine synthesis, despite the normal level of phosphatidylserine synthase activity . These results, together with previous observations, indicate that phosphatidylserine synthesis requires the membrane-associated form of phosphatidylserine synthase, which is related to the membrane-levels of acidic phospholipids, thus yielding balanced compositions of zwitterionic and acidic phospholipids. Mol Microbiol, 1996 Jan, 19(1), 145 - 58 In vitro selection of optimal AbrB-binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures; Xu K et al.; The AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters . Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination . Using in vitro selection techniques, we have isolated optimal AbrB-binding sites from oligonucleotides containing 22 or 44 random base pairs . The best of these sites have an apparent in vitro Kd which is fivefold lower than a similar-sized DNA fragment containing the sequence corresponding to the AbrB-binding site on the spo0E gene . We tested one of the sites in vivo and found that it confers AbrB-mediated control upon a promoter not normally regulated by AbrB . In each of four separate trials, the selected sites possess motifs that converge to a simple consensus . It is argued that the nature and spacing of these motifs produce a type of three-dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant. Zh Mikrobiol Epidemiol Immunobiol, 1996 Jan-Feb, (1), 75 - 7 {The efficacy of the new bacterial preparation biosporin in treating acute intestinal infections}; Gracheva NM et al.; The clinico-laboratory study of the new probiotic Biosporin revealed its effectiveness in the treatment of patients with acute enteric infections . The pronounced curative action of the preparation, manifested by the rapid normalization of stool, the disappearance of abdominal pains and the decrease of dysbiosis in the intestine, was demonstrated . The best results were registered after the administration of Biosporin containing 2 x 10(9) microbial cells (Bacillus subtilis and Bacillus licheniformis) . Biosporin was well tolerated by the patients, no side effects were observed . Biosporin is recommended for use in medical practice for the treatment of patients with acute enteric infections. Crit Rev Microbiol, 1996, 22(2), 101 - 38 Alkaline-fermented foods: a review with emphasis on pidan fermentation; Wang J et al.; Alkaline-fermented foods constitute a group of less-known food products that are widely consumed in Southeast Asia and African countries . They can be made from different raw ingredients . For instance, Japanese natto, Thai thua-nao, and kinema are made from cooked soybeans, dawadawa from African locust beans, ogiri from melon seeds, ugba from African oil beans, kawal from fresh legale leaves, owoh from cotton seeds, and pidan from fresh poultry eggs . In alkaline-fermented foods, the protein of the raw materials is broken down into amino acids and peptides; ammonia is released during the fermentation, raising the pH of the final products and giving the food a strong ammoniacal smell . Most alkaline fermentations are achieved spontaneously by mixed bacteria cultures, principally dominated by Bacillus subtilis . In other cases, pure cultures can be used . For example, Japanese natto is inoculated with a pure culture of B . subtilis var natto . Pidan is a special example of alkaline fermentation . Instead of using microorganisms, pidan is made using an alkali-treated fermentation . Sodium hydroxide (NaOH) is produced from the reaction of sodium carbonate (Na2CO3), water (H2O), and calcium oxide (CaO) of pickle or coating mud . NaOH penetrates into the eggs, causing the physicochemical changes, color changes, and gelation . The appearance of pidan differs from fresh eggs in that the white becomes a semitransparent tea-brown color, and the yolk is solid or semisolid with a dark-green color . The nutritional value of pidan is slightly decreased compared with fresh eggs, but pidan has an extremely long shelf life and a pleasant, fragrant taste that is preferred by most people in Southeast Asian countries . In a small-scale laboratory study conducted by the authors, B . subtilis was not found in pidan . Four Staphylococcus spp . (S . cohnii, S . epidermidis, S . haemolyticus, and S . warneri) and two strains of Bacillus spp . (B . cereus and B . macerans) were isolated from pidan . Staphylococcus spp . did not contribute to the fermentation and were considered contaminants. Adv Exp Med Biol, 1996, 379, 171 - 82 Random mutagenesis of the weak calcium binding site in subtilisin Carlsberg and screening for thermostability by temperature-gradient gel electrophoresis; Sattler A et al.; A random mutagenesis approach was directed to the weak calcium binding site of subtilisin Carlsberg in order to enhance the thermal stability of the enzyme by changing its calcium affinity . The structural motif of the binding site was altered by two strategies, the ligand strategy, which was directed to the amino acid ligands of the calcium ion and the conformation strategy, by which a part of the calcium cave was redesigned . Subtilisin mutants were expressed in Bacillus subtilis and screened for enhanced thermostability by a filter assay and by temperature-gradient gel electrophoresis (TGGE) . Characterization of selected mutants and application of TGGE to investigate the thermal stability of proteases and protease-inhibitor complexes in general is described. Biosens Bioelectron, 1996, 11(4), 443 - 8 Covalent enzyme immobilization on paramagnetic polyacrolein beads; Varlan AR et al.; An optimized procedure of covalent glucose oxidase, urease, Bacillus subtilis alpha-amylase and Bacillus licheniformis alpha-amylase immobilization on paramagnetic, non-porous, polyacrolein beads is presented . The resulting insolubilized enzymes can be employed for extended periods of time without loss of activity . The conditions were optimized for maximizing the activity of the linked enzyme . Coated beads bearing up to 15 micrograms active enzyme/mg(beads) were obtained on reproducible basis . The paramagnetic feature of the particles facilitates the enzyme handling . In the magnetic field, the enzyme separation is fast and complete . Thus, the paramagnetic beads represent an excellent carrier for immobilized enzymes. Trends Genet, 1996 Jan, 12(1), 31 - 4 Determination of cell fate in Bacillus subtilis; Errington J; On starvation, the soil bacterium Bacillus subtilis stops dividing and initiates sporulation, a simple developmental process involving the differentiation of two cell types . Sporulation begins with a reorganization of the cell cycle, to produce cells with the size and chromosome content appropriate for the developmental process . The central division that would normally occur, to produce a pair of identical daughter cells, is blocked and the cell divides asymmetrically to produce a small, polar prespore cell and a much larger mother cell . The developmental fates of the two cells are dictated by the localized activation of cell-specific transcription factors, which are controlled by mechanisms that respond to the cellular asymmetry. J Biomed Mater Res, 1996 Summer, 33(2), 101 - 5 Glutaraldehyde retains its disinfectant properties in presence of hydroxypropylmethyl cellulose (HPMC) gel; Matchette LS et al.; Explanted medical devices are routinely sent to laboratories at the Center for Devices and Radiological Health for analysis . The shipping of these devices presents potential hazards to personnel as well as an opportunity for damage to the devices . In an effort to address these concerns, a viscous disinfecting shipping medium that would limit splashing and cushion a suspended device was proposed . Consequently, we investigated the disinfectant properties of adding a gelling agent, hydroxypropylmethyl cellulose (HPMC) to common disinfectants . We found that the germicidal effectiveness of 2.5% glutaraldehyde in 0.05 M borax when tested against Bacillus subtilis spores was not changed by the addition of 2% HPMC . In addition, HPMC appears to be compatible with 70% ethanol and at least one commercial disinfectant containing a quaternary ammonium compound . Preliminary experiments indicate that an HPMC-disinfectant gel is a potentially useful packaging agent for minimizing the hazards to personnel and materials during shipping of explanted medical devices . The use of such a medium would be subject to guidelines within the context of a program for handling biologically contaminated materials. Mikrobiologiia, 1996 Jan-Feb, 65(1), 74 - 8 {Physiological effects of sunlight on Bacillus subtilis strains with varying capacity to repair photoproducts}; Lotareva OV; Nonlethal doses of sunlight were established to cause a growth delay in Bacillus subtilis strain 1201 possessing an unimpaired repair system . This effect was not observed with the 1201-10 uvr1 mutant deficient in DNA excision repair; however, addition of an extract of uvr1+ cells restorated the phenomenon of growth delay by sunlight in this mutant . Experiments with glass light filters showed that a weakened growth delay in strain 1201 could also be produced by rays of sunlight with wavelengths greater than 380 nm . Maximum growth delay was observed after irradiation with the complete spectrum of sunlight . Experiments with the protein synthesis inhibitor chloramphenicol indicated that sunlight promotes synthesis of proteins preventing the dying of cells . The presence of Casamino acids is favorable for the synthesis of protective proteins. Radiats Biol Radioecol, 1996 Jan-Feb, 36(1), 63 - 7 {Effects of single and fractionated irradiation on Bacillus subtilis spores under different incubation conditions}; Mal'tsev VN et al.; Effect of 60Co gamma-rays on inactivation of Bac . subtilis spores was studied . In the course of investigation, the dose of gamma-rays was divided in to two parts with an interval of 4 or 24 hours between irradiation . Fractional irradiation was found to be more effective for decontamination of Bac . subtilis spores than single irradiation. Plasmid, 1996 Jan, 35(1), 14 - 30 A positive selection vector for the analysis of structural plasmid instability in Bacillus subtilis; Meima R et al.; A system for the positive selection of structural plasmid rearrangements in Bacillus subtilis was developed . Random deletions removing a transcription terminator structure in the assay plasmid, designated pGP100, resulted in expression of the cat-86 gene, under control of a constitutive bacteriophage promoter . The resulting chlorampenicol-resistant colonies were analyzed for plasmid contents and were shown, by restriction analysis, to contain initially both the intact parental plasmid and a deletion variant . Sequence analysis of deletion derivatives revealed a consensus target site (5'-A-T-T-A-A/T-3') at or near deletion termini, which resembles topoisomerase I target sites . Endpoints on one side of the deletions were found to be clustered in the promoter region of the tetracycline resistance gene present on pGP100, the gene product of which is an integral membrane protein . Furthermore, deletion of the genes encoding the ATP-dependent exonuclease, AddAB, severely reduced the structural stability of pGP100 . The data indicate that similar mechanisms underlie deletion formation in pGP100, and a different plasmid-based system, pGP1, which we have analyzed previously. Plasmid, 1996 Jan, 35(1), 1 - 13 Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria; Metcalf WW et al.; We describe several new cloning vectors for mutagenesis and allele replacement experiments . These plasmids have the R6K gamma DNA replication origin (oriR(R6K gamma) so they replicate only in bacteria supplying the pi replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of pi . They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria . Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA . Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker . One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants . Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones . Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker . Plasmid-free segregants with an allele replacement can be subsequently selected as TetS or SucR recombinants . A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well. FEMS Microbiol Lett, 1996 Jan 1, 135(1), 9 - 15 The role of CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis; Henkin TM; The CcpA protein has been identified as a key regulator of carbon metabolism in Bacillus subtilis . CcpA is a DNA binding protein in the LacI/GalR transcriptional repressor family, and genes which respond to CcpA contain common cis-acting target sequences (Ccp boxes) . A number of pathways involved in carbon source utilization are repressed by CcpA, while at least one gene which is involved in excretion of excess carbon is activated by CcpA . Genes repressed by CcpA generally contain Ccp boxes within or downstream of the promoter, while ackA, which is activated by CcpA, contains Ccp boxes upstream of the promoter . It therefore appears that CcpA acts globally to direct carbon flow in B . subtilis. Nucleic Acids Res, 1996 Jan 1, 24(1), 41 - 5 NRSub: a non-redundant database for Bacillus subtilis; Perriere G et al.; In the context of the international project aimed at sequencing the whole genome of Bacillus subtilis we have developed a non-redundant, fully annotated database of sequences from this organism . Starting from the B.subtilis sequences available in the EMBL, GenBank and DDBJ collections we have removed all encountered duplications and then added extra annotations to the sequences (e.g . accession numbers for the genes, locations on the genetic map, codon usage, etc.) We have also added cross-references to the EMBL, MEDLINE, SWISS-PROT and ENZYME data banks . The present system results from merging of the NRSub and SubtiList databases and the sequence contigs used in the two systems are identical . NRSub is distributed as a flatfile in EMBL format (which is supported by most sequence analysis software packages) and as an ACNUC database, while SubtiList is distributed as a relational database under 4th Dimension . It is possible to access the data through two dedicated World Wide Web servers located in France and Japan. Microbiology, 1996 Jan, 142 ( Pt 1), 71 - 7 Molecular analysis of an operon in Bacillus subtilis encoding a novel ABC transporter with a role in exoprotein production, sporulation and competence; Leskela S et al.; The levels of exoamylase and other exoenzymes of Bacillus subtilis are pleiotropically decreased by the ecs-26 (prs-26) and ecs-13 (prs-13) mutations . These mutations also cause a competence- and sporulation-deficient phenotype . In the present work, the ecs locus, which has been defined by the ecs-26 and ecs-13 mutations, was cloned and sequenced . Sequence analysis revealed a putative operon of three ORFs (ecsA, ecsB and ecsC) . ecsA can encode a putative polypeptide of 248 amino acid residues containing an ATP-binding site . The polypeptide shows about 30% sequence similarity with the ATP-binding components of numerous membrane transporters of the ABC-type (ATP-binding cassette transporters or traffic ATPases) . The ecs-26 mutation was found to result from a transition of one base pair chaning the glycine164 of EcsA to a glutamic acid residue in the vicinity of the putative ATP-binding pocket . ecsB was predicted to encode a hydrophobic protein with six membrane-spanning helices in a pattern found in other hydrophobic components of ABC transporters . The properties deduced for the ecsA and ecsB gene products are consistent with the interpretation that ecs encodes a novel ABC-type membrane transporter of B . subtilis . The third ORF, ecsC, can encode a putative polypeptide of 237 amino acid residues . The polypeptide does not resemble components of ABC transporters. Photochem Photobiol, 1996 Jan, 63(1), 74 - 8 Experimental correspondence between spore dosimetry and spectral photometry of solar ultraviolet radiation; Munakata N et al.; The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores . Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer . To obtain the effective spectrum, the irradiance for each 1 nm wavelenght interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples . Integration of the effective spectrum provided the estimate for ID/min . The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993 . The average ratio (+/- SD) between them was 1.24 (+/- 0.16) for 14 data points showing high inactivation rates (> 0.05 ID/min) . Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation. J Bacteriol, 1996 Jan, 178(2), 560 - 3 A Bacillus subtilis malate dehydrogenase gene; Jin S et al.; A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase . Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation . In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation. J Bacteriol, 1996 Jan, 178(2), 546 - 9 Exchange of precursor-specific elements between Pro-sigma E and Pro-sigma K of Bacillus subtilis; Carlson HC et al.; sigma E and sigma K are sporulation-specific sigma factors of Bacillus subtilis that are synthesized as inactive proproteins . Pro-sigma E and pro-sigma K are activated by the removal of 27 and 20 amino acids, respectively, from their amino termini . To explore the properties of the precursor-specific sequences, we exchanged the coding elements for these domains in the sigma E and sigma K structural genes and determined the properties of the resulting chimeric proteins in B . subtilis . The pro-sigma E-sigma K chimera accumulated and was cleaved into active sigma K, while the pro-sigma K-sigma E fusion protein failed to accumulate and is likely unstable in B . subtilis . A fusion of the sigE "pro" sequence to an unrelated protein (bovine rhodanese) also formed a protein that was cleaved by the pro-sigma E processing apparatus . The data suggest that the sigma E pro sequence contains sufficient information for pro-sigma E processing as well as a unique quality needed for sigma E accumulation. J Bacteriol, 1996 Jan, 178(2), 424 - 34 Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein; Saxild HH et al.; The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined . Sequence analysis showed that the genes were localized immediately downstream of the hut operon . Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp . A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure . In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine . By the use of lacZ fusions, the regulation was found to be at the level of transcription . The operon expression was subject to glucose repression . Upstream of the dra gene an open reading frame of 313 amino acids was identified . Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides . The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator). J Bacteriol, 1996 Jan, 178(1), 61 - 7 The gntP gene of Escherichia coli involved in gluconate uptake; Klemm P et al.; The gntP gene, located between the fim and uxu loci in Escherichia coli K-12, has been cloned and characterized . Nucleotide sequencing of a region encompassing the gntP gene revealed an open reading frame of 447 codons with significant homology to the Bacillus subtilis gluconate permease . Northern (RNA) blotting indicated that the gntP gene was monocistronic and was transcribed as an mRNA with an apparent molecular size of 1.54 kb . The transcriptional start point was determined by primer extension analysis . The gntP gene was found to be under catabolite repression and was not induced by gluconate . Also, expression seemed to be stringently controlled . Several observations indicated that the GntP protein is an inner membrane protein; it contains characteristic membrane-spanning regions and was isolated predominantly from the inner-membrane fraction of fractionated host cells . A topology analysis predicted a protein with 14 membrane-spanning segments . The inability of a mutant strain to grow on gluconate minimal medium could be relieved by introduction of a plasmid encoding the gntP gene . Finally, the kinetics of GntP-mediated gluconate uptake were investigated, indicating an apparent Km for gluconate of